HK40015700B - Liquid protein formulations containing viscosity-lowering agents - Google Patents

Description

Liquid protein formulations comprising viscosity reducing agents

The application has application date of 2014, 9 and 11, and application numbers of: 2014800615057, title of invention: a divisional application of the invention patent application of "liquid protein formulation comprising viscosity reducing agent".

Cross reference to related applications

The present application claims U.S. provisional application 62/030,521 entitled "Low-Viscisity Protein Formulations connections Hypophobic Salts" filed on 7/29/2014, U.S. provisional application 62/026,497 entitled "Low-Viscisity Protein Formulations connections GRAS Viscosurfacity-Reducing Agents" filed on 7/18/2014, U.S. provisional application 62/008,050 entitled "Low-Viscity Protein Formulations connections proteins" filed on 5/2014, U.S. provisional application 61/988,005 entitled "filed on 5/2/2014, U.S. provisional application 20142 entitled" Low-Viscity Protein Formulations connections 2014, U.S. provisional application 54 entitled "Low-Visity Protein Formulations connections 2014 temporary application 2014 2 filed on 2/28/2014, U.S. provisional application 2014 2 filed on 28/3/54, U.S. provisional application 2014 for" Protein Formulations proteins connections 2014-10 "filed on 7/24/3/42, and U.S. provisional application 3621 entitled" filed on 3/3, The priority and benefits of U.S. provisional application 61/940,227 entitled "centralized, Low-visual High-Molecular-Weight Protein Formulations" filed on 14.2.2014 and U.S. provisional application 61,876,621 entitled "centralized, Low-visual, High-Molecular-Weight Protein Formulations" filed on 11.9.2013, the disclosures of which are expressly incorporated herein by reference.

Technical Field

The present invention is generally in the field of injectable low viscosity pharmaceutical formulations of highly concentrated proteins and methods of making and using the same.

Background

Monoclonal antibodies (mabs) are important protein-based therapeutics for the treatment of various human diseases such as cancer, infectious diseases, inflammation, and autoimmune diseases. Over 20 monoclonal antibody products have been approved by the U.S. Food and Drug Administration (FDA) and about 20% of all biopharmaceuticals currently being evaluated in clinical trials are monoclonal antibodies (Daugherty et al, adv. drug Deliv. Rev.58: 686. 706, 2006; and Buss et al, curr. opinion in Pharmacol.12: 615. 622, 2012).

Monoclonal antibody-based therapeutics are usually administered repeatedly over extended periods of time and require several mg/kg doses. Antibody solutions or suspensions can be administered by parenteral routes, such as by Intravenous (IV) infusion and Subcutaneous (SC) or Intramuscular (IM) injection. The subcutaneous or intramuscular routes reduce treatment costs during administration, increase patient compliance and improve convenience for both the patient and the healthcare provider compared to the intravenous route. To be effective and pharmaceutically acceptable, parenteral formulations should preferably be sterile, stable, injectable (e.g., via syringe), and non-irritating at the site of injection, in order to comply with FDA guidelines. These routes of administration for high dose protein therapeutics require concentrated protein solutions because of the small volumes required for subcutaneous (typically less than about 2mL) and intramuscular (typically less than about 5mL) injections. These high concentrations often result in very viscous formulations that are difficult to administer by injection, cause pain at the injection site, are often imprecise and/or may have reduced chemical and/or physical stability.

These characteristics lead to manufacturing, storage and use requirements that may not be easily met, particularly for formulations with high concentrations of high molecular weight proteins such as monoclonal antibodies. All protein therapeutics are affected to some extent by physical and chemical instabilities, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation and tailoring (Wang et al, j.pharm.sci.96:1-26,2007). Therefore, the development of optimized formulations is crucial in the development of commercial protein drugs.

High protein concentrations pose challenges associated with the physical and chemical stability of the protein and difficulties in the production, storage and delivery of protein formulations. One problem is the tendency of proteins to aggregate and form particles during processing and/or storage, which makes handling during further processing and/or delivery difficult. Concentration-dependent degradation and/or aggregation is a major challenge in developing higher concentration protein formulations. Reversible self-association in aqueous solution can occur unless the native protein aggregates and the possibility of particle formation, which also contributes to viscosity increases that complicate injection delivery (see, e.g., Steven J.Shire et al, J.Pharm. Sci.93: 1390-. Viscosity increase is one of the major challenges encountered in concentrated protein compositions, affecting both the production process and the ability to easily deliver such compositions by conventional methods (see, e.g., J. Jezek et al, Advanced Drug Delivery Reviews 63: 1107-.

Highly viscous liquid formulations are difficult to prepare, draw into syringes and inject subcutaneously or intramuscularly. The use of force in handling viscous formulations can lead to excessive foaming, which can further denature and inactivate the therapeutically active protein. High viscosity solutions also require larger diameter injection needles and cause more pain at the injection site.

Currently marketed monoclonal antibody products for administration by subcutaneous or intramuscular injection are typically formulated to prevent aggregation and improve stability in aqueous buffers such as phosphate or L-histidine buffers and excipients or surfactants such as mannitol, sucrose, lactose, trehalose, sodium chloride, and sodium chloride, or sodium chloride,(a nonionic triblock copolymer consisting of a central hydrophobic chain, i.e. polyoxypropylene (poly (propylene oxide)), and two hydrophilic chains, i.e. polyoxyethylene (poly (ethylene oxide)), on both sides) or80(PEG (80) sorbitan monolaurate). The reported concentrations of antibody formulated as described above are typically up to about 100mg/mL (Wang et al, J.Pharm.Sci.96:1-26,2007).

U.S. Pat. No. 7,758,860 describes the use of buffers and viscosity-reducing inorganic salts such as calcium chloride or magnesium chloride to reduce viscosity in the formulation of low molecular weight proteins. However, these same salts showed little effect on the viscosity of the high molecular weight antibody (IMA-638) formulation. The viscosity of aqueous formulations of high molecular weight proteins has been reduced by the addition of salts such as arginine hydrochloride, sodium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride or sodium acetate at concentrations greater than about 100mM as described in U.S. patent 7,666,413 or by the addition of organic or inorganic acids as described in U.S. patent 7,740,842. However, these salts do not reduce the viscosity to the required level and in some cases make the formulation sufficiently acidic to likely cause pain at the injection site.

U.S. Pat. No. 7,666,413 describes formulations with reduced viscosity containing specific salts and reconstituted anti-IgE monoclonal antibodies, but the maximum antibody concentration is only about 140mg/mL at the most. U.S. Pat. No. 7,740,842 describes E25 anti-IgE monoclonal antibody preparations containing acetate/acetate buffer where the antibody concentration is up to 257 mg/mL. Salts such as NaCl, CaCl₂Or MgCl₂The addition of (a) was confirmed to reduce the dynamic viscosity under high shear conditions; however, the salts produce an undesirable and significant increase in dynamic viscosity at low shear. Furthermore, inorganic salts such as NaCl can reduce solution viscosity and/or reduce aggregation (EP 1981824).

Non-aqueous antibody or protein formulations have also been described. WO2006/071693 describes non-aqueous suspensions of up to 100mg/mL of monoclonal antibody in a formulation with a viscosity enhancing agent (polyvinylpyrrolidone, PVP) and a solvent (benzyl benzoate or PEG 400). WO2004/089335 describes 100mg/mL non-aqueous lysozyme suspension formulations containing PVP, glycofurol, benzyl benzoate, benzyl alcohol or PEG 400. US2008/0226689a1 describes a 100mg/mL single phase three vehicle component (polymer, surfactant and solvent) non-aqueous viscous formulation of human growth hormone (hGH). Us patent 6,730,328 describes non-aqueous, hydrophobic, non-polar, low reactivity vehicles such as perfluorodecalin for protein formulations. These formulations are not optimal and have high viscosities that hinder processing, preparation and injection; results in the presence of multiple vehicle components in the formulation; and present potential regulatory challenges associated with the use of FDA unapproved polymers.

Alternative non-aqueous protein or antibody formulations have been described using organic solvents such as benzyl benzoate (Miller et al, Langmuir 26: 1067-1754, 2010), benzyl acetate, ethanol or methyl ethyl ketone (Srinivasan et al, pharm. Res.30:1749-1757, 2013). In both cases, a viscosity of less than 50 centipoise (cP) is achieved when the protein concentration is configured to be at least about 200 mg/mL. U.S. Pat. No. 6,252,055 describes monoclonal antibody preparations in a concentration range of 100mg/mL up to 257 mg/mL. Formulations at concentrations greater than about 189mg/mL showed significantly increased viscosity, low recovery and processing difficulties. U.S. patent application publication 2012/0230982 describes antibody formulations at concentrations of 100mg/mL to 200 mg/mL. None of these formulations have a low viscosity sufficient to ease injection.

Du and Klibanov (Biotechnology and Bioengineering 108:632-636,2011) describe a viscosity reduction of concentrated aqueous solutions of bovine serum albumin at maximum concentrations up to 400mg/mL and bovine gamma globulin at maximum concentrations up to 300 mg/mL. Guo et al (Pharmaceutical Research 29:3102-3109,2012) describe the use of hydrophobic salts to achieve low viscosity aqueous solutions of four model monoclonal antibodies. The monoclonal antibody formulation used by Guo has an initial viscosity of no greater than 73cP prior to salt addition. In addition, the viscosity of many pharmaceutically important monoclonal antibodies can exceed 1,000cP at therapeutically relevant concentrations.

It is not an trivial matter to control aggregation and viscosity in high concentration monoclonal antibody solutions (EP 2538973). This is confirmed by several monoclonal antibody products currently on the market in the form of high concentration formulations (>100mg/mL) (EP 2538973).

The references cited above indicate that while various groups have attempted to prepare low viscosity formulations of monoclonal antibodies and other therapeutically important proteins, truly useful formulations have not been obtained for a variety of proteins. Notably, many of the above reports use substances whose safety and toxicity characteristics have not been fully established. Thus, these formulations may face a higher regulatory burden prior to approval than formulations containing compounds that are known to be safe. Indeed, even if the compound is demonstrated to substantially reduce viscosity, the compound may eventually be unsuitable for use in formulations intended for injection into the human body.

Due to the problems associated with the high viscosity and other properties of concentrated solutions of large proteins, a variety of pharmaceutically important high molecular weight proteins, such as monoclonal antibodies, are currently administered via intravenous infusion to deliver therapeutically effective amounts of the protein. For example, to provide a therapeutically effective amount of various high molecular weight proteins, such as monoclonal antibodies, in a volume of less than about 2mL, a protein concentration of greater than 150mg/mL is typically required.

It is therefore an object of the present invention to provide concentrated low viscosity liquid formulations of pharmaceutically important proteins, in particular high molecular weight proteins such as monoclonal antibodies.

It is another object of the present invention to provide concentrated, low viscosity liquid formulations of proteins, particularly high molecular weight proteins such as monoclonal antibodies, which are capable of delivering therapeutically effective amounts of these proteins in volumes useful for subcutaneous and intramuscular injections.

It is another object of the present invention to provide concentrated low viscosity liquid formulations of proteins, particularly high molecular weight proteins such as monoclonal antibodies, having low viscosities which may improve injectability and/or patient compliance, convenience and comfort.

It is also an object of the present invention to provide a method for preparing and storing a concentrated low viscosity formulation of a protein, in particular a high molecular weight protein such as a monoclonal antibody.

It is another object of the present invention to provide a method of administering a concentrated, low viscosity liquid formulation of a protein, particularly a high molecular weight protein such as a monoclonal antibody. It is another object of the present invention to provide a method for processing a high concentration of a reduced viscosity biological agent using concentration and filtration techniques known to those skilled in the art.

Disclosure of Invention

Concentrated low viscosity low volume liquid pharmaceutical formulations of proteins have been developed. Such formulations may be administered quickly and conveniently by Subcutaneous (SC) or Intramuscular (IM) injection rather than by prolonged intravenous infusion. These formulations comprise low and/or high molecular weight proteins such as monoclonal antibodies and viscosity reducing agents, typically bulky polar organic compounds such as a wide range of compounds in GRAS (a list of compounds generally recognized as safe by the U.S. food and drug administration), inactive injectable ingredients and FDA approved therapeutics.

The concentration of the protein is from about 10mg/mL to about 5,000mg/mL, more preferably from about 100mg/mL to about 2,000 mg/mL. In some embodiments, the concentration of the protein is from about 100mg/mL to about 500mg/mL, more preferably from about 300mg/mL to about 500 mg/mL. The formulations containing the protein and viscosity reducing agent are stable for a period of at least one month, preferably at least two months and most preferably at least three months when stored at a temperature of 4 ℃. The viscosity of the formulation is less than about 75cP, preferably less than 50cP and most preferably less than 20cP at about 25 ℃. In some embodiments, the viscosity is less than about 15cP or even less than or about 10cP at about 25 ℃. In some embodiments, the viscosity of the formulation is about 10 cP. Formulations containing protein and viscosity reducing agent are typically at about 0.6s when measured using a cone and plate viscometer^-1To about 450s^-1And preferably about 2s^-1To about 400s^-1Shear rate measurement of (2). Formulations containing protein and viscosity reducing agent are typically about 3s when measured using a microfluidic viscometer^-1To about 55,000s^-1And preferably about 20s^-1To about2,000s^-1Shear rate measurement of (2).

The viscosity of the protein formulation is reduced by the presence of one or more viscosity reducing agents. Unless otherwise specifically stated, the term "viscosity reducing agent" includes a single compound and mixtures of two or more compounds. Preferably, the viscosity reducing agent is present in the formulation at a concentration of less than about 1.0M, preferably less than about 0.50M, more preferably less than about 0.30M and most preferably less than about 0.15M. In some embodiments, the viscosity-lowering agent is present in the formulation at a concentration as low as 0.01M. The formulation may have a viscosity that is at least about 30% lower, preferably at least about 50% lower, and most preferably at least about 75% lower than the viscosity of a corresponding formulation under the same conditions except that the viscosity-lowering agent is replaced with an appropriate buffer or salt at about the same concentration. In some embodiments, low viscosity formulations are provided, wherein the viscosity of the corresponding formulation without the viscosity-lowering agent is greater than about 200cP, greater than about 500cP or even greater than about 1,000 cP. In a preferred embodiment, the shear rate of the formulation is at least about 0.5s when measured using a cone and plate viscometer^-1Or at least about 1.0s when measured using a microfluidic viscometer^-1。

For embodiments in which the protein is a "high molecular weight protein," the high molecular weight protein may have a molecular weight of from about 100kDa to about 1,000kDa, preferably from about 120kDa to about 500kDa and most preferably from about 120kDa to about 250 kDa. The high molecular weight protein may be an antibody such as a monoclonal antibody or pegylated or other derivatized form thereof. Preferred monoclonal antibodies include natalizumab (natalizumab)Cetuximab (cetuximab)Bevacizumab (bevacizumab)Trastuzumab (trastuzumab)Infliximab (infliximab)Rituximab (rituximab)Panitumumab (panitumumab)Oxamumumab (ofatumumab)And biological analogs thereof. The optionally pegylated high molecular weight protein may be an enzyme. Other proteins and mixtures of proteins may also be formulated to reduce their viscosity.

In some embodiments, the protein and viscosity-lowering agent are provided in a lyophilized dosage unit sized for reconstitution with a sterile aqueous pharmaceutical vehicle to produce a concentrated low viscosity liquid formulation. The presence of one or more viscosity-lowering agents aids and/or accelerates reconstitution of the lyophilized dosage unit as compared to a lyophilized dosage unit that does not contain a viscosity-lowering agent.

Provided herein are methods for preparing concentrated low viscosity liquid formulations of high molecular weight proteins, such as monoclonal antibodies, and methods for storing and administering low viscosity high concentration protein formulations to a patient. In another embodiment, viscosity reducing agents are added to aid processing (e.g., pumping, concentrating, and/or filtering) by reducing the viscosity of the protein solution.

Drawings

FIG. 1 depicts the bio-analog cetuximab at 25 ℃ and a final pH of 7.0The viscosity in cP of the solution in 0.25M phosphate buffer (PB; rhombus) and in a solution containing 0.25M Camphoresulfonic acid L-lysine (CSAL; squares), which is the function of the protein concentration (in mg/mL)And (4) counting. Data points contain standard deviations, however they are often smaller than symbols.

FIG. 2 depicts the biological analog bevacizumab at 25 ℃ and a final pH of 7.0The viscosity in cP of the solutions in 0.25M phosphate buffer (PB; diamonds) and 0.25M CSAL (squares) as a function of protein concentration (in mg/mL). Data points contain standard deviations, however they are often smaller than symbols.

FIG. 3 is a graph of bevacizumab, a biological analog of 200. + -.9 mg/mL, containing 0.25M phosphate-citrate buffer or camphorsulfonate arginine (CSAA)As a function of pH along the x-axis.

FIG. 4 shows the results of the analysis of the biological analogs bevacizumab (B)About 200mg/mL or 226mg/mL) and 0.25M camphorsulfonic acid arginine (CSAA) as a function of pH. The fold reduction was calculated as the ratio of the viscosity (cP) of the phosphate-citrate buffer to the viscosity (cP) of the 0.25M CSAA solution.

FIG. 5 shows the bio-analog cetuximab (C) containing 0.25M CSAA202 + -5 mg/mL, 229 + -5 mg/mL, or 253 + -4 mg/mL) at 25 deg.C as a function of pH along the x-axis.

FIG. 6 is a size exclusion chromatographic trace depicting storage at 4 ℃ for up to 100 days compared to a fresh reconstituted commercial drug product220mg/mL of aqueous solution as a function of elution time in minutesIntensity (at 280 nm).

FIG. 7 depicts the biological analog bevacizumabViscosity (cP) of aqueous solutions in 0.25M phosphate buffer, 0.10M or 0.25M APMI x 2HCl ((1- (3-aminopropyl) -2-methyl-1H-imidazole dihydrochloride), as a function of protein concentration (mg/mL).

FIG. 8 depicts the biological analog bevacizumabViscosity (cP) of aqueous solutions in 0.25M phosphate buffer, 0.10M thiamine pyrophosphate (TPP) or 0.10M TPP1- (3-aminopropyl) -2-methyl-1H-imidazole (APMI) as a function of protein concentration (mg/mL).

FIG. 9 depicts golimumab with 0.15M phosphate buffer or 0.15M thiamine HClThe viscosity (cP) of the aqueous solution as a function of protein concentration (mg/mL).

Detailed Description

I. Definition of

The term "protein" as generally used herein refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide of sufficient chain length to produce at least a detectable tertiary structure. Proteins with a molecular weight (expressed in kDa, where "Da" stands for "dalton" and 1kDa to 1,000Da) greater than about 100kDa may be designated as "high molecular weight proteins", while proteins with a molecular weight less than about 100kDa may be designated as "low molecular weight proteins". The term "low molecular weight protein" does not include small peptides that lack at least the tertiary structure, which is considered an essential condition required for a protein. Protein molecular weight can be determined using standard methods known to those skilled in the art, including but not limited to mass spectrometry (e.g., ESI, MALDI) or calculated from known amino acid sequences and glycosylation. The protein may be naturally occurring or non-naturally occurring, synthetic or semi-synthetic.

"substantially pure protein" and "substantially pure protein" are used interchangeably herein and refer to a composition comprising at least about 90 wt% pure protein, preferably at least about 95 wt% pure protein. "substantially homogeneous" and "substantially homogeneous" are used interchangeably herein and refer to compositions wherein at least about 90 wt%, preferably at least about 95 wt%, of the proteins present are a combination of monomeric and reversible dimeric and oligomeric associations (not irreversible aggregates).

The term "antibody" as generally used herein broadly encompasses monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies and single chain antibody molecules and antibody fragments (e.g., Fab ', F (ab') 2, and Fv), single domain antibodies, multivalent single domain antibodies, Fab fusion proteins, and fusions thereof.

The term "monoclonal antibody" or "mAb" as generally used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single epitope. For example, these antibodies are typically synthesized by culturing hybridoma cells as described by Kohler et al (Nature 256:495,1975) or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) or isolated from phage antibody libraries using the techniques described by Clackson et al (Nature 352: 624-. "monoclonal antibodies" as used herein specifically includes derivatized antibodies, antibody-drug conjugates, and "chimeric" antibodies (where a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855,1984).

An "antibody fragment" comprises a portion of an intact antibody, including the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng.8:1057-1062, 1995); a single chain antibody molecule; a multivalent single domain antibody; and multispecific antibodies formed from antibody fragments.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof of mostly human sequence (such as Fv, Fab ', F (ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin (see, e.g., Jones et al, Nature 321: 522. sup. 525, 1986; Reichmann et al, Nature 332: 323. sup. 329, 1988; and Presta, curr. Op. struct. biol.2: 593. sup. 596, 1992).

"rheology" refers to the study of deformation and flow of a substance.

"viscosity" refers to the resistance of a substance (typically a liquid) to flow. Viscosity is related to the concept of shear; which is understood to be the effect of different layers of fluid exerting a shear force on each other or on other surfaces as they move relative to each other. There are several viscosity measurement methods. The unit of viscosity is Ns/m²Known as pascal-seconds (Pa-s). The viscosity may be "dynamic" or "absolute". Dynamic viscosity is a measure of the rate at which momentum is transferred through a fluid. It is measured as Stator (St). Kinetic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume but different viscosities are placed in the same capillary viscometer and allowed to flow by gravity, a higher viscosity fluid takes longer to flow through the capillary than a lower viscosity fluid. For example, if one fluid takes 200 seconds(s) to complete its flow and another fluid takes 400s, the viscosity of the second fluid is said to be twice that of the first fluid on a dynamic viscosity scale. The dimension of dynamic viscosity is length²Time/time. Typically, kinematic viscosity is expressed in centistokes (cSt). SI unit of dynamic viscosity is mm²S, which is equal to 1 cSt. "AbsoluteViscosity "(sometimes referred to as" dynamic viscosity "or" simple viscosity ") is the product of dynamic viscosity and fluid density. Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s), where 1cP is 1 mPa-s.

Viscosity can be measured at a given shear rate or multiple shear rates using, for example, a viscometer. The "extrapolated zero shear" viscosity can be determined as follows: a best fit line is created for the four highest shear points on the absolute viscosity versus shear rate curve and the viscosity is linearly extrapolated back to zero shear. Alternatively, for newtonian fluids, viscosity may be determined by an average viscosity value at a plurality of shear rates. Viscosity can also be measured using a microfluidic viscometer at single or multiple shear rates (also known as flow rates), where absolute viscosity is derived from the change in pressure as the liquid flows through the channel. Viscosity is equal to shear stress versus shear rate. In some embodiments, the viscosities measured with a microfluidic viscometer can be directly compared to extrapolated zero shear viscosities, such as those extrapolated from viscosities measured at multiple shear rates using a cone and plate viscometer.

"shear rate" refers to the rate of change in viscosity of a layer of fluid as it passes over an adjacent layer. The velocity gradient is the rate of change of velocity with distance from the plate. This simple case shows the shear rate (v) where the unit is (cm/sec)/(cm)₁-v₂) 1/sec. Thus, the unit of shear rate is the reciprocal seconds or generally the reciprocal time. For microfluidic viscometers, changes in pressure and flow rate are related to shear rate. "shear rate" refers to the rate at which deformation of a material occurs. Formulations containing protein and viscosity reducing agent typically have a shear rate of about 0.5s when measured using a cone and plate viscometer and a spindle appropriately selected by one of skill in the art^-1To about 200s^-1Time measurement to accurately measure viscosity over the range of viscosities of the samples of interest (i.e., 20cP samples were most accurately measured with a CPE40 spindle fixed to a DV2T viscometer (Brookfield)); a shear rate greater than about 20s when measured using a microfluidic viscometer^-1To about 3,000s^-1。

For the classical "newtonian" fluids commonly used herein, viscosity is substantially independent of shear rate. However, for "non-newtonian fluids," the viscosity decreases or increases with increasing shear rate, e.g., the fluid is "shear thinning" or "shear thickening," respectively. In the case of concentrated (i.e. high concentration) protein solutions, this may manifest as pseudoplastic shear thinning behavior, i.e. viscosity decreases with shear rate.

The term "chemical stability" as generally used herein refers to the ability of a protein component in a formulation to resist degradation by chemical pathways such as oxidation, deamidation, or hydrolysis. Protein formulations are generally considered to be chemically stable if less than about 5% of the components degrade after 24 months storage at 4 ℃.

The term "physical stability" as generally used herein refers to the ability of a protein formulation to resist physical deterioration such as polymerization. Physically stable formulations form only an acceptable percentage of irreversible aggregates (e.g., dimers, trimers, or other aggregates) of the biologically active protein. The presence of aggregates can be assessed in a number of ways, including by measuring the average particle size of the protein in the formulation by means of dynamic light scattering. The formulation is considered to be physically stable if the irreversible aggregates formed are less than about 5% after 24 months storage at 4 ℃. An acceptable level of agglomerated impurities should ideally be less than about 2%. While levels as low as about 0.2% are achievable, about 1% is more typical.

The term "stable formulation" as generally used herein refers to a formulation that is both chemically and physically stable. A stable formulation may be one in which more than about 95% of the bioactive protein molecules in the formulation retain bioactivity after 24 months of storage at 4 ℃ or after 1 month of storage at high temperature equivalent processing conditions, such as at 40 ℃. Various analytical techniques for measuring Protein stability are available in the art and are described, for example, in Peptide and Protein Drug Delivery, 247-. Stability may be measured at a selected temperature for a period of time. For example, for rapid screening, the formulation may be stored at 40 ℃ for 2 weeks to 1 month, and then the remaining biological activity measured and compared to the initial conditions to assess stability. When the formulation is stored at 2-8 ℃, the formulation should generally be stable at 30 ℃ or 40 ℃ for at least 1 month and/or at 2-8 ℃ for at least 2 years. When the formulation is stored at room temperature, i.e., about 25 ℃, the formulation should generally be stable at about 25 ℃ for at least 2 years and/or at 40 ℃ for at least about 6 months. The extent of aggregation after lyophilization and storage can be used as an indicator of protein stability. In some embodiments, stability is assessed by measuring the particle size of the protein in the formulation. In some embodiments, stability can be evaluated as follows: the activity of the formulation is measured using standard biological activity or binding assays known to those skilled in the art.

The term "protein particle size" as generally used herein refers to the average diameter of the major population of particles of biologically active molecules in a formulation, or the particle size distribution thereof, as determined by using known particle sizers such as dynamic light scattering, SEC (size exclusion chromatography), or other methods known to those skilled in the art.

The term "concentrated" or "high concentration" as generally used herein describes a liquid formulation having a final concentration of protein of greater than about 10mg/mL, preferably greater than about 50mg/mL, more preferably greater than about 100mg/mL, more preferably greater than about 200mg/mL or most preferably greater than about 250 mg/mL.

As generally used herein, "reconstituted formulation" refers to a formulation prepared as follows: the protein is dissolved or dispersed in an aqueous solution for administration by dissolving the protein in a dry powder, i.e., lyophilized, spray-dried or precipitated from a solvent, in a diluent.

A "lyoprotectant" is a substance that, when combined with a protein, significantly reduces the chemical and/or physical instability of the protein during lyophilization and/or subsequent storage. Exemplary lyoprotectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and combinations thereof. Other exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethyl cellulose. Preferred sugar alcohols are those compounds obtained by reduction of mono-and disaccharides such as lactose, trehalose, maltose, lactulose and maltulose. Other examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Lyoprotectants are typically added to the pre-lyophilized formulation in a "lyoprotecting amount". This means that the protein substantially retains its physical and chemical stability and integrity after lyophilization in the presence of a lyoprotectant amount of the lyoprotectant.

As generally used herein, a "diluent" or "carrier" is an ingredient that is pharmaceutically acceptable (i.e., safe and non-toxic for administration to humans or other mammals) and that can be used to prepare liquid formulations, such as aqueous formulations that are reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution, and combinations thereof.

"preservatives" are compounds that can be added to the formulations of the present invention to reduce contamination and/or effects caused by bacteria, fungi, or other infectious agents. For example, the addition of a preservative may aid in the production of a multiple use (multi-dose) formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, chlorhexidine di-ammonium, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides in which the alkyl group is long chain) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol, and benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol.

As generally used herein, a "bulking agent" is a compound that increases the quality of the lyophilized mixture and promotes the physical structure of the lyophilized cake (e.g., helps to produce a substantially uniform lyophilized cake that retains an open cell structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starches, poly (ethylene glycol), and sorbitol.

A "therapeutically effective amount" is the minimum concentration required to achieve a measurable improvement or prevention of any symptom or particular condition or disorder, a measurable extension of life expectancy, or a substantial improvement in the quality of life of the patient. A therapeutically effective amount will depend on the particular bioactive molecule and the particular condition or disorder being treated. Therapeutically effective amounts of various proteins, such as the monoclonal antibodies described herein, are known in the art. A therapeutically effective amount of a protein that has not yet been determined or a therapeutically effective amount of a known protein, such as a monoclonal antibody, for the treatment of a particular disorder and clinically for the treatment of other disorders can be determined by standard techniques known to those skilled in the art, such as physicians.

The term "injectability" or "needle-through" as generally used herein refers to the injectability of a pharmaceutical formulation through a syringe equipped with an optionally thin-walled 18-32 gauge needle. Injectability depends on a number of factors such as the pressure or force required for injection, uniformity of flow, amount of aspiration, and avoidance of clogging. Injectability of a liquid pharmaceutical formulation can be evaluated by comparing the injection force of the viscosity-lowering formulation with that of a standard formulation without the addition of a viscosity-lowering agent. The reduction in the injection force of the formulation containing the viscosity-lowering agent reflects the improved injectability of the formulation. The viscosity-lowering formulation has improved injectability when the injection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50% and most preferably at least 75% as compared to a standard formulation having the same protein concentration under the same conditions except that the viscosity-lowering agent is replaced with an appropriate buffer at about the same concentration. Alternatively, the injectability of a liquid pharmaceutical formulation can be evaluated as follows: the time required to inject the same volume of a different liquid protein formulation, such as 0.5mL or more preferably about 1mL, when the syringe is depressed with the same amount of force is compared.

The term "injection force" is generally used herein to refer to the force required to push a given liquid formulation through a given syringe equipped with a needle of a given size at a given injection speed. Injection force is usually reported in newtons. For example, the injection force can be measured as the force required to push the liquid formulation through a 0.50 inch 27 gauge needle equipped 1mL plastic syringe with an inner diameter of 0.25 inch at an injection speed of 250 mm/min. The test equipment may be used to measure the injection force. Formulations with lower viscosities will generally require an overall lower injection force when measured under the same conditions.

As used herein, "viscosity gradient" refers to the rate of change in the viscosity of a protein solution as the protein concentration increases. The viscosity gradient can be approximated by a plot of viscosity as a function of protein concentration for a series of formulations that are otherwise identical but differ in protein concentration. As the protein concentration increases, the viscosity increases in an approximately exponential manner. The viscosity gradient at a particular protein concentration can be approximated by the slope of the tangent of the plot of viscosity as a function of protein concentration. The viscosity gradient can be approximated by a linear approximation of the plot of viscosity as a function of any protein concentration or at a narrow window of protein concentration. In some embodiments, a formulation is considered to have a reduced viscosity gradient when the function of viscosity versus protein concentration is approximated as an exponential function having an index that is less than the index obtained for an otherwise identical formulation that does not contain a viscosity-lowering agent. In a similar manner, a formulation is considered to have a lower/higher viscosity gradient if its index is lower/higher than that of the second formulation when compared to the second formulation. The viscosity gradient can be numerically approximated from a plot of viscosity as a function of protein concentration by other methods known to the skilled formulation researcher.

The term "viscosity-reducing formulation" as generally used herein refers to a liquid formulation having a high concentration of a high molecular weight protein, such as a monoclonal antibody or a low molecular weight protein, which is modified by the presence of one or more viscosity-reducing additives as compared to a corresponding formulation without the one or more viscosity-reducing additives.

The term "osmolarity" as generally used herein refers to the total number of components dissolved per liter. Osmolarity is similar to molarity, but includes the total number of moles of various species dissolved in the solution. Molar ratio of 1Osm/LOsmolarity refers to 1 mole of dissolved component per liter of solution. Some solutes, such as ionic solutes that dissociate in solution, will contribute more than 1 mole of dissolved component per mole of solute in solution. For example, NaCl dissociates to Na in solution⁺And Cl^-And thereby provide 2 moles of dissolved component per 1 mole of dissolved NaCl in the solution. The physiological osmolality is typically from about 280mOsm/L to about 310 mOsm/L.

The term "tonicity," as generally used herein, refers to the osmotic pressure gradient resulting from the separation of two solutions by a semi-permeable membrane. Specifically, tonicity is used to describe the osmotic pressure that is generated on both sides of the cell membrane when the cell is exposed to an external solution. Solutes that can pass through the cell membrane do not contribute to the final osmotic pressure gradient. Only dissolved substances that cannot cross the cell membrane will contribute to the osmotic pressure difference and thus to the tonicity.

The term "hypertonic" as generally used herein means that the solution has a higher concentration of solutes than is present inside the cell. When cells are immersed in hypertonic solutions, the tendency is for water to flow out of the cells to balance the concentration of solutes.

The term "hypotonic" as generally used herein means that a solution has a lower concentration of solutes than is present inside the cell. When the cells are immersed in the hypotonic solution, water flows into the cells to balance the concentration of solutes.

The term "isotonic" as generally used herein refers to a solution in which the osmotic pressure gradient across the cell membrane is substantially balanced. Isotonic preparations are preparations having substantially the same osmotic pressure as human blood. Isotonic preparations will generally have an osmotic pressure of about 250 to 350 mOsm/kg.

The term "liquid formulation" as used herein is a protein provided in an acceptable pharmaceutical diluent or reconstituted in an acceptable pharmaceutical diluent prior to administration to a patient.

The terms "brand" and "reference" when used in reference to a protein or a biological product are used interchangeably herein and refer to the individual biological product as permitted under the united states public health service act (42u.s.c. § 262) article 351 (a).

The term "biological analog" as used herein is generally used interchangeably with "general equivalent" or "second generation product". For example, a "biosimilar monoclonal antibody" refers to a subsequent variant of the innovator's monoclonal antibody that is typically produced by another company. "biological analogs," when used in reference to a branded protein or branded biological, can refer to a biological that is evaluated against the branded protein or branded biological and licensed under article 351(k) of the U.S. public health service act (42u.s.c. § 262). The bioanalog monoclonal antibody can be a bioanalog monoclonal antibody that satisfies one or more guidelines (ref. EMA/CHMP/BMWP/403543/2010) that are passed on by the european drug administration pharmaceutical Commission (CHMP) on day 5, 30 of 2012 and are published by the european union as "guiding on pharmaceutical biological products relating to monoclonal antibodies-non-clinical and clinical issues".

Biological analogs can be produced by microbial cells (prokaryotic, eukaryotic), cell lines of human or animal origin (e.g., mammalian, avian, insect), or tissues derived from animals or plants. The expression construct of the proposed biosimilar product will typically encode the same primary amino acid sequence as its reference product. There may be minor modifications such as N-or C-terminal truncations that do not have an impact on safety, purity or efficacy.

The bio-analog monoclonal antibody is physico-chemical or biologically similar in safety and efficacy to the reference monoclonal antibody. The bioanalog monoclonal antibodies can be evaluated relative to a reference monoclonal antibody using one or more in vitro studies including assays detailed as follows: binding to one or more antigens of interest; isoforms that bind to Fc γ receptors (Fc γ RI, Fc γ RII, and Fc γ RIII), FcRn, and complement (C1 q); fab-related functions (e.g., neutralization of soluble ligands, receptor activation or blocking); or Fc-related functions (e.g., antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, complement activation). In vitro comparison with in vivo comparisons demonstrating similarity of pharmacokinetics, pharmacodynamics and/or safetyAnd (6) data combination. Clinical evaluation of a bioanalog monoclonal antibody relative to a reference monoclonal antibody can include comparing pharmacokinetic properties (e.g., AUC)_0-inf、AUC_0-t、C_max、t_max、C_trough) (ii) a A pharmacodynamic endpoint; or similarity of clinical effects (e.g., using a random parallel group comparative clinical trial). Quality comparisons between bioanalogue monoclonal antibodies and reference monoclonal antibodies can be evaluated using established procedures, including those described in "guidelines on biological reagent products relating to biological activity as active substtate" ("EMEA/CHMP/BWP/49348/2005") and "guidelines on development, production, characterization and characterization for monoclonal antibodies and related substtate" ("EMEA/CHMP/BWP/157653/2007").

Differences between the bio-analog monoclonal antibody and the reference monoclonal antibody can include post-translational modifications, for example, by attaching other biochemical groups such as phosphate esters, various lipids, and carbohydrates to the monoclonal antibody; by proteolytic cleavage after translation; by altering the chemical nature of the amino acid (e.g., formylation); or by a variety of other mechanisms. Other post-translational modifications may be the result of manufacturing process manipulations, for example glycosylation may occur upon exposure of the product to a reducing sugar. In other cases, storage conditions may allow for some degradation pathways such as oxidation, deamidation, or aggregation. All of these product-associated variants can be included in the bio-analog monoclonal antibody.

The term "viscosity reducing agent" as used herein refers to a compound that acts to reduce the viscosity of a solution in the absence of the viscosity reducing agent as compared to the viscosity of the solution. The viscosity reducing agent may be a single compound or may be a mixture of one or more compounds. When the viscosity-reducing agent is a mixture of two or more compounds, the concentrations listed refer to each individual agent unless otherwise specified. For example, a formulation containing about 0.25M camphorsulfonic acid arginine as a viscosity reducing agent is a solution wherein the concentration of camphorsulfonic acid is 0.25M and the concentration of arginine is 0.25M.

Some viscosity reducing agents contain acidic or basic functional groups. Whether these functional groups are fully or partially ionized depends on the pH of the formulation in which they are present. Unless otherwise indicated, when referring to a formulation containing a viscosity reducing agent having an ionizable functional group, both the parent compound and any possible ionization state are included.

The term "hydrogen bond donor" as used herein refers to a hydrogen atom attached to an atom that is relatively electronegative, forming a partial positive charge on the hydrogen atom.

The term "hydrogen bond acceptor" as used herein refers to a relatively electronegative atom or functional group capable of interacting with a hydrogen atom bearing a partial positive charge.

The term "free rotating bond" as used herein refers to any singly bonded pair of non-hydrogen atoms.

The term "molecular polar surface area" as used herein refers to the total polar area exposed on the surface of the molecule of interest.

The term "molar volume" as used herein refers to the total volume occupied by a mole of the molecule of interest in its native state (i.e., solid, liquid).

The term "polarizability" as used herein refers to the dipole moment induced when a molecule of interest is subjected to an electric field of unit intensity.

The term "pharmaceutically acceptable salt" as used herein refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids and bases and organic acids and bases. Suitable non-toxic acids include inorganic and organic acids such as acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Suitable positively charged counterions include sodium, potassium, lithium, calcium and magnesium.

The term "ionic liquid" as used herein refers to a crystalline or amorphous salt, zwitterion, or mixture thereof that is a liquid at or near a temperature at which most conventional salts are solids, said temperature being less than 200 ℃, preferably less than 100 ℃ or more preferably less than 80 ℃. Some ionic liquids have a melting temperature near room temperature, e.g., 10 ℃ to 40 ℃ or 15 ℃ to 35 ℃. The term "zwitterion" is used herein to describe a molecule that is generally neutral in charge, bearing both a formal positive charge and a formal negative charge on different chemical groups of the molecule. Examples of ionic liquids are found in ridean et al, chem.soc.rev.,42: 9055-; rantwijk et al, chem.Rev.,107:2757-2785, 2007; earle et al, Pure appl. chem.,72(7), 1391-; and Sheldon et al, Green chem.,4:147-151, 2002.

The term "organophosphate ester" as used herein refers to a compound containing one or more phosphoryl groups, at least one of which is covalently linked to an organic group by a phosphoester bond.

As used herein, a "water-soluble organic dye" is an organic molecule that has a molar solubility of at least 0.001M at 25 ℃ and pH 7 and absorbs some wavelengths of light, preferably the visible to infrared portion of the electromagnetic spectrum, while transmitting or reflecting other wavelengths of light.

The term "chalcogen" as used herein refers to group 16 elements, including oxygen, sulfur, and selenium in any oxidation state. For example, unless otherwise specified, the term "chalcogen" also includes SO₂。

The term "alkyl" as used herein refers to straight, branched and cyclic hydrocarbon groups. Unless otherwise indicated, the term "alkyl" includes hydrocarbon groups containing one or more double or triple bonds. Alkyl groups containing at least one ring system are "cycloalkyl groups". An alkyl group containing at least one double bond is an "alkenyl" and an alkyl group containing at least one triple bond is an "alkynyl".

The term "aryl" as used herein refers to an aromatic carbocyclic ring system including fused ring systems. In an "aryl group," each atom forming the ring is a carbon atom.

The term "heteroaryl" as used herein refers to an aromatic ring system, including fused ring systems, wherein at least one atom forming the ring is a heteroatom.

The term "heterocycle" as used herein refers to a non-aromatic ring system including fused ring systems wherein at least one atom forming the ring is a heteroatom.

The term "heteroatom" as used herein is any non-carbon or non-hydrogen atom. Preferred heteroatoms include oxygen, sulfur and nitrogen. Exemplary heteroaryl and heterocyclyl rings include benzimidazolyl, benzofuranyl, benzothienyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4 aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5, 2-dithiazinyl, dihydrofuro [2,3-b ] tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, 3H-indolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatoiyl, isobenzofuranyl, isochromanyl, isoindolyl, isoindolinyl, dihydroindolyl, dihydrooxazolyl, and pharmaceutically acceptable salts thereof, Isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, decahydroisoquinolinyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyrazolooxazolyl, pyrazoloimidazolyl, pyrazolothiazolyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, Pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazinyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, and xanthenyl.

Preparation II

Biocompatible low viscosity protein solutions those biocompatible low viscosity protein solutions, such as monoclonal antibodies, can be used to deliver therapeutically effective amounts of protein in volumes suitable for Subcutaneous (SC) and Intramuscular (IM) injections, typically less than or about 2mL for subcutaneous injections and less than or about 5mL for intramuscular injections, more preferably less than or about 1mL for subcutaneous injections and less than or about 3mL for intramuscular injections. The protein may generally have any molecular weight, although in some embodiments, high molecular weight proteins are preferred. In other embodiments, the protein is a low molecular weight protein.

The formulation may have a protein concentration of about 10mg/mL to about 5,000 mg/mL. Formulations, including monoclonal antibody formulations, can have a protein concentration of greater than 100mg/mL, preferably greater than 150mg/mL, more preferably greater than about 175mg/mL, more preferably greater than about 200mg/mL, more preferably greater than about 225mg/mL, more preferably greater than about 250mg/mL and most preferably greater than or about 300 mg/mL. In the absence of a viscosity-lowering agent, the viscosity of the protein formulation increases exponentially with increasing concentration. Such protein formulations may have a viscosity of greater than 100cP, greater than 150cP, greater than 200cP, greater than 300cP, greater than 500cP or even greater than 1,000cP when measured at 25 ℃ in the absence of a viscosity-lowering agent. Such formulations are generally not suitable for subcutaneous or intramuscular injection. The use of one or more viscosity reducing agents allows for the preparation of formulations having a viscosity of less than or about 100cP, preferably less than or about 75cP, more preferably less than or about 50cP, more preferably less than or about 30cP, more preferably less than or about 20cP or most preferably less than or about 10cP when measured at 25 ℃.

While viscosity reducing agents may be used to reduce the viscosity of concentrated protein formulations, they may also be used in less concentrated formulations. In some embodiments, the formulation may have a protein concentration of about 10mg/mL to about 100 mg/mL. The formulation may have a protein concentration greater than about 20mg/mL, greater than about 40mg/mL, or greater than about 80 mg/mL.

For some proteins, formulations without viscosity-lowering agents may have a viscosity greater than about 20cP, greater than about 50cP, or greater than about 80 cP. The use of one or more viscosity reducing agents allows for the preparation of formulations having a viscosity of less than or about 80cP, preferably less than or about 50cP, more preferably less than about 20cP or most preferably less than or about 10cP when measured at 25 ℃.

In some embodiments, the aqueous protein formulation has a viscosity that is at least about 30% lower than a similar formulation without the one or more viscosity-lowering agents when measured under the same conditions. In other embodiments, the formulation has a viscosity that is 40% lower, 50% lower, 60% lower, 70% lower, 80% lower, 90% lower, or even more than 90% lower than a similar formulation without one or more viscosity-lowering agents. In a preferred embodiment, the formulation contains a therapeutically effective amount of one or more high molecular weight proteins, such as monoclonal antibodies, in a volume of less than about 2mL, preferably less than about 1mL, or more preferably less than about 0.75 mL.

The reduced viscosity formulation has improved injectability and requires less injection force than an otherwise identical formulation without the viscosity-reducing agent (e.g., in phosphate buffer). In some embodiments, the injection force is reduced by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 2-fold as compared to a standard formulation otherwise identical but lacking the one or more viscosity-lowering agents. In some embodiments, the formulation has "newtonian flow characteristics," which is defined as having a viscosity that is substantially independent of shear rate. The protein formulation can be easily injected through a needle having a size of about 18-32 gauge. Preferred needle sizes for delivery of the low viscosity formulations include 27, 29 and 31 gauge, optionally thin walled.

The formulation may contain one or more other excipients such as buffers, surfactants, sugars and sugar alcohols, other polyols, preservatives, antioxidants and chelating agents. The formulation has a pH and osmolarity suitable for administration without causing significant adverse side effects. In some embodiments, the pH of the concentrated low viscosity formulation is 5 to 8, 5.5 to 7.6, 6.0 to 7.6, 6.8 to 7.6, or 5.5 to 6.5.

Low viscosity protein formulations may allow greater flexibility in formulation development. A low viscosity formulation may exhibit a viscosity change that is less dependent on protein concentration than an otherwise identical formulation that does not contain a viscosity-lowering agent. Low viscosity protein formulations may allow for increased protein concentrations and reduced frequency of protein administration. In some embodiments, the low viscosity protein formulation contains 2 or more, 3 or more, or 4 or more different proteins. For example, a combination of 2 or more monoclonal antibodies may be provided in a single low viscosity protein formulation.

Because a protein (such as a monoclonal antibody) formulation can be administered to a patient at a higher protein concentration than an otherwise similar protein formulation that does not contain a viscosity-lowering agent, the frequency of administration of the protein can be reduced. For example, when the protein is formulated with a viscosity-lowering agent, the protein that previously needed to be administered once a day may be administered once every two days, once every three days, or even less frequently. Proteins that currently require multiple administrations on the same day (at the same or different times of the day) can be administered in fewer injections per day. In some cases, the frequency may be reduced to a single injection once per day. By increasing the dose administered per injection by a multiple, the frequency of administration can be reduced, for example from once every 2 weeks to once every 6 weeks.

In some embodiments, the liquid formulation has an osmolality of, for example, about 280mOsm/L to about 310 mOsm/L. In some embodiments, the liquid formulation has an osmolality greater than about 250mOsm/L, greater than about 300mOsm/L, greater than about 350mOsm/L, greater than about 400mOsm/L, or greater than about 500 mOsm/L. In some embodiments, the formulation has an osmolality of about 200 to about 2,000mOsm/L or about 300 to about 1,000 mOsm/L. In some embodiments, the liquid formulation is substantially isotonic with human blood. In some cases, the liquid formulation may be hypertonic.

Additives, including viscosity reducing agents, can be introduced in any amount to achieve a desired viscosity level of the liquid formulation, so long as the amount is not toxic or harmful and does not substantially interfere with the chemical and/or physical stability of the formulation. In some embodiments, the one or more viscosity reducing agents may be present independently at the following concentrations: less than about 1.0M, preferably less than about 0.50M, less than or equal to about 0.30M or less than or equal to 0.15M. Particularly preferred concentrations include about 0.15M and about 0.30M. For some embodiments having two or more viscosity-lowering agents, the substances are preferably, but not necessarily, present at the same concentration.

The viscosity reducing agent allows for faster reconstitution of the lyophilized dosage unit. The dosage unit is a lyophilized cake of protein, viscosity-lowering agent, and other excipients to which water, saline, or other pharmaceutical fluid is added. In the absence of a viscosity-lowering agent, it typically takes 10 minutes or more to completely dissolve the high protein concentration lyophilized cake. When the lyophilized cake contains one or more viscosity-lowering agents, the time required to completely dissolve the lyophilized cake is typically reduced by one-half, one-fifth, or one-tenth of what it took to completely dissolve. In some embodiments, less than 1 minute is required to completely dissolve a lyophilized cake having a protein concentration greater than or about 150, 200, or even 300 mg/mL.

Low viscosity protein formulations allow greater flexibility in formulation development. The low viscosity formulation exhibits a viscosity that changes less as the protein concentration increases compared to an otherwise identical formulation that does not contain one or more viscosity-lowering agents. The low viscosity formulation exhibits a reduced viscosity gradient compared to an otherwise identical formulation without the viscosity-lowering agent.

The viscosity gradient of the protein formulation may be one-half, one-third, or even less than one-third, as compared to the viscosity gradient of an otherwise identical protein formulation without the one or more viscosity-lowering agents. For protein formulations having a protein concentration of 10mL/mg to 2,000mL/mg, the protein formulation can have a viscosity gradient of less than 2.0cPML/mg, less than 1.5cPML/mg, less than 1.0cPML/mg, less than 0.8cPML/mg, less than 0.6cPML/mg, or less than 0.2 cPML/mg. By reducing the viscosity gradient of the formulation, the protein concentration can be increased to a higher level before an exponential increase in viscosity is observed.

A. Protein

Any protein may be formulated, including recombinant, isolated or synthetic proteins, glycoproteins or lipoproteins. These proteins may be antibodies (including antibody fragments and recombinant antibodies), enzymes, growth factors or hormones, immunomodulators, anti-infective factors, antiproliferative factors, vaccines or other therapeutic, prophylactic or diagnostic proteins. In some embodiments, the protein has a molecular weight greater than about 150kDa, greater than 160kDa, greater than 170kDa, greater than 180kDa, greater than 190kDa, or greater than 200 kDa.

In some embodiments, the protein may be a pegylated protein. The term "pegylated protein" as used herein refers to a protein having one or more poly (ethylene glycol) or other stealth polymer groups covalently attached to the protein, optionally through a chemical linker that may be different from the one or more polymer groups. Pegylated proteins are characterized by their generally reduced renal filtration, reduced uptake by the reticuloendothelial system and reduced enzymatic degradation, resulting in, for example, extended half-life and increased bioavailability. Stealth polymers include poly (ethylene glycol); poly (propylene glycol); poly (amino acid) polymers such as poly (glutamic acid), poly (hydroxyethyl-L-asparagine), and poly (hydroxyethyl-L-glutamine); poly (glycerol); poly (2-oxazoline) polymers such as poly (2-methyl-2-oxazoline) and poly (2-ethyl-2-oxazoline); poly (acrylamide); poly (vinyl pyrrolidone); poly (N- (2-hydroxypropyl) methacrylamide); and copolymers and mixtures thereof. In a preferred embodiment, the stealth polymer in the pegylated protein is poly (ethylene glycol) or a copolymer thereof. The pegylated protein may be randomly pegylated, i.e., a stealth polymer having one or more non-specific sites covalently attached to the protein or may be pegylated in a site-specific manner by covalently attaching the stealth polymer to one or more specific sites on the protein. Site-specific pegylation can be achieved, for example, using an activated stealth polymer having one or more reactive functional groups. See, e.g., Hoffman et al, Progress in Polymer Science,32: 922-.

In a preferred embodiment, the protein is high molecular weight and is an antibody (most preferably a monoclonal antibody) and has a high viscosity in a buffered aqueous solution when concentrated enough to inject a therapeutically effective amount in a volume of no more than 1.0 to 2.0mL for subcutaneous injection and in a volume of no more than 3.0 to 5.0mL for intramuscular injection. High molecular weight proteins may include those described in: scolnik, mAbs 1:179-184, 2009; beck, mAbs 3: 107-; baumann, curr. drug meth.7:15-21,2006; or Federici, Biologicals 41: 131-. The protein used in the formulations described herein is preferably substantially pure and substantially homogeneous (i.e., substantially free of contaminating proteins and/or irreversible aggregates thereof).

Preferred monoclonal antibodies herein include natalizumabCetuximabBevacizumabTrastuzumabInfliximabRituximabPanitumumabOlympic single antibodyAnd biological analogs thereof. Exemplary high molecular weight proteins may include toclizumabAlemtuzumab (marketed under several trade names), brodalumab (developed by Amgen, Inc. ("Amgen"), denosumab (r) ("alemtuzumab"))And) And biological analogs thereof.

Exemplary molecular targets of the antibodies described herein include CD proteins such as CD3, CD4, CD8, CD19, CD20, and CD 34; HER receptor family members such as the EGF receptor, HER2, HER3, or HER4 receptor; cell adhesion molecules such as LFA-1, Mo1, p150,95, VLA-4, ICAM-1, VCAM, and α v/β 3 integrins, including their α or β subunits (e.g., anti-CD 11a, anti-CD 18, or anti-CD 11b antibodies); growth factors, such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptors; protein C; PCSK9 and the like.

Antibody therapeutics currently on the market

Many protein therapeutics, especially the antibodies defined herein, currently on the market are administered via intravenous infusion due to the high doses required. The formulation may comprise one of the antibody therapeutics currently on the market or a biological analog thereof. Some protein therapeutics currently on the market are not high molecular weight, but are still administered via intravenous infusion due to the high doses required for therapeutic efficacy. In some embodiments there is provided a liquid formulation of these low molecular weight proteins as defined herein at a concentration suitable for delivering a therapeutically effective amount for subcutaneous or intramuscular injection.

Antibody therapeutics currently on the market include belimumab (belimumab)Golimumab (Simponi)) Abciximab, and pharmaceutically acceptable salts thereofTo be provided withMarketed combination of tositumomab (tositumomab) and iodo-131 tositumomab, alemtuzumabPalivizumab (palivizumab)Basiliximab (basiliximab)ado-trastuzumab emtansinePertuzumab (pertuzumab)Caromab pentosan (capromab pendentide)caclizumabIbritumomab tiuxetan (ibritumomab tiuxetan)Aikuiti monoclonal antibody (eculizumab)Epipilimumab (ipil)imumab)Moluomab (muromonab) -CD3 (Orthoclone)) Rexibacumab (raxibacumab), nimotuzumab (nimotuzumab)Berentuzumab vitamin A (brentuximabvedotin)Adalimumab (adalimumab)GollimumabPalivizumabOmalizumab (omalizumab)Heyoutke monoclonal antibody (ustekinumab)

Natalizumab is used for the treatment of multiple sclerosis and crohn's disease as a humanized monoclonal antibody against the cell adhesion molecule α 4-integrin. Natalizumab previously under the trade nameMarketed and currently marketed by Biogen Idec ("Biogen") and Elan Corp. ("Elan") toAre listed together.Produced in murine myeloma cells. Each 15mL dose contains 300mg natalizumab; 123mg sodium chloride USP; 17.0mg sodium dihydrogen phosphate monohydrate USP; 7.24mg disodium phosphate heptahydrate USP; 3.0mg polysorbate 80 USP/NF; water for intravenous injection USP pH 6.1. Natalizumab is typically administered by monthly Intravenous (IV) infusion and has been shown to be effective in treating the symptoms of multiple sclerosis and crohn's disease and for preventing relapse, vision loss, cognitive decline, and significantly improving the quality of life of patients.

The term "NATALIZUMAB" as used herein includes monoclonal antibodies or antigen-binding portions thereof against the cell adhesion molecule α 4-integrin known by the international non-proprietary name "NATALIZUMAB". Natalizumab includes antibodies described in U.S. patent 5,840,299, U.S. patent 6,033,665, U.S. patent 6,602,503, U.S. patent 5,168,062, U.S. patent 5,385,839, and U.S. patent 5,730,978. Natalizumab includes the trade name Biogen Idec and Elan CorporationActive agents or their bio-analogue products in marketed products.

Cetuximab is an Epidermal Growth Factor Receptor (EGFR) inhibitor useful for the treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab is a chimeric (murine/human) monoclonal antibody that is typically administered by intravenous infusion. Cetuximab is available under the trade nameMarketed by Bristol-Myers Squibb Company (North America; "Bristol-Myers Squibb"), Eli Lilly and Company (North America; "Eli Lilly"), and Merck KGaA for intravenous use only.Produced in mammalian (murine myeloma) cell culture.Each single use 50mL vial of (a) contains 100mg cetuximab at a concentration of 2mg/mL and is formulated in a preservative-free solution containing 8.48mg/mL sodium chloride, 1.88mg/mL sodium phosphate dibasic heptahydrate, 0.42mg/mL sodium phosphate monobasic monohydrate, and water for intravenous injection USP.

Cetuximab is indicated for the treatment of patients with KRAS wild-type metastatic colorectal cancer (mCRC) expressing Epidermal Growth Factor Receptor (EGFR) in combination with chemotherapy and as a single agent in patients who are not able to be treated or who are intolerant to irinotecan with oxaliplatin and irinotecan-based therapies. Cetuximab is useful for treating patients with squamous cell carcinoma of the head and neck in combination with platinum-based chemotherapy for first-line treatment of recurrent and/or metastatic disease and in combination with radiotherapy for locally advanced disease. Approximately 75% of patients with metastatic colorectal cancer have EGFR-expressing tumors and are thus believed to be suitable for treatment with cetuximab or panitumumab according to FDA guidelines.

The term "CETUXIMAB" as used herein includes monoclonal antibodies or antigen-binding portions thereof known by the international non-proprietary name "CETUXIMAB". Cetuximab includes antibodies described in U.S. patent No. 6,217,866. Cetuximab includes the trade nameActive agents and their bio-analogue products are marketed.The biological analogs of (a) may include those currently being developed by Amgen, AlphaMab co., Ltd. ("AlphaMab") and Actavis plc ("Actavis").

Bevacizumab, a humanized monoclonal antibody that inhibits vascular endothelial growth factor a (VEGF-a), acts as an anti-angiogenic agent. Under the trade name ofFrom Gendentech, Inc. ("Genentech") and f.hoffmann-La Roche, LTD ("Roche") are marketed. It is approved for the treatment of a variety of cancers, including colorectal, lung, breast (except in the united states), glioblastoma (in the united states only), renal, and ovarian cancers.Approved by the FDA in 2004 for metastatic colorectal cancer when used with standard chemotherapy (as first-line therapy) and for second-line metastatic colorectal cancer when used with 5-fluorouracil-based therapies. FDA approval in 2006In combination with carboplatin/paclitaxel chemotherapy for first-line advanced non-squamous non-small cell lung cancer.Administered by intravenous infusion once every three weeks at a dose of 15mg/kg or 7.5 mg/kg. Higher doses are typically administered with carboplatin-based chemotherapy, while lower doses are administered with cisplatin-based chemotherapy. FDA approval in 2009For metastatic renal cell carcinoma (a form of renal carcinoma). FDA in 2009 also allowed accelerated approvalFor the treatment of recurrent glioblastoma multiforme. Treatment for initial growth is still in phase III clinical trials.

The national comprehensive cancer network ("NCCN") recommends bevacizumab as a standard first-line therapy in combination with any platinum-based chemotherapy, and then holds bevacizumab until disease progression. NCCN updated its clinical practice guidelines in oncology for breast cancer (NCCN guidelines) in 2010 to confirm the use of bevacizumab in the treatment of metastatic breast cancer: (NCCN guidelines)Genentech/Roche).

The term "BEVACIZUMAB" as used herein includes monoclonal antibodies or antigen-binding portions thereof that inhibit vascular endothelial growth factor a (VEGF-a) known by the international non-proprietary/generic name "BEVACIZUMAB". Bevacizumab is described in U.S. Pat. No. 6,054,297. Bevacizumab includes the trade name bevacizumabActive agents and their bio-analogue products are marketed.The biological analogs of (a) can include those currently being developed by Amgen, Actavis, AlphaMab, and Pfizer, Inc ("Pfizer").The biological analogs of (a) can include a biological analog known as BCD-021, produced by Biocad and currently being tested clinically in the United states.

Trastuzumab is a monoclonal antibody that interferes with the HER2/neu receptor. Trastuzumab is available under the trade nameMarketed by Genentech, Inc.Produced by a mammalian cell (chinese hamster ovary (CHO)) line.Is a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous administration. Each one of which isVial contained 440mg trastuzumab, 9.9mg L-histidine HCl, 6.4mg L-histidine, 400mg a, a-trehalose dihydrate and 1.8mg polysorbateAlcohol esters 20 USP. Reconstitution with 20mL of water provided a multi-dose solution containing 21mg/mL trastuzumab.Currently, they are administered via intravenous infusion at a frequency of once weekly and at a dose of about 2mg/kg to about 8 mg/kg.

Trastuzumab is used primarily for the treatment of some breast cancers. The HER2 gene is amplified in 20-30% of early breast cancers, which allows it to overexpress the Epidermal Growth Factor (EGF) receptor in the cell membrane. Trastuzumab is typically administered as a maintenance therapy for patients with HER2 positive breast cancer, usually for one year following chemotherapy. Trastuzumab is currently administered via intravenous infusion at a frequency of once a week and at a dose of about 2mg/kg to about 8 mg/kg.

The term "TRASTUZUMAB" as used herein includes monoclonal antibodies known by the international non-proprietary/common name "TRASTUZUMAB" to interfere with the HER2/neu receptor or antigen-binding portions thereof. Trastuzumab is described in U.S. patent No. 5,821,337. Trastuzumab includes tradenameActive agents and their biological analogs in marketed products. The term "trastuzumab" includes the trade name trastuzumabMarketed and under the trade name Mylan, Inc. (the name "Mylan")Marketed by Biocon, Ltd. ("Biocon")Active agents in bio-analog products. Trastuzumab may include that being developed by Amgen and PlantForm Corporation, CanadaActivity in Bioanalogue productsA sex agent.

Infliximab is a monoclonal antibody directed against tumor necrosis factor alpha (TNF-a) for the treatment of autoimmune diseases. Under the trade name ofFrom Janssen Global Services, LLC ("Janssen") (USA), Mitsubishi Tanabe Pharma (Japan), Xian Janssen (China), and Merck&Co ("Merck") (other countries and regions) are on the market. Infliximab is a chimeric mouse/human monoclonal antibody with a high molecular weight, i.e., about 144 kDa. In some embodiments, the formulation containsBiological analogs such as remimas^TMOr INFLECTRA^TM. REMIMA developed by Celltrion, Inc. (a. ("Celltrion"))^TMAnd INFLECTRA developed by Hospira Inc., UK^TMHas been recommended for administrative approval in europe. Celltrion has submitted REMIMMA to the FDA^TM. Infliximab is currently administered via intravenous infusion at a dose of about 3mg/kg to about 10 mg/kg.

Infliximab contains about 30% murine variable region amino acid sequences that confer antigen binding specificity to human TNF α. The remaining 70% corresponded to the human IgG1 heavy chain constant region and the human kappa light chain constant region. Infliximab has high affinity for human TNF α, a cytokine with a variety of biological effects including mediating inflammatory responses and modulating the immune system.

Infliximab is a recombinant antibody that is typically produced and secreted by mouse myeloma cells (SP2/0 cells). The antibodies are currently produced by continuous pre-perfusion of cell cultures. Infliximab is expressed using a chimeric antibody gene that constitutes: the variable region sequence cloned from mouse anti-TNF alpha hybridoma cell A2 and the human antibody constant region sequence provided by plasmid expression vector. Murine anti-TNF α hybridomas are generated by immunizing BALB/c mice with purified recombinant human TNF α. The heavy and light chain vector constructs were linearized and transfected into Sp2/0 cells by electroporation. Standard purification steps may include chromatographic purification, viral inactivation, nanofiltration and ultrafiltration/diafiltration.

The term "INFLIXIMAB" as used herein includes chimeric mouse/human monoclonal antibodies or antigen-binding portions thereof known by the international non-proprietary name "INFLIXIMAB". Infliximab neutralizes the biological activity of TNF α and inhibits its binding to its receptor by binding with high affinity to the soluble, transmembrane form of TNF α. See us patent 5,698,195 for infliximab. The term "infliximab" includes the trade name infliximabBy multiple companies, with REMIMA^TMFrom Celltrion and at INFLECTRA^TMAn active agent in a product marketed or to be marketed by Hospira, Inc ("Hospira"). Infliximab is provided in the form of a sterile lyophilized cake for reconstitution and dilution. Each vial of infliximab contains 100mg infliximab and excipients such as sodium phosphate monobasic monohydrate, sodium phosphate dibasic dihydrate, sucrose, and polysorbate 80.

denosumab(And) Is a human monoclonal antibody and is the first RANKL inhibitor approved for postmenopausal women at risk of osteoporosis and patients with bone metastases from solid tumors. denosumab is undergoing phase II clinical trials for the treatment of rheumatoid arthritis.

Panitumumab is a fully human monoclonal antibody approved by the FDA for the treatment of EGFR-expressing metastatic cancer with disease progression. Panitumumab is available under the trade nameMarketed by Amgen.In the form of a 20mg/ml panitumumab concentratePackaged in 5ml, 10ml and 15ml vials for intravenous infusion. The final concentration of panitumumab, when formulated according to the package insert, does not exceed 10 mg/ml.Administered by intravenous infusion at a dose of 6mg/kg once every 14 days. The term "PANITUMUMAB" as used herein includes anti-human epidermal growth factor receptors known by the international non-proprietary name "PANITUMUMAB". The term "panitumumab" includes the trade namesActive agents and their biological analogs in the products marketed by Amgen. The term "panitumumab" includes monoclonal antibodies described in U.S. Pat. No. 6,235,883. The term "panitumumab" includesBioanalogue products, including those being developed by BioXpress, SA ("BioXpressA biological analog.

BelimumabIs a human monoclonal antibody inhibiting B cell activating factor (BAFF) having a molecular weight of about 151.8 kDa. Belimumab is approved in the united states, canada, and europe for the treatment of systemic lupus erythematosus. Belimumab is currently administered to lupus patients by intravenous infusion at a dose of 10 mg/kg. The high molecular weight, low viscosity protein formulation may comprise belimumab, preferably at a concentration of about 400mg/mL to about 1,000 mg/mL. Preferred ranges are based on 40-100kg (about 80-220 pounds) of body weight in a volume of 1 mL.

AbciximabManufactured by Janssen Biologics BV and by Eli Lilly&Company ("Eli Lilly") distribution. Abciximab is a Fab fragment of the chimeric human/murine monoclonal antibody 7E 3. Abciximab binds to the Glycoprotein (GP) IIb/IIIa receptor of human platelets and inhibits platelet aggregation by preventing the binding of fibrinogen, von Willebrand factor and other adhesion molecules. It also binds to the vitronectin (α v β 3) receptor found on platelets, vascular wall endothelial cells and smooth muscle cells. Abciximab is a platelet aggregation inhibitor used primarily during and after coronary procedures. Abciximab was administered via intravenous infusion, first as a bolus injection at 0.25mg/kg, followed by continuous intravenous infusion at 0.125 mcg/kg/min for 12 hours.

TositumomabIs a medicament for treating follicular lymphoma. It is an IgG2a anti-CD 20 monoclonal antibody derived from immortalized mouse cells. Tositumomab was administered by sequential infusion: cold monoclonal antibody followed by iodine: (¹³¹I) Tositumomab is the same antibody that is covalently bound to the radionuclide iodine-131. Clinical trials have established the efficacy of the tositumomab/idotositumomab dosing regimen in patients with relapsed refractory follicular lymphoma.Currently, administration is via intravenous infusion at a dose of 450 mg.

Alemtuzumab (withOrAre on the market and are currently availableDeveloped) are monoclonal antibodies for the treatment of Chronic Lymphocytic Leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma. It is also used to treat some autoimmune diseases such asMultiple sclerosis. Alemtuzumab has a molecular weight of about 145.5 kDa. It was administered by intravenous infusion at 30mg per day to patients with B-cell chronic lymphocytic leukemia.

PalivizumabIs a humanized monoclonal antibody directed against an epitope in the antigenic site A of the F protein of respiratory syncytial virus. In two phase III clinical trials conducted on the pediatric population, palivizumab reduced the risk of hospitalization due to respiratory syncytial virus infection by 55% and 45%. Palivizumab was administered once a month via an intramuscular injection of 15 mg/kg.

Ofatumumab is a human anti-CD 20 monoclonal antibody that appears to inhibit early B lymphocyte activation. Aframumab is available under the trade nameMarketed by GlaxoSmithKline, plc ("GlaxoSmithKline").Distributed in single use vials containing 100mg/5mL and 1,000mg/50mL ofatumumab for intravenous infusion. Ofatumumab is approved by the FDA for the treatment of chronic lymphocytic leukemia and has also shown potential in the treatment of follicular non-hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing remitting multiple sclerosis. The molecular weight of ofatumumab is about 149 kDa. It is currently administered by intravenous infusion at an initial dose of 300mg, followed by 2,000mg by intravenous infusion once a week. The term "OFATUMUMAB" as used herein includes the anti-CD 20 monoclonal antibody known by the international non-proprietary name "OFATUMUMAB". The term "ofatumumab" includes the termActive agents and their biological analogs in marketed products. The term "ofatumumab" includes those being developed by BioexpressActive agents in bio-analog products. The high molecular weight, low viscosity liquid protein formulation may comprise ofatumumab, preferably at a concentration of about 300mg/mL to about 2,000 mg/mL.

trastuzumab emtansine (ado-trastuzumab emtansine in the United states, toMarketed) are prepared by reaction with a cytotoxic agent mertansineAn antibody-drug conjugate comprised of the linked monoclonal antibody trastuzumab. Trastuzumab stops the growth of cancer cells by binding to HER2/neu receptor, while mertansine enters cells and destroys cells by binding to tubulin. trastuzumab emtansine is approved in the united states for the treatment of particularly recurrent HER2 positive metastatic breast cancer. A number of phase III clinical trials on trastuzumab emtansine were planned or underway in 2014. trastuzumab emtansine is currently administered by intravenous infusion at 3.6 mg/kg. The high molecular weight, low viscosity liquid formulation may comprise trastuzumab emtansine, preferably at a concentration of about 144mg/mL to about 360 mg/mL.

PertuzumabIs a monoclonal antibody that inhibits dimerization of HER 2. Pertuzumab was FDA approved in 2012 for the treatment of HER2 positive metastatic breast cancer. The currently recommended dose of pertuzumab is 420mg to 840mg by intravenous infusion. The high molecular weight, low viscosity liquid formulation may comprise pertuzumab, preferably at a concentration of about 420mg/mL to about 840 mg/mL.

Daclizumab (daclizumab) is a humanized anti-CD 25 monoclonal antibody and is used to prevent rejection in organ transplants, particularly kidney transplants. The drug is also being investigated for the treatment of multiple sclerosis. The molecular weight of daclizumab is about 143 kDa. Daclizumab is available in the United statesMarketed by Hoffmann-La Roche, Ltd. ("Roche") and dosed by intravenous infusion of 1 mg/kg. Daclizumab high production methods (DAC HYP; BIIB 019; Biogen Idec ("Biogen") and AbbVie, Inc. ("AbbVie")) are in phase III clinical trials for monthly subcutaneous injections of 150mg for the treatment of relapsing-remitting multiple sclerosis. The high molecular weight, low viscosity liquid formulation may comprise daclizumab, preferably at a concentration of about 40mg/mL to about 300 mg/mL.

Aikume monoclonal antibodyAre humanized monoclonal antibodies approved for the treatment of rare blood disorders such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Ekutemab with a molecular weight of about 148kDa was developed by Alexion Pharmaceuticals, Inc ("Alexion"). It is administered by intravenous infusion in an amount of about 600mg to about 1,200 mg. The high molecular weight, low viscosity liquid formulation may comprise eculizumab, preferably at a concentration of about 500mg/mL to about 1,200 mg/mL.

Tuzhu monoclonal antibodyIs a humanized monoclonal antibody directed against the interleukin-6 receptor. It is an immunosuppressive drug used mainly for the treatment of Rheumatoid Arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form of rheumatoid arthritis in children. Tolizumab is typically administered by intravenous infusion at a dose of about 6mg/kg to about 8 mg/kg. The high molecular weight, low viscosity liquid formulation may comprise tositumumab, preferably at a concentration of about 240mg/mL to about 800 mg/mL.

RituximabIs a chimeric anti-CD 20 monoclonal antibody for use in the treatment of a variety of diseases characterized by an excess number of B cells, overactive B cells, or dysfunctional B cells. Rituximab for the treatment of leukopeniaCancers of the cellular system such as leukemias and lymphomas, including hodgkin's lymphoma and its lymphocyte-predominant subtype. It has been shown to be an effective rheumatoid arthritis therapy. Rituximab is widely used outside the specification for the treatment of difficult cases of multiple sclerosis, systemic lupus erythematosus and autoimmune anemia.

Rituximab is available in the United states under the trade name RituximabMarketed by Biogen and Genentech in combination and under the trade name outside the united statesMarketed by Roche.Distributed in single use vials containing 100mg/10mL and 500mg/50 mL.Typically about 375mg/m by intravenous infusion²To administer the drug. The term "RITUXIMAB" as used herein includes the anti-CD 20 monoclonal antibody known by the international non-proprietary/common name "RITUXIMAB". Rituximab includes monoclonal antibodies described in U.S. patent No. 5,736,137. Rituximab is included under the trade nameAndactive agents and their biological analogs in marketed products.

The high molecular weight, low viscosity liquid formulation may comprise rituximab, preferably at a concentration of about 475mg/mL to about 875mg/mL (approximated using the body surface area range obtained from Mosteller's equation for a human of 5 feet 40kg to6 feet 100kg, i.e., 1.3 to 2.3 square meters). The concentration was calculated for 1mL of formulation.

Epiperca is available from Bristol-Myers Squibb Company ("Bristol-Myer)s Squibb ") was used. Which is provided withAre marketed and used for the treatment of melanoma and are undergoing clinical trials for the treatment of non-small cell lung cancer (NSCLC), Small Cell Lung Cancer (SCLC) and metastatic hormone refractory prostate cancer. Epimedium antibody is currently administered by intravenous infusion at 3 mg/kg. The high molecular weight, low viscosity liquid formulation may contain the epirubicin, preferably at a concentration of about 120mg/mL to about 300 mg/mL.

Rebaudibus monoclonal antibodyAre human monoclonal antibodies directed to the prevention and treatment of inhaled anthrax. It is currently administered by intravenous infusion. The recommended dose in adults and children over 50kg is 40 mg/kg. The high molecular weight, low viscosity liquid formulation may comprise resibazumab, preferably at a concentration of about 1,000mg/mL to about 4,000 mg/mL.

Nimotuzumab (A), (B)BIOMAB) Is a humanized monoclonal antibody with molecular weight of about 151kDa for treating head and neck squamous cell carcinoma, recurrent or refractory high-grade malignant glioma, anaplastic astrocytoma, glioblastoma, diffuse intrinsic pontine glioma. Nimotuzumab is typically administered by weekly intravenous infusions of about 200 mg. The high molecular weight, low viscosity liquid formulation may comprise nimotuzumab, preferably at a concentration of about 200 mg/mL.

Betuzumab vitamineIs an antibody-drug conjugate directed against the protein CD30 expressed in classical hodgkin lymphoma and systemic anaplastic large cell lymphoma. It is administered by intravenous infusion at about 1.8mg/kgAnd (4) administration. The high molecular weight, low viscosity liquid formulation may comprise a bevacizumab visfate, preferably at a concentration of about 80mg/mL to about 200 mg/mL.

itolizumabIs a humanized IgG1 monoclonal antibody developed by Biocon. itolizumab successfully completed phase III clinical trials in patients with moderate to severe psoriasis. itolizumab has received marketing approval in india; no approval application has been filed with the FDA.

Obinutuzumab originally developed by Roche and being further developed according to a cooperative agreement with BiogenIs a humanized anti-CD 20 monoclonal antibody approved for the treatment of chronic lymphocytic leukemia. It is also being studied in phase III clinical trials on patients with various lymphomas. A dose of about 1,000mg is administered via intravenous infusion.

Pegylated certolizumab (certolizumab pegol)Is a recombinant humanized antibody Fab' fragment conjugated with polyethylene glycol of about 40kDa (PEG2MAL40K) having specificity for human tumor necrosis factor alpha (TNF alpha). The molecular weight of the pegylated certolizumab ozogamicin is about 91 kDa.

Other antibody therapeutics that can be formulated with the viscosity-lowering agent include CT-P6 from Celltrion, Inc.

Antibody therapeutics in later trials and development

Antibody therapeutics that progress to late clinical development and administrative approval are evolving at a rapid pace. Over 300 monoclonal antibodies were in clinical trials in 2014 and 30 commercially-initiated antibody therapeutics are being evaluated in late-stage studies. The FDA was recently filed a first time for marketing with two monoclonal antibodies, vedolizumab (vedolizumab) and ramucirumab (ramucirumab). Amgen is currently initiating multiple phase III trials with additional trial plans or patient recruitment for brodalumab use in patients with plaque psoriasis. Two phase I clinical trials of mab 1(Xilonix) against patients with advanced cancer or type 2 diabetes have been initiated by XBiotech, inc. Other trials with MABp1 are recruiting patients. For the treatment of leukemia with moxetumomab pasudotox, multiple trials were initiated by MedImmune, LLC ("MedImmune") and patients were ongoing or enrolled. Studies are ongoing on the long-term safety and efficacy of treatment of chronic plaque psoriasis using tiltrakizumab. Multiple phase II trials for the treatment of various cancers with rilotumumab have recently been completed.

At least 28 monoclonal antibodies currently in progress or recently completed phase III studies directed to the treatment of inflammatory or immune disorders, cancer, high cholesterol, osteoporosis, alzheimer's disease and infectious diseases are high molecular weight proteins. Monoclonal antibodies currently undergoing or recently completing phase III trials include AMG145, eltotuzumab, Epatuzumab (Epratuzumab), farlettuzumab (MORAB-003), gantenezumab (RG1450), gevokizumab, Ezeuzumab ozogamicin (inotub ozogamicin), itolizumab, Ixekizumab, lebrikizumab, mepolizumab (mepolizumab), naptumomab estafatenox, necitumumab, nivolumab, ocrelizumab, onartuzumab, racutuzumab, ramucirumab, reslizumab, romosozumab, sariluzumab, secuumumab, sirukuzumab, soeuzumab, tabauzumab and dolizumab. Monoclonal antibody mixtures (actoxumab and bezlotoxumab) are also being evaluated in phase III assays. See, e.g., Reichert, MAbs 5:1-4,2013.

Victorizumab is a monoclonal antibody developed by Millennium Pharmaceuticals, Inc ("Millennium"; subsidiary of Takeda Pharmaceuticals Company, Ltd. ("Takeda")). Vedolizumab was found to be safe and highly effective in terms of inducing and maintaining clinical remission in patients with moderate to severe ulcerative colitis. Phase III clinical trials have shown that it achieves the goal of inducing clinical responses and maintaining remission in patients with crohn's disease and ulcerative colitis. Studies evaluating long-term clinical outcomes indicate that nearly 60% of patients achieve clinical remission. A common dose of Vidolizumab is 6mg/kg for intravenous infusion.

Ramucirumab is a human monoclonal antibody developed for the treatment of solid tumors. Phase III clinical trials are ongoing for the treatment of breast cancer, metastatic gastric adenocarcinoma, non-small cell lung cancer and other types of cancer. Ramucirumab was administered by intravenous infusion at about 8mg/kg in some phase III trials.

rilotumumab is a human monoclonal antibody that inhibits the action of hepatocyte growth factor/scatter factor. It was developed by Amgen and in phase III trials for the treatment of solid tumors. An open phase III study for treatment with rilotumumab in advanced or metastatic esophageal cancer patients would administer rilotumumab at about 15mg/kg via intravenous infusion.

evolocumab (AMG 145) was also developed by Amgen and is a monoclonal antibody that binds to PCSK 9. The indications for evolocumab are hypercholesterolemia and hyperlipidemia.

alirocumab (REGN727) is a human monoclonal antibody from Regeneron Pharmaceuticals, Inc. ("Regeneron") and Sanofi-Aventis u.s.llc ("Sanofi"), which is indicated for hypercholesterolemia and acute coronary syndrome.

Naptumomab estafenatox, ABR-217620, from Active Biotech AB ("Active Biotech") is a monoclonal antibody suitable for renal cell carcinoma.

From CIMAB, SA ("CIMAB"); rackatumomab of laboratory Elea s.a.c.i.f.y a. is a monoclonal antibody suitable for non-small cell lung cancer.

Other antibodies that may be formulated with viscosity-lowering agents include bococizumab (PF-04950615) and tanezumab; ganitumab, blinatumomab, trebanib from Amgen; anthrax immunoglobulin from Cangene Corporation; teplizumab from macrogenetics, inc; MK-3222, MK-6072 from Merck & Co ("Merck"); girentuximab from Wilex AG; RIGScan from Navidea Biopharmaceuticals ("Navidea"); PF-05280014 from Pfizer; SA237 from Chugai Pharmaceutical co.ltd. ("Chugai"); guselkumab from Janssen/Johnson and Johnson Services, Inc. ("J & J"); antithrombin gamma (KW-3357) from Kyowa; and CT-P10 from Celltrion.

Antibodies in early clinical trials

A variety of monoclonal antibodies have recently entered or are entering clinical trials. It may include proteins currently administered via intravenous infusion, preferably those with a molecular weight greater than about 120kDa, typically from about 140kDa to about 180 kDa. It may also include high molecular weight proteins such as drugs or peptides conjugated to albumin that are also entering clinical trials or have been approved by the FDA. A number of monoclonal antibodies from Amgen are currently in clinical trials. These monoclonal antibodies may be high molecular weight proteins such as AMG 557, a human monoclonal antibody developed by Amgen in combination with AstraZeneca and currently in phase I trials for the treatment of lupus. Similarly, AMG 729 is a humanized monoclonal antibody developed by Amgen and is currently in phase I trials for the treatment of lupus and rheumatoid arthritis. Furthermore, AMG 110 is a monoclonal antibody directed against epithelial cell adhesion molecules; AMG 157, developed by Amgen in combination with AstraZeneca, is a human monoclonal antibody currently in phase I trials for the treatment of asthma; AMG 167 is a humanized monoclonal antibody that has been evaluated in multiple phase I trials for the treatment of osteoporosis; AMG 334, which has been studied for phase I administration and is currently in phase II studies directed to the treatment of migraine and hot flashes, is a human monoclonal antibody that inhibits calcitonin gene-related peptide; AMG 780 is a human anti-angiogenin monoclonal antibody that inhibits the interaction of endothelial cell selective Tie2 receptor with its ligands Ang1 and Ang2 and recently completed phase I trials as cancer therapy; AMG 811 is a human monoclonal antibody that inhibits interferon gamma and is being studied as a therapy for systemic lupus erythematosus; AMG 820 is a human monoclonal antibody that inhibits c-fms and reduces tumor-associated macrophage (TAM) function and is being investigated as a cancer therapy; AMG 181 developed by Amgen in combination with AstraZeneca is a human monoclonal antibody that inhibits the action of α 4/β 7 and is in phase II trials as a therapy for ulcerative colitis and crohn's disease.

A variety of monoclonal antibodies are currently in clinical trials directed at treating autoimmune disorders. These monoclonal antibodies may be contained in a low viscosity high molecular weight liquid formulation. RG7624 is a fully human monoclonal antibody designed to specifically and selectively bind to cytokines of the human interleukin-17 family. A phase I clinical trial to evaluate RG7624 against autoimmune disease is ongoing. BIIB033 is an anti-LINGO-1 monoclonal antibody developed by Biogen that is currently in phase II trials for the treatment of multiple sclerosis.

High molecular weight proteins may also include AGS-009, a monoclonal antibody targeted to IFN-alpha developed by Argos Therapeutics, inc. AGS-009 up to 30mg/kg is administered to the patient via intravenous infusion. BT-061, developed by AbbVie, is in a phase II trial for patients with rheumatoid arthritis. Pegylated certolizumabIs a monoclonal antibody in a phase II test against ankylosing spondylitis and juvenile rheumatoid arthritis. clazakizumab is an anti-IL 6 monoclonal antibody in a phase II assay performed by Bristol-Myers Squibb.

CNTO-136(sirukumab) and CNTO-1959 are monoclonal antibodies that have recently been tested by Janssen in stages II and III. Daclizumab (previously withMarketed by Roche) several phase III trials for the treatment of multiple sclerosis are currently or recently completed by AbbVie. Epratuzumab is a humanized monoclonal antibody in a phase III assay directed to the treatment of lupus. Carnacumab (canakinumab)Is a human monoclonal antibody targeted to interleukin-1 beta. It is approved for the treatment of cryopyrin-associated cycle syndrome. Carnacumab is in phase I trial as a possible therapy for chronic obstructive pulmonary disease, gout, and coronary artery diseaseAnd (6) performing an experiment. mavrilimumab is a human monoclonal antibody designed for the treatment of rheumatoid arthritis. Mavrilimumab, which is found by Cambridge Antibody Technology as CAM-3001, is being developed by MedImmune.

MEDI-546 and MEDI-570 are monoclonal antibodies currently in phase I and II trials by AstraZeneca for the treatment of lupus. MEDI-546 was administered in the phase II study by conventional intravenous infusion of 300-1,000 mg. MEDI-551 is another monoclonal antibody developed by AstraZeneca for various indications and is also currently administered by intravenous infusion. NN8209 is a monoclonal antibody developed by Novo Nordisk a/S ("Novo Nordisk") for blocking the C5aR receptor and phase II dosing studies have been completed for the treatment of rheumatoid arthritis. NN8210 is another anti-C5 aR monoclonal antibody being developed by Novo Nordisk and is currently in phase I testing. IPH2201(NN8765) is a humanized monoclonal antibody targeted to NKG2A being developed by Novo Nordisk for the treatment of patients with inflammatory and autoimmune disorders. NN8765 recently completed phase I trials.

olokizumab is a humanized monoclonal antibody that targets the cytokine IL-6 potently. IL-6 is involved in several autoimmune and inflammatory pathways. olokizumab has completed phase II trials for the treatment of rheumatoid arthritis. otelixizumab (also known as TRX4) is a monoclonal antibody that is being developed for the treatment of type 1 diabetes, rheumatoid arthritis, and other autoimmune diseases. ozoralizumab is a humanized monoclonal antibody that has completed phase II trials.

Pfizer currently performs phase I trials directed to monoclonal antibodies PD-360324 and PF-04236921 for the treatment of lupus. The rituximab biosimilar PF-05280586 has been developed by Pfizer and is in phase I/phase II trials against rheumatoid arthritis.

The ontalizumab is a humanized monoclonal antibody under development by Genentech. It recently completed phase II trials for treatment of lupus. SAR113244 (anti-CXCR 5) is a monoclonal antibody developed by Sanofi in phase I trials. The sifalimumab (anti-IFN- α monoclonal antibody) is a monoclonal antibody in a phase II trial directed at treating lupus.

The high molecular weight, low viscosity liquid formulation may comprise one of the monoclonal antibodies in early clinical development directed to the treatment of various blood disorders. For example, belimumsPhase I trials have recently been completed for patients with vasculitis. Other monoclonal antibodies in early trials against hematological diseases include BI-655075 from Boehringer Ingelheim GmbH ("Boehringer Ingelheim"), ferroportin and hepcidin monoclonal antibodies from Eli Lily, and SelG1 from Selexs Pharmaceuticals, Corp. ("Selexs").

One or more monoclonal antibodies in early development directed to the treatment of various cancers and related conditions may be included in a low viscosity, high molecular weight liquid formulation. United Therapeutics, Corporation has two monoclonal antibodies in phase I trial, the 8H9 monoclonal antibody and the ch14.18 monoclonal antibody. Monoclonal antibodies ABT-806, enavatuzumab and volociximab from AbbVie were under early development. Early experiments were performed with the monoclonal antibodies Actimab-A (M195mAb), anti-CD 45mAb and Iomab-B. Seattle Genetics, Inc. ("Seattle Genetics") has several monoclonal antibodies in early trials against cancer and related disorders, including anti-CD 22ADC (RG 7593; pinatuzumab vedotin), anti-CD 79b ADC (RG7596), anti-STEAP 1ADC (RG7450), ASG-5ME and ASG-22ME from Agensys, Inc. ("Agensys"), antibody-drug conjugate RG7458 and voretuzumab. Early cancer therapeutics from Genentech may be included in low viscosity formulations, including ALT-836, antibody-drug conjugates RG7600 and DEDN6526A, anti-CD 22ADC (RG7593), anti-EGFL 7mAb (RG7414), anti-HER 3/EGFR DAFmAb (RG7597), anti-PD-L1 mAb (RG7446), DFRF4539A, MINT 1526A. Bristol-Myers Squibb is developing early monoclonal antibodies against cancer therapeutics, including those identified as anti-CXCR 4, anti-PD-L1, IL-21(BMS-982470), lirilumab, and urelumab (anti-CD 137). Other monoclonal antibodies in early trials as cancer therapeutics include APN301 from Apeiron Biologics AG (hu14.18-IL2), AV-203 from AVEO Pharmaceuticals, Inc. ("AVEO"), AVX701 and AVX901 from AlphaVax, BAX-69 from Baxter International, Inc. (the term "Baxter"), BAY 79-4620 and BAY 20-10112 from Bayer HealthCare AG, BHQ880 from Novartis AG, 212-TCtrastuzumab from AREVA Med, Ab-7 from Abomics International Inc., and ABIO-0501(TALL-104 Pb) from Abiogen Pharma S.p.A.

Other antibody therapeutics that may be formulated with the viscosity-lowering agent include alzumab, GA101, daratummab, siltuximab, ALX-0061, ALX-0962, ALX-0761, bimagumamab (BYM338), CT-011(pidilizumab), actoxaumab/bezotuximab (MK-3515A), MK-3475(pembrolizumab), dalotuzumab (MK-0646), icrucumab (IMC-18F1, LY3012212), AMG139(MEDI 0), SAR339658, dupimolumab (REGN), SAR156597, 252512, 279356, SAR3419, SAR153192(REGN421, enotumumab), 307746 (nesvacuumab), 650984, 6586, 069386, SAR 9319, SAR3419, SAR 34142, SAR 34133, SGuizumab (SG7458-33), SG7458-33, SGuizumab), SG587420, GCE 33, SG7420-33, SGuizumab (SG7420), SG7458-33, GCE), SG7420-33, GCE III, GCE-D-33, GCT-E, GCT-D-III, GCE 3, GCE, XmAb7195, cixutuzumab (LY3012217), LY2541546 (desozumab), olaratumab (LY3012207), MEDI4893, MEDI573, MEDI0639, MEDI3617, MEDI4736, MEDI6469, MEDI0680, MEDI5872, PF-05236812(AAB-003), PF-05082566, BI 1034020, RG7116, RG7356, RG7155, RG7212, RG7599, RG7636, RG7221, RG7652(MPSK3169A), RG7686, HuxTFADC, MOR103, BT061, MOR208, OMP59R 58 (anti-notch 2/3), VAY736, MOR202, BAY 94-43, KHJM 716, OMP52M51, GSK933776, GSK 93320, GSK 241076, GSK 08093655, VAY 30188655, LY 37959-685 38413, AGS 387538, AGS 247538, AGS 247533 and AGS 2475316.

Other early monoclonal antibodies that could be formulated with viscosity reducing agents include benralizumab, MEDI-8968, anifrozumab, MEDI7183, sifalimumab, MEDI-575, tralokinumab from AstraZeneca and MedImmune; BAN2401 from Biogen Idec/Eisai co.ltd ("Eisai")/BioArctic Neuroscience AB; CDP7657 from Biogen (an anti-CD 40L monovalent pegylated antibody Fab fragment), STX-100 (an anti-avB 6 monoclonal antibody), BIIB059, anti-TWEAK (BIIB023) and BIIB 022; fulranumab from Janssen and Amgen; BI-204/RG7418 from BioInvent International/Genentech; BT-062(indatuximab ravtansine) from Biotest Pharmaceuticals Corporation; XmAb from Boehringer Ingelheim/Xencor; anti-IP 10 from Bristol-Myers Squibb; j591 Lu-177 from BZL Biologics LLC; CDX-011(glembatumumab vedotin), CDX-0401 from Celldex Therapeutics; foravirumab from brucell; tigatuzumab from Daiichi Sankyo Company Limited; MORAB-004, MORAB-009(amatuximab) from Eisai; LY2382770 from Eli Lilly; DI17E6 from EMD Serono inc; zanolimumab from Emergent BioSolutions, inc; FG-3019 from fibrigen, inc; catumaxomab from Fresenius SE & co.kgaa; pateclizumab, ontalizumab from Genentech; fresolimumab from Genzyme and Sanofi; GS-6624(simtuzumab) from Gilead; CNTO-328 from Janssen, bapineuzumab (AAB-001), carlumab, CNTO-136; KB003 from KaloBios Pharmaceuticals, inc; ASKP1240 from Kyowa; RN-307 from Labrys Biologics Inc.; ecromeximab from Life Science Pharmaceuticals; LY2495655, LY2928057, LY3015014, LY2951742 from Eli Lilly; MBL-HCV1 from MassBiologics; AME-133v from MENTRIK Biotech, LLC; abituzumab from Merck KGaA; MM-121 from Merrimack Pharmaceuticals, inc; MCS110, QAX576, QBX258, QGE031 from Novartis AG; HCD122 from Novartis AG and XOMA Corporation ("XOMA"); NN8555 from Novo Nordisk; bavituximab, cotara, from Peregrine Pharmaceuticals, inc; PSMA-ADC from Progenics Pharmaceuticals, inc; oregoviamab from Quest Pharmatech, inc; faminumab (REGN475), REGN1033, SAR231893, REGN846 from Regeneron; RG7160, CIM331, RG7745 from Roche; ibalizumab from TaiMed Biologics inc (TMB-355); TCN-032 from Theralclone Sciences; TRC105 from TRACON Pharmaceuticals, inc; UB-421 from United Biomedical inc; VB4-845 from Viventia Bio, Inc.; ABT-110 from AbbVie; caplacizumab, ozoralizumab from Ablynx; PRO 140 from CytoDyn, inc; GS-CDA1, MDX-1388 from Medarex, inc; AMG 827, AMG 888 from Amgen; ublituximab from TG Therapeutics inc; TOL101 from Tolera Therapeutics, inc; huN901-DM1(lorvotuzumab mertansine) from ImmunoGen inc; epratuzumab Y-90/veltuzumab combination from immunolics, inc (IMMU-102); anti-fibrin monoclonal antibody/3B 6/22Tc-99m from Agenix, Limited; ALD403 from Alder biopharmaceutics, inc; RN6G/PF-04382923 from Pfizer; CG201 from CG Therapeutics, inc; KB001-A from KaloBios Pharmaceuticals/Sanofi; KRN-23 from kyowa; y-90hPAM 4 from Immunodics, Inc.; tarextumab from Morphosys AG and OncoMed Pharmaceuticals, inc; LFG316 from Morphosys AG and Novartis AG; CNTO3157, CNTO6785 from Morphosys AG and Jannsen; RG6013 from Roche and Chugai; MM-111 from Merrimac Pharmaceuticals, Inc. (a "Merrimac"); GSK2862277 from GlaxoSmithKline; AMG 282, AMG 172, AMG 595, AMG 745, AMG 761 from Amgen; BVX-20 from Biocon; CT-P19, CT-P24, CT-P25, CT-P26, CT-P27, CT-P4 from Celltrion; GSK284933, GSK2398852, GSK2618960, GSK1223249, GSK933776A from GlaxoSmithKline; anetumab ravtansine from Morphosys AG and Bayer AG; BI-836845 from Morphosys AG and Boehringer Ingelheim; NOV-7, NOV-8 from Morphosys AG and Novartis AG; MM-302, MM-310, MM-141, MM-131, MM-151 from Merrimack; RG7882 from Roche and Seattle Genetics; RG7841 from Roche/Genetch; PF-06410293, PF-06438179, PF-06439535, PF-04605412, PF-05280586 from Pfizer; RG7716, RG7936, genenerumab, RG7444 from Roche; MEDI-547, MEDI-565, MEDI1814, MEDI4920, MEDI8897, MEDI-4212, MEDI-5117, MEDI-7814 from Astrazeneca; ulocuplumab, PCSK9adnectin from Bristol-Myers Squibb; FPA009, FPA145 from filprime Therapeutics, inc; GS-5745 from Gilead; BIW-8962, KHK4083, KHK6640 from Kyowa Hakko Kirin; MM-141 from Merck KGaA; REGN1154, REGN1193, REGN1400, REGN1500, REGN1908-1909, REGN2009, REGN2176-3, REGN728 from Regeneron; SAR307746 from Sanofi; SGN-CD70A from Seattle Genetics; ALX-0141, ALX-0171 from Ablynx; matuzumab-DOX, matuzumab, TF2 from immamedics, inc; MLN0264 from Millennium; ABT-981 from AbbVie; AbGn-168H from AbGenomics International inc; ficlatuzumab from AVEO; BI-505 from BioInvent International; CDX-1127, CDX-301 from Celldex Therapeutics; CLT-008 from Cellerant Therapeutics Inc.; VGX-100 from Circadian; u3-1565 from Daiichi Sankyo Company Limited; DKN-01 from Dekkun Corp; flanvotumab (TYRP1 protein), IL-1 β antibody, IMC-CS4 from Eli Lilly; VEGFR3 monoclonal antibody, IMC-TR1(LY3022859) from Eli Lilly and Imclone, LLC; anthiim from Elusys therapeutics Inc.; HuL2G7 from Galaxy Biotech LLC; IMGB853, IMGN529 from ImmunoGen inc; CNTO-5, CNTO-5825 from Janssen; KD-247 from Kaketsuken; KB004 from kalobis Pharmaceuticals; MGA271, MGAH22 from macrogenetics, inc; XmAb5574 from MorphoSys AG/Xencor; (ii) the nsituximab (NPC-1C) from Neogenix Oncology, inc; LFA102 from Novartis AG and XOMA; ATI355 from Novartis AG; SAN-300 from Santarus inc; SelG1 from Selexys; HuM195/rGel from Targa Therapeutics, corp; VX15 from Teva Pharmaceuticals, Industries Ltd. ("Teva") and Vaccinex inc; TCN-202 from Theralclone Sciences; XmAb2513, XmAb5872 from xenor; XOMA 3AB from XOMA and national institute for allergy and infectious disease; neuroblastoma antibody vaccine from MabVax Therapeutics; cytolin from CytoDyn, Inc.; thravixa from emergentobio solutions inc; FB 301 from cytopnce Biologics; rabies monoclonal antibodies from Janssen and Sanofi; an influenza monoclonal antibody from Janssen and funded by the national institutes of health of the United states; MB-003 and ZMAP from Mapp Biopharmaceutical, Inc.; and ZMAb from Defyrus inc.

Other protein therapeutics

The protein may be an enzyme, a fusion protein, a stealth or pegylated protein, a vaccine, or other biologically active protein (or mixture of proteins). The term "enzyme" as used herein refers to a protein or functional fragment thereof that catalyzes the biochemical conversion of a target molecule to a desired product.

Enzymes as drugs have at least two important features, namely i) generally bind and act on their targets with high affinity and specificity, and ii) catalyze and convert multiple target molecules into desired products. In some embodiments, the protein may be pegylated as defined herein.

The term "fusion protein" as used herein refers to a protein produced from two different genes encoding two separate proteins. Fusion proteins are typically produced by recombinant DNA techniques known to those skilled in the art. The two proteins (or protein fragments) are covalently fused together and exhibit the properties of the two parent proteins.

There are a variety of fusion proteins on the market.

(Etanercept) is a fusion protein that competitively inhibits TNF marketed by Amgen.

Namely, the antihemophilic factor (recombinant) Fc fusion protein is an antihemophilic factor produced from recombinant DNA and is suitable for the control and prevention of bleeding episodes in adults and children with haemophilia a (congenital coagulation factor VIII deficiency), perioperative management, routine prophylaxis aimed at preventing or reducing the frequency of bleeding episodes.

(aflibercept) is a recombinant fusion protein composed of portions of the extracellular domains of human VEGF receptor 1 and 2 fused to the Fc portion of human IgG1 formulated as an isotonic solution for intravitreal administration.(Aflibercept) is a recombinant fusion protein composed of portions of the extracellular domains of human VEGF receptor 1 and 2 fused to the Fc portion of human IgG1 formulated as an isotonic solution for intravitreal administration. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, making up another 15% of the total molecular weight, resulting in a total molecular weight of 115 kDa. affibercept was produced in recombinant Chinese Hamster Ovary (CHO) cells and marketed by Regeneron.

ALPROLIX^TMThat is, the coagulation factor IX (recombinant) Fc fusion protein is a coagulation factor IX concentrate produced from recombinant DNA suitable for use in the control and prevention of bleeding episodes in adults and children with hemophilia b, perioperative management, routine prophylaxis aimed at preventing or reducing the frequency of bleeding episodes.

pegloticaseIs a drug developed by Savient Pharmaceuticals, inc. for the treatment of severe refractory chronic gout and is the first drug approved for this indication. pegloticase is a pegylated recombinant porcine uricase with a molecular weight of about 497 kDa. Pegloticase is currently administered by intravenous infusion at 8 mg/kg. The high molecular weight, low viscosity liquid formulation may comprise pegloticase, preferably at a concentration of about 300mg/mL to about 800 mg/mL.

Alteplase (alteplase)Is a tissue plasminogen activator produced by recombinant DNA techniques. It is a purified glycoprotein comprising 527 amino acids and was synthesized using complementary dna (cdna) from a native human tissue-type plasminogen activator from a human melanoma cell line. Alteplase intravenously immediately following stroke symptomsAbout 100mg was infused for administration. In some embodiments, a low viscosity formulation containing alteplase is provided, preferably at a concentration of about 100 mg/mL.

glucarpidaseIs a drug approved by the FDA for the treatment of elevated methotrexate levels (defined as at least 1 μmol/L) during the treatment of cancer patients with impaired renal function. glucarpidase is administered intravenously in a single dose of about 50 IU/kg. In some embodiments, low viscosity formulations containing glucarpidate are provided.

alglucosidaseαIs an enzyme replacement therapy orphan drug for treating Pompe disease (glycogen storage disease type II), a rare lysosomal storage disease. It has a molecular weight of about 106kDa and is currently administered by intravenous infusion at about 20 mg/kg. In some embodiments, low viscosity pharmaceutical formulations of alglucosidase alpha are provided, preferably at a concentration of about 100mg/mL to about 2,000 mg/mL.

pegdamase bovineIs a modified enzyme for use in enzyme replacement therapy for the treatment of Severe Combined Immunodeficiency Disease (SCID) associated with adenosine deaminase deficiency. pegdamase bone is a conjugate of multiple strands of monomethoxypolyethylene glycol (PEG) of molecular weight 5,000Da covalently attached to adenosine deaminase already available from bovine intestine.

Alpha-galactosidase is a lysosomal enzyme that catalyzes the hydrolysis of glycolipids, namely ceramide trihexoside (GL-3), to galactose and ceramide dihexoside. Fabry disease is a rare inherited lysosomal storage disease characterized by α -galactosidase and the resulting accumulation of GL-3 that is less than normal enzymatic activity. Galactosidase alphaIs human alpha-galactosidase a produced by a human cell line. Galactosidase betaIs a recombinant human alpha galactosidase expressed in a CHO cell line.Was administered by intravenous infusion at a dose of 0.2mg/kg every other week to treat fabry's disease and was used outside the instructions for the treatment of gaucher's disease.Administered by intravenous infusion at a dose of 1.0mg/kg body weight every other week. Other lysosomal enzymes may also be used. For example, the protein may be a lysosomal enzyme as described in US 2012/0148556.

Labriase (rasburicase)Is a recombinant urate oxidase suitable for initial control of plasma uric acid levels in children and adult patients suffering from malignant leukemias, lymphomas and solid tumors undergoing anti-cancer therapy expected to result in tumor lysis and subsequent increase in plasma uric acid.Administered by daily intravenous infusion at a dose of 0.2 mg/kg.

Imiglucerase (imiglucerase)Is a recombinant analog of human β -glucocerebrosidase. The initial dose is 2.5U/kg body weight 3 times a week to 60U/kg 1 time per 2 weeks.Administration is by intravenous infusion.

abraxane, albumin conjugated with paclitaxel, is approved for metastatic breast cancer, non-small cell lung cancer, and advanced pancreatic cancer.

taligluceraseαIs an enzyme specific for lysosome glucocerebroside hydrolytically suitable for long-term enzyme replacement therapy of gaucher type 1. The recommended dose is given as 60U/kg body weight once every 2 weeks via intravenous infusion.

laronidaseIs a polymorphic variant of human alpha-L-iduronidase produced by a CHO cell line.The recommended dosage regimen of (a) is to be administered by intravenous infusion once a week at 0.58 mg/kg.

elosulfaseαHuman N-acetylgalactosamine-6-sulfatase produced by a CHO cell line was BioMarin Pharmaceuticals Inc ("BioMarin"). It was FDA approved for the treatment of type IVA mucopolysaccharidosis on day 2, 14 of 2014. It was administered via intravenous infusion at a dose of 2mg/kg weekly.

Other biologicals that may be formulated with viscosity reducing agents include asparaginase erwinia chrysanthemiiincobotulinumtoxin A(alfa-epoetin),(alfa epoetin),(alfa bepoting) alpha-amino acids,(abatacept),(interferon beta-1 b),(thiolase);(idursulfase);(alpha-glucosidase);(velaglucerase)、abobotulinumtoxin ABAX-326, octine α from Baxter; syncria from GlaxoSmithKline; liprotamase from Eli Lilly; xiaflex (clostridium histolyticum collagenase) from auxium and biospeciics Technologies corp; anakinra from Swedish Orphan Biovitrum AB; metreleptin from Bristol-Myers Squibb; avonex, plegridy (BIIB017) from Biogen; NN1841, NN7008 from Novo Nordisk; KRN321 (Afavabepotin), AMG531 (romiplosmistim), KRN125(pegfilgrastim), KW-0761(mogamulizumab) from Kyowa; IB1001 from InspirantionBiopharmaceuticals; iprivask from the Canyon Pharmaceuticals Group.

Protein therapeutics under development

VRS-317 of Versartis, inc. is a recombinant human growth hormone (hGH) fusion protein using XTEN half-life extension technology. It is intended to reduce the frequency of hGH injections required by patients suffering from hGH deficiency. VRS-317 has completed phase II studies comparing its efficacy with daily injections of non-derivatized hGH with positive results. A phase III study was planned.

Vibrio hemolysin is a proteolytic enzyme secreted by the gram-negative marine microorganism Vibrio proteolyticus. The endoprotease has specific affinity for hydrophobic regions of the protein and is capable of cleaving the protein adjacent to the hydrophobic amino acids. Vibrio hemolysin is currently being studied by Biomarin for the clearance and/or treatment of burns. The vibrio hemolysin preparation is described in patent WO 02/092014.

PEG-PAL (pegylated recombinant phenylalanine ammonia lyase or "PAL") is a research enzyme replacement therapy for the treatment of Phenylketonuria (PKU), an inherited metabolic disease caused by a deficiency in phenylalanine hydroxylase (PAH). PEG-PAL is being developed as a drug for blood phenylalanine (Phe) levels that have not been exploitedA potential therapy for the patient under adequate control. PEG-PAL is now in the treatment pairPhase 2 clinical development in patients with inadequate response.

Other protein therapeutics that can be formulated with the viscosity-lowering agent include Alprolix/rFIXFC, Elockate/rFVIIIFc, BMN-190; BMN-250; lamazyme; galazyme; ZA-011; sebelispase alpha; SBC-103; and HGT-1110. In addition, fusion proteins containing XTEN half-life extension technology can be formulated with viscosity-lowering agents, including but not limited to VRS-317 GH-XTEN; factor VIIa, factor VIII, factor IX; PF05280602, VRS-859; exenatide-XTEN; AMX-256; GLP 2-2G/XTEN; and AMX-179 folate-XTEN-DM 1.

Other late stage protein therapeutics that can be formulated with the viscosity-lowering agent include CM-AT from CureMark LLC; NN7999, NN7088, Liraglutide (Liraglutide) (NN8022), NN9211, somaglutide (NN9535) from Novo Nordisk; AMG 386 from Amgen, Filgrastim (Filgrastim); CSL-654 from CSL Behring, coagulation factor VIII; LA-EP2006 (PEGylated filgrastim bioanalogue) from Novartis AG; multikine (interleukin) from CEL-SCI Corporation; LY2605541 from Eli Lilly, Teriparatide (recombinant PTH 1-34); NU-100 from Nuron Biotech, inc; calaspragase Pegol from Sigma-Tau Pharmaceuticals, Inc.; ADI-PEG-20 from Polaris Pharmaceuticals, inc; BMN-110, BMN-702 from BioMarin; NGR-TNF from Molmed s.p.a.; recombinant human C1 esterase inhibitor from Pharming Group/Santarus inc; a growth hormone biosimilar from LG Life Sciences LTD; natpara from NPS Pharmaceuticals, inc; ART123 from Asahi Kasei Corporation; BAX-111 from Baxter; OBI-1 from Inspiration Biopharmaceuticals; wilate from Octapharma AG; talctoferrin alpha from Agennix AG; desmoprase (Desmoteplase) from Lundbeck; cinryze from Shire; RG7421 from Roche and Exelixis, inc; midostaurin (Midostaurin) from Novartis AG (PKC 412); damococog α pegol, BAY 86-6150, BAY 94-9027 from Bayer AG; pegylated interferon lambda-1 a, Nulojix (Belatacept) from Bristol-Myers Squibb; pergoveris, follitropin alpha (MK-8962) from Merck KGaA; recombinant coagulation factor IX Fc fusion protein (rFIXFfc; BIIB029) and recombinant coagulation factor VIII Fc fusion protein (rFVIIIFc; BIIB031) from Biogen; and Myalept from AstraZeneca.

Other early protein biologicals that can be formulated with viscosity reducing agents include Alferon LDO from Hemispherx BioPharma, inc; SL-401 from Stemline Therapeutics, inc; PRX-102 from Protalix Biotherapeutics, inc; KTP-001 from Kaketsuken/Teijin Pharma Limited; vericiguat from Bayer AG; BMN-111 from BioMarin; ACC-001 from Janssen (PF-05236806); LY2510924, LY2944876 from Eli Lilly; NN9924 from Novo Nordisk; INGAP peptide from Exsulin; ABT-122 from Abbvie; AZD9412 from AstraZeneca; NEUBLASTIN from Biogen (BG 00010); from CelgeneLuspatercept (ACE-536), Sotatercept (ACE-011) from Corporation; PRAME immunotherapeutics from GlaxoSmithKline; plovemer acetate from Merck KGaA (PI-2301); PREMIPLEX from fire (607); BMN-701 from BioMarin; ontak from Eisai; rHuPH 20/insulin from Halozyme, inc; PB-1023 from PhaseBio Pharmaceuticals, inc; ALV-003 from Alvine Pharmaceuticals Inc. and Abbvie; NN8717 from Novo Nordisk; PRT-201 from Proteon Therapeutics inc; PEGPH20 from Halozyme, inc; from Astellas Pharma Incalefacept; f-627 from Regeneron; AGN-214868(senrebotase) from Allergan, Inc.; BAX-817 from Baxter; PRT4445 from Portola Pharmaceuticals, inc; VEN100 from Ventria Bioscience; onconase/ranpirnase from Tamir Biotechnology Inc.; an interferon alpha-2 b fusion protein from Medtronic, inc; seblipase α from syneva BioPharma; IRX-2 from IRX Therapeutics, Inc; GSK2586881 from GlaxoSmithKline; SI-6603 from Seikagaku Corporation; ALXN1101 from Alexion, asfotase α; SHP611, SHP609(Elaprase, idursufase) from Shire; PF-04856884, PF-05280602 from Pfizer; ACE-031, Dalatercept from Acceleron Pharma; ALT-801 from Altor BioScience corp; BA-210 from BioAxone Biosciences, Inc.; WT1 immunotherapeutics from GlaxoSmithKline; GZ402666 from Sanofi; MSB0010445, Atacicept from Merck KGaA; leukine (sargramostim) from Bayer AG; KUR-211 from Baxter; fibroblast growth factor-1 from cardiovasular BioTherapeutics inc; SPI-2012 from Hanmi Pharmaceuticals Co., LTD/Spectrum Pharmaceuticals; FGF-18(sprifermin) from Merck KGaA; MK-1293 from Merck; interferon- α -2b from HanAll Biopharma; CYT107 from cytheis SA; RT001 from innovation Therapeutics, inc; MEDI6012 from AztraZeneca; e2609 from Biogen; BMN-190, BMN-270 from BioMarin; ACE-661 from Acceleron Pharma; AMG 876 from Amgen;GSK3052230 from GlaxoSmithKline; RG7813 from Roche; SAR342434, Lantus from Sanofi; AZ01 from Allozyne inc; ARX424 from Ambrx, inc; FP-1040, FP-1039 from FivePrime Therapeutics, Inc.; ATX-MS-1467 from Merck KGaA; XTEN fusion proteins from Amunix Operating inc; enterimod (CBLB502) from Cleveland BioLabs, inc; HGT2310 from Shire; HM10760A from Hanmi Pharmaceuticals co., LTD; ALXN1102/ALXN1103 from Alexion; CSL-689, CSL-627 from CSL Behring; glial growth factor 2 from Acorda Therapeutics, inc; NX001 from Nephrx Corporation; NN8640, NN1436, NN1953, NN9926, NN9927, NN9928 from Novo Nordisk; NHS-IL 12 from EMD Serono; 3K3A-APC from ZZ Biotech LLC; PB-1046 from PhaseBiopharmaceuticals, Inc.; RU-101 from R-Tech Ueno, Ltd.; insulin lispro/BC 106 from Adocia; hl-con1 from Iconic Therapeutics, inc; PRT-105 from Protalix BioTherapeutics, inc; PF-04856883, CVX-096 from Pfizer; ACP-501 from AlphaCore Pharma LLC; BAX-855 from Baxter; CDX-1135 from Celldex Therapeutics; PRM-151 from Promedior, inc; TS01 from Thrombolytic Science International; TT-173 from Thrombotargets Corp.; QBI-139 from Quessence Biosciences, Inc.; vatelizumab, GBR500, GBR600, GBR830 and GBR900 from Glenmark Pharmaceuticals; and CYT-6091 from Cytimmune Sciences, Inc.

Other biopharmaceuticals

Other biopharmaceuticals that may be formulated with viscosity-lowering agents include PF-05285401, PF-05231023, RN317(PF-05335810), PF-06263507, PF-05230907, Dekavil, PF-06342674, PF06252616, RG7598, RG7842, RG7624D, OMP54F28, GSK1995057, BAY1179470, IMC-3G3, IMC-18F1, IMC-35C, IMC-20D7S, PF-06480605, PF-06647263, PF-06650808, PF-05335810(RN317), PD-0360324, PF-00547659; MK-8237 from Merck; BI033 from Biogen; GZ402665, SAR438584/REGN2222 from Sanofi; IMC-18F 1; ibruumab, IMC-3G3 from Imclone LLC; ryzodeg, Tresiba, Xultophy from Novo Nordisk; toujeo (U300), LixiLan, Lyxumia (lixisenatide) from Sanofi; MAGE-a3 immunotherapeutics from GlaxoSmithKline; tecemotide from Merck KGaA; sereleaxin (RLX030) from Novartis AG; erythropoietin; pegylation of filgrastim; LY2963016, Dulaglutide (dilaglutide) from Eli Lilly (LY 2182965); and insulin glargine from Boehringer Ingelheim.

B. Viscosity reducing agent

The viscosity of a liquid protein formulation comprising low molecular weight and/or high molecular weight proteins is reduced by the addition of one or more viscosity reducing agents. The pharmaceutical formulation may be converted from a non-newtonian fluid to a newtonian fluid by the addition of an effective amount of one or more viscosity reducing agents.

When used in formulations intended for administration to humans or other mammals, the viscosity-lowering agent must be pharmaceutically acceptable as the formulation itself. The viscosity reducing agent is typically an organic compound containing at least one non-carbon, non-hydrogen atom. Preferably, the viscosity reducing agent contains hydrogen, carbon, oxygen, and at least one other type of atom. In some embodiments, the viscosity reducing agent is characterized by at least one of the following properties:

1) an organic compound having at least four carbon and four hydrogen atoms and at least one sulfur, oxygen, nitrogen, or phosphorus atom;

2) molecular weight of about 85 to 1,000 Da;

3) the presence of at least one charged or other hydrophilic moiety;

4) there is at least one, preferably two and more preferably three free-rotating keys;

5) at least one substituted ring is present;

6) a molecular polar surface area of at leastPreferably at leastAnd more preferably at least

7) Molar volume of at least 75cm³Preferably at least 85cm³More preferably at least 100cm³And most preferably at least 120cm³；

8) Polarizability of at least 10cm³Preferably at least 15cm³More preferably at least 20cm³And most preferably at least 25cm³(ii) a And

9) at least one, preferably two and more preferably three hydrogen bond donors and/or hydrogen bond acceptors are present.

In some embodiments, the viscosity reducing agent is characterized by at least two, three, four, five, six, seven, eight, or all nine of the above properties. In some embodiments, the viscosity reducing agent is further characterized in that it does not contain aldehyde or carbon-carbon triple bond functionality.

In other embodiments, the viscosity-lowering agent is a combination of two or more compounds, each of which is characterized by at least two, three, four, five, six, seven, eight, or all nine of the above properties.

In some embodiments, the viscosity-reducing agent is a GRAS listed by the U.S. food and drug administration ("FDA") as of 11 days 9 month 2014. "GRAS" is the phrase "generally considered safeGenerally Recognized As Safe) ". According to the U.S. federal food, drug and cosmetic act (the act) sections 201(s) and 409, any substance intended to be added to food is a food additive and requires pre-market review and approval by the FDA unless qualified experts generally believe that the substance has been sufficiently proven safe under the conditions of its intended use or unless the use of the substance is otherwise excluded from the definition of a food additive. Another source of compounds is the FDA Inactive Ingredients Guide (IIG) by 2014, 11 months, and equivalents listed by the international pharmaceutical excipients association (IPEC) and the European Medicines Administration (EMA). The substances used in the formulations must be safe for injection. Preferably, the GRAS listed viscosity reducing agents are characterized byAt least two, three, four, five, six, seven, eight, or all nine of the above characteristics.

In other embodiments, the viscosity-reducing agent is a drug product approved by the FDA or EMA by 11 days 9 month 2014. Toxicity and safety of FDA and EMA approved drug products are well established as are compounds cited in the GRAS and IIG lists. In addition to reducing the viscosity of the protein solution, the use of FDA or EMA approved drug products provides an opportunity to administer combination therapies. Preferably, the FDA or EMA approved drug product viscosity reducing agent is characterized by at least two, three, four, five, six, seven, eight, or all nine of the above properties.

In some embodiments, the viscosity-lowering agent comprises at least one compound of formula (I):

whereinRepresents a single bond or a double bond, A is selected from O, S, SO₂、NR³、C(R³)₂Or

Wherein R is³Independently selected from hydrogen, R²、-OH、NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aand-N (R)^4a)₂；

Wherein R is²Is independently selected from C_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl and C_2-12A heterocyclic group;

wherein each C_1-12The alkyl group may be substituted one or more times with: c_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl group, C_2-12Heterocyclyl, -OH, NH₂、(＝O)、(＝NR^4a)、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein each C_3-12The cycloalkyl group may be substituted one or more times by: c_1-12Alkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl, C_2-12Heterocyclyl, -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein each C_6-12The aryl group may be substituted one or more times with: c_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_1-12Heteroaryl group, C_2-12Heterocyclyl, -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein each C_1-12Heteroaryl groups may be substituted one or more times with: c_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_6-12Aryl radicals、C_2-12Heterocyclyl, -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein each C_2-12The heterocyclic group may be substituted one or more times with: c_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl, -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein R is⁴Is independently selected from C_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl and C_2-12Heterocyclyl, each of which may be substituted one or more times by: -OH, -NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)OH、-SO₃H、-PO₃H₂or-C (═ O) NH₂；

Wherein R is^4aCan be R⁴Or hydrogen;

wherein R is²、R³、R⁴And R^4aAny two or more of the groups may together form a ring;

wherein when two R are³When radicals are bound to the same carbon atom, the two R' s³The radicals may together form (═ O), (═ NR)^4a) Or (═ C (R)^4a)₂)；

Wherein z is independently selected at each occurrence from 1 or2, with the proviso that when (R)³)_zSubstituents and sp²When the hybridized carbon is bonded, z is 1, and when (R)³)_zSubstituents and sp³When the hybrid carbons are attached, z is 2.

When substituent-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂When present, preferably R^4aSelected from hydrogen, thereby obtaining-NHC (═ NH) NH₂。

In some embodiments, the compound of formula (1) contains at least one substituent selected from the group consisting of: -C (═ O) OH, -SO₃H、-SO₂NHC(＝O)R⁴and-PO₃H₂. In one embodiment, the compound of formula (1) contains at least one-SO₃And (4) an H group.

In some embodiments, one or more R is³The substituents may be:

wherein R is^3aAnd R^3bIndependently selected from hydrogen, C_1-12Alkyl radical, C_3-12Cycloalkyl, C_6-12Aryl radical, C_1-12Heteroaryl, C_2-12Heterocyclyl, C (═ O) R^4a、-C(＝O)OH、-C(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NHC(＝O)R⁴、C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR⁴and-N (R)^4a)₂And when any two R are^3bWhen bound to the same carbon atom, the two R' s^3bThe radicals together may form (═ O), (═ NR)^4a) Or (═ C (R)^4a)₂)；

Wherein each C_1-12Alkyl radical, C_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl and C_2-12The heterocyclic group may be substituted one or more times with: -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

Wherein R is⁴And R^4aAs defined above;

wherein x is selected from 1,2,3, 4, 5,7, 8, 9 or 10; and is

Wherein R is³、R^3a、R⁴And R^4aAny two or more of the groups may together form a ring.

In some embodiments, the compound of formula (1) may be represented by a compound of formula (1a) or (1 b):

wherein R is³Have the above-mentioned meanings.

In some embodiments, the compound of formula (1a) may be represented by a compound of formulae (1a-i to 1 a-iv):

wherein R is³Independently selected from hydrogen, NH₂、CH₃、Cl、OR⁴And NHR⁴；

Wherein x is 1 or 2;

wherein R is^3aAnd R^3bIndependently selected from hydrogen and C_1-12An alkyl group;

wherein said C_1-12The alkyl group may be substituted one or more times with: c_3-12Cycloalkyl radical, C_6-12Aryl radical, C_1-12Heteroaryl, C_2-12Heterocyclyl, -OH, NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R^4a、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-OC(＝O)R⁴、-OC(＝O)OR⁴、-SO₃H、-SO₂N(R^4a)₂、-SO₂R⁴、-SO₂NR^4aC(＝O)R⁴、-PO₃H₂、-R^4aC(＝NR^4a)N(R^4a)₂、-NHC(＝NR^4a)NH-CN、-NR^4aC(＝O)R⁴、-NR^4aSO₂R⁴、-NR^4aC(＝NR^4a)NR^4aC(＝NR^4a)N(R^4a)₂、-NR^4aC(＝O)N(R^4a)₂、-C(＝O)NH₂、-C(＝O)N(R^4a)₂、-OR⁴、-SR^4aor-N (R)^4a)₂；

R⁴And R^4aAs defined above; and is

Wherein R is^3a、R^3b、R⁴、R^4aAny two or more of which may together form a ring.

The compound of formula (1) may be represented by a compound of formula (1a-v, vi or vii):

wherein R is^3fSelected from-C (═ O) OH, -SO₃H、-SO₂NHC(＝O)R⁴and-PO₃H₂And R is³As defined above, in some preferred embodiments, R³Independently selected from hydrogen, OH, NH₂、C_1-6Alkyl and COOH.

In other embodiments, the compound of formula (1) may be represented by any one of compounds of formula (1c), (1d), (1e), or (1 f):

wherein R is³Have the above-mentioned meanings.

In other embodiments, the compound of formula (1) may be represented by a compound of formula (1 g):

wherein R is^3cIndependently selected from hydrogen and R²Wherein R is²Have the above-mentioned meanings;

wherein R is^3dIndependently selected from hydrogen, OH, NH₂、NH(C_1-6Alkyl group), N (C)_1-6Alkyl radical)₂、NHC(＝O)(C_1-6Alkyl), COOH and CH₂OH；

Or any two R attached to the same carbon atom^3cAnd R^3dThe radicals may together form oxo (═ O), imino (═ NR)^4a) Or alkenes (═ C (R)^4a)₂) Wherein R is^4aHave the above-mentioned meanings;

wherein R is^3eSelected from hydrogen, -OH OR OR⁴(ii) a And is

Wherein R is⁴Have the above-mentioned meanings.

In some embodiments, the viscosity reducing agent comprises a compound of formula (1 g-i):

wherein R is^3eSelected from OH and-OC_1-12An alkyl group further substituted with at least one OH and at least one COOH; and is

Wherein R is^3dSelected from COOH and CH₂OH。

In some embodiments, the viscosity-lowering agent comprises a compound of formula (2):

whereinRepresents a single bond or a double bond;

x is independently selected from chalcogen, N (R)³)_zAnd C (R)³)_z；

X¹Is absent or presentIs a chalcogen element, N (R)³)_z、C(R³)_zOr

Wherein R is³Have the meanings given for the compounds of formula (1); with the proviso that when (R)³)_zSubstituents and sp²When the hybridized nitrogen is attached, z is 0 or1, when (R)³)_zSubstituents and sp²Hybridized carbon or sp³When the hybridized nitrogen is attached, z is 1, and when (R)³)_zSubstituents and sp³Z is 2 when the hybrid carbons are attached;

wherein X or X¹Is a chalcogen or N (R)³)_z。

In some embodiments, the compound may be an aromatic ring. Exemplary aromatic rings include compounds of formula (2 a-e):

wherein R is³And X has the above-mentioned meaning, and X²Is selected from N (R)³)_zAnd C (R)³)_z。

In some embodiments, the viscosity reducing agent is a compound of formula (2 a-i):

wherein R is⁴As defined above and is preferably hydrogen or CH₃；

Wherein R is⁶Is C_1-12Heteroaryl, which may be substituted by C_1-6Alkyl substitution one or more times;

wherein said C_1-6Alkyl groups may be substituted one or more times with the following substituents: OH, -NH₂、-F、-Cl、-Br、-I、-NO₂、-CN、-C(＝O)R⁴、-C(＝NR^4a)R⁴、-C(＝O)OH、-C(＝O)OR⁴、-SO₃H、-SO₂NR⁴-、-SO₂R₄、-PO₃H₂、-NHC(＝O)R⁴、-NHC(＝O)N(R⁴)₂、-C(＝O)NH₂、-C(＝O)N(R⁴)₂、-OR^4b、-SR^4b、-N(R^4b)₂Wherein R is⁴Have the above-mentioned meanings; or

Wherein R is⁴As defined above, and R⁷Selected from SR⁴and-C (═ O) R⁴. The double bonds in the above groups may be in either the E or Z geometry.

In a preferred embodiment, R⁶Is a heterocycle having the structure:

wherein X⁴Is a chalcogen, and R^6aIs hydrogen or C_1-6Alkyl radical, wherein said C_1-6The alkyl group may be substituted one or more times with: -OH, -NH₂、-F、-Cl、-Br、-I、-NO₂-CN, -C (═ O) OH. In a more preferred embodiment, R⁶Is a heterocycle having the structure:

wherein R is^6aSelected from unsubstituted C_1-6Alkyl and C substituted one or more times by-OH_1-6An alkyl group.

The viscosity reducing agent may be an imidazole of formula (2 b-i):

wherein R is³As defined above. In some embodiments, R³Independently selected from hydrogen, NO₂And R⁴. In some preferred embodiments, the compounds of formula (2b-i) have the following structure:

wherein R is³Is independently selected from C_1-6An alkyl group which may be unsubstituted or substituted one or more times by a group selected from: OH, NH₂、SR⁴F, Cl, Br and I; and is

R^3gIs hydrogen or NO₂。

In other embodiments, the viscosity reducing agent has the structure of formula (2a-ii) or formula (2 c-i):

wherein R is³Independently selected from OH, Cl, Br, F, I, N (R)^4a)₂、C(＝O)OH、C(＝O)NH₂。

In other embodiments, at least one R is³The substituent is NHR⁴Wherein R is⁴Is C_1-6Alkyl, optionally substituted with one or more groups selected from: cl, Br, F, I, OH, C (═ O) OH, NH₂、NH(C_1-6Alkyl) and N (C)_1-6Alkyl radical)₂。

In other embodiments, the viscosity reducing agent is a pyridinium salt of formula (2 a-iii):

wherein R is³And R⁴As defined above.

In other embodiments, the heterocyclic ring is not a heteroaryl ring. Exemplary non-aromatic rings include compounds of formula (2 f-k):

wherein R is⁵And X has the above-mentioned meaning, and X³Is a chalcogen or N (R)³)_z。

In some embodiments, the compound of formula (2f) is a β -lactam of formula (2 f-i):

the beta-lactams of formula (2f-i) include penicillin-type compounds and cephalosporin-type and cephamycin-type compounds of formulae (2f-ii) and (2 f-iii):

wherein X and R3 are as defined above. In a preferred embodiment, X is sulfur.

In some embodiments, the compound of formula (2i) is a compound of formula (2 i-i):

wherein X and R³As defined above. In some embodiments, X is NR in both cases⁴Wherein R is⁴Has the above-mentioned meaning, and R³In both cases hydrogen.

In other embodiments, the compound of formula (2) may be represented by a compound of formula (2 i-ii):

x, X therein¹And R³As aboveAs defined herein.

The compound of formula (2j) may be represented by a compound of formula (2 j-i):

wherein X³And R³As defined above, and R⁸Is selected from NHC (═ O) R²And OC (═ O) R². In a preferred embodiment, X³Is N⁺(CH₃)₂，R³Are each hydrogen, or R³Together form an epoxide or double bond.

The compound of formula (2k) may be represented by a compound of formula (2 k-i):

wherein X³And R⁸As defined above.

In other embodiments, the viscosity reducing agent comprises a compound having the structure of formula (3):

wherein R is⁵Independently at each occurrence selected from hydrogen and R²；

R^5’Is R⁵Or is absent;

provided that at least one R⁵The substituent being other than hydrogen, wherein R²Have the same meaning as given for the compound of formula (1).

In some embodiments, the viscosity reducing agent is a mixture of two or more compounds selected from formula (1), formula (2), and formula (3).

In a preferred embodiment, the viscosity reducing agent is camphorsulfonic acid (CSA) or a pharmaceutically acceptable salt thereof such as an alkali metal or alkaline earth metal salt. Combining camphorsulfonic acid or a salt thereof with one or more compounds of formula (1), (2) or (3) to give a mixture such as CSA-piperazine, CSA-TRIS, CSA-4-aminopyridine, CSA-1- (o-tolyl) biguanide, CSA-procaine, CSA-Na-aminocyclohexanecarboxylic acid, CSA-Na-creatinine and CSA-Na-ornidazole. Other preferred viscosity reducing agents include thiamine, procaine, biotin, creatinine, metoclopramide, scopolamine, cimetidine, chloroquine phosphate, mepivacaine, granisetron, sucralose, HEPES-TRIS, nicotinamide, lactobionic acid-TRIS, glucuronic acid-TRIS, sulphamoyl, CSA-4-aminopyridine, CSA-piperazine and cefazolin. Any two or more of the viscosity reducing agents listed above may be further combined in the same formulation.

In other embodiments, the viscosity reducing agent is an organic sulfonic acid. Exemplary organic sulfonic acids include, but are not limited to, camphorsulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, toluenesulfonic acid, cyclohexylsulfonic acid, xylenesulfonic acid (including p-xylene-2-sulfonic acid, m-xylene-4-sulfonic acid, and o-xylene-3-sulfonic acid), methanesulfonic acid, 1, 2-ethanedisulfonic acid, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 2-hydroxyethane-1-sulfonic acid, 3-hydroxypropane-1-sulfonic acid, cymene sulfonic acid, 4-hydroxybutane-1-sulfonic acid, and pharmaceutically acceptable salts thereof. The organic sulfonic acid may be in the form of an alkali or alkaline earth metal salt, such as lithium, sodium, potassium, magnesium and calcium salts. The organic sulfonic acid (or salt thereof) may be combined with one or more compounds of formula (2) or formula (3).

In some embodiments, the viscosity reducing agent comprises at least one carboxylic acid. The carboxylic acid may be in the form of an alkali or alkaline earth metal salt, such as lithium, sodium, potassium, magnesium and calcium salts. Exemplary carboxylic acid compounds include lactobionic acid, glucuronic acid, 1-aminocyclohexanecarboxylic acid, biotin, bromcrine, cyclopentanepropionic acid, hydroxynaphthoic acid, phenylpropionic acid, gentisic acid, salicylic acid, camphoric acid, mandelic acid, sulfosalicylic acid, hydroxybenzoylbenzoic acid, phenylacetic acid, acetylsalicylic acid, cinnamic acid, t-butylacetic acid, phthalic acid, trimethylacetic acid, meglitinide, and pharmaceutically acceptable salts thereof. The carboxylic acid (or salt thereof) may be combined with one or more compounds of formula (2) or formula (3).

The following compounds may also be used as viscosity reducing agents: colistin, articaine, tetracaine, propoxymetacaine, metoclopramide, procaine, lidocaine, cyclomethiylcaine, perocaine, chloroprocaine, etidocaine, benzocaine, phenylephrine, bupivacaine, mepivacaine, cinchocaine, mixtures thereof, and pharmaceutically acceptable salts thereof.

Other materials that may be used as viscosity reducers include 1-aminocyclohexane carboxylic acid, 1- (o-tolyl) biguanide, benzethonium chloride, benzoic acid, bromcrine, calcium carrageenan, calcium cyclamate, cobutramine calcium, caro's acid, camphorsulfonic acid, creatinine, dalfampridine, dehydroacetic acid, diazolidinyl imidazole urea, dichlorobenzyl alcohol, dimethyl isosorbide, epimeridine, ethylmaltol, ethylvanillin, ornidazole, ethanolamine gentisic acid, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), gentisic acid, glucuronic acid, iodixanoic acid, menthol, galactose, methylene phosphate, m-cresol, glutathione, lactobionic acid, maltol, caprylyl salicylate, hydroxyquinoline, pentetic acid, piperazine, propenyl guaethol, propyl gallate, propenyl carbonate, propyl paraben, lactic acid, Protamine sulfate, quaternary ammonium-15, quaternary ammonium-52, satialgine H, 1, 2-ethanedisulfonic acid sodium, cocoyl sarcosine sodium, lauroyl sarcosine sodium, sodium polymetaphosphate, sodium pyrophosphate, pyroglutamic acid, trimetaphosphate-3 Na, sodium tripolyphosphate, sorbitan, tartaric acid, lactic acid, iodofinamide, sucralose, 1- (4-pyridyl) pyridinium chloride, aminobenzoic acid, sodium sulfacetamide, naphthalene-2-sulfonic acid, t-butylhydroquinone, thimerosal, tromethamine, trojanine, vanillin, vessemine, 1, 2-cyclohexanedione dioxime, niacinamide, methylisothiazolinone, mannose D, maltose, lidonine, lactose, lactitol, isomalt, imidazolidinyl urea, gluconolactone, methanesulfonic acid, xylenesulfonic acid, sulfobutyl ether β -cyclodextrin and pharmaceutically acceptable salts thereof.

In some embodiments, the viscosity reducing agent comprises an organic base. Exemplary organic bases include N-methylglucamine, morpholine, piperidine, primary amines, secondary amines, tertiary amines, quaternary amines, substituted amines, and cyclic amines. For example, it may be isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, lidocaine, hydrabamine, choline, betaine, ethylenediamine, theobromine, purine, piperazine, N-ethylpiperidine, N-methylpiperidine polyamine. Particularly preferred organic bases are arginine, histidine, lysine, ethanolamine, thiamine, 2-amino-2-hydroxymethylpropane-1, 3-diol (TRIS), 4-aminopyridine, aminocyclohexanecarboxylic acid, 1-o-tolylbiguanide, ornidazole, urea, nicotinamide, benzethonium chloride, 5-amino-1-pentanol, 2- (2-aminoethoxy) ethanol, trans-cyclohexane-1, 4-diamine, trans-cyclohexane-1R, 2R-diamine, ethylenediamine, propane-1, 3-diamine, butane-1, 4-diamine, pentane-1, 5-diamine, hexane-1, 6-diamine, octane-1, 8-diamine, 5-amino-1-pentanol, thionine, pentanes, thionine, pentanes, thionine, and the like, 2- (2-aminoethoxy) ethylamine, 2- (2- (2-aminoethoxy) -ethoxy) ethylamine, 3- (4- (3-aminopropoxy) -butoxy) propan-1-amine, 3- (2- (2- (3-aminopropoxy) -ethoxy) propan-1-amine, N- (2- (2-aminoethylamino) ethyl) ethane-1, 2-diamine, N- (2-aminoethyl) ethane-1, 2-diamine, N-1- (2- (2- (2-aminoethylamino) ethylamino) -ethyl) ethane-1, 2-diamine, mixtures thereof, and mixtures thereof, N, N-dimethylhexane-1, 6-diamine, N, N-tetramethylbutane-1, 4-diamine, phenyltrimethylammonium salt, isopropylamine, diethylamine, ethanolamine, tromethamine, choline, 1- (3-aminopropyl) -2-methyl-1H-imidazole, piperazine, 1- (2-aminoethyl) piperazine, 1- [3- (dimethylamino) propyl ] piperazine, 1- (2-aminoethyl) piperidine, 2- (2-aminoethyl) -1-methylpyrrolidine, mixtures thereof and pharmaceutically acceptable salts thereof.

Exemplary beta-lactams include benzylpenicillin (penicillin G), phenoxymethylpenicillin V (penicillin V), cloxacillin, dicloxacillin, flucloxacillin, methicillin, nevucillin, oxacillin, temocillin, amoxicillin, ampicillin, mecillin, carbenicillin, ticarcillin, azlocillin, mezlocillin, piperacillin, cefoxitin, cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefixime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, pivalo, cefpiramide, biapenem, doripenem, faropenem, imipenem, meropenem, panipenem, aprepipenem, tipipenem, tiamidem, thienamycin, tejimoman, tezomib, te, Nocardiin a, alpha, epsilon-diamino-beta-hydroxyheptanedioic acid, clavulanic acid, tazobactam, sulbactam and pharmaceutically acceptable salts thereof.

Other viscosity reducing agents include tropane N-heterocycles such as atropine, hyoscyamine, scopolamine and its salts, tiotropium and ipratropium salts, thiamine, allithiamine, prosultiamine, thiofuran, benfotiamine, sulbuthionine, quaternium 15, 1- (3-aminopropyl) -2-methyl-1H-imidazole dihydrochloride, creatinine, biotin, cimetidine, perocaine, cyclomethiycaine, granisetron, moxifloxacin, chloroquine, mepivacaine, levetiracetam, bupivacaine, cinchocaine, clindamycin and pharmaceutically acceptable salts thereof. Thiamine is a particularly preferred viscosity reducing agent.

In some formulations, the following compounds are not preferred and are excluded from the structural formulae and definitions of the useful viscosity reducers described above: creatinine, cadaverine, lidocaine, arginine, and lysine.

C. Excipient(s)

A wide variety of pharmaceutical excipients useful in liquid protein formulations are known to those skilled in the art. Which includes one or more additives, such as a liquid solvent or a co-solvent; sugars or sugar alcohols such as mannitol, trehalose, sucrose, sorbitol, fructose, maltose, lactose or dextran; surfactants, such as20. 60 or 80 (polysorbate 20, 60 or 80); a buffering agent; preservatives, such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, and chlorhexidine diacetate; carriers such as poly (ethylene glycol) (PEG); antioxidants such as ascorbic acid, sodium metabisulfite, and methionine; chelating agents such as EDTA or citric acid; or biodegradable polymers such as water-soluble polyesters; a cryoprotectant; a freeze-drying protective agent; a filler; and a stabilizer.

Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington: "The Science and Practice of Pharmacy", 20 th edition, Alfonso R.Gennaro, Ed., Lippincott Williams & Wilkins (2000) may also be included in The protein formulations described herein, provided that they do not adversely affect The desired properties of The formulation.

The viscosity-lowering agents described herein may be combined with one or more other types of viscosity-lowering substances, such as organic phosphates described in the PCT application entitled "LIQUID products FORMULATIONS compositions CONTAINING organic salts," filed concurrently with Arsia Therapeutics; WATER-SOLUBLE DYES described in the PCT application entitled "LIQUID PROTEIN FORMULATIONS compositions contacting Water dye" co-filed by Arsia Therapeutics; and IONIC LIQUIDs as described in the PCT application entitled "LIQUID products FORMULATIONS contents IONIC LIQUIDs" co-filed by Arsia Therapeutics.

Preparation method

The protein to be formulated, such as a monoclonal antibody, can be produced by any known technique, such as by culturing cells transformed or transfected with a vector containing one or more nucleic acid sequences encoding the protein as known in the art or by synthetic techniques (such as recombinant techniques and peptide synthesis or a combination of these techniques) or can be isolated from endogenous sources of the protein.

Purification of the protein to be formulated may be carried out by any suitable technique known in the art, such as ethanol or ammonium sulfate precipitation, reverse phase HPLC, chromatography on silica gel or on cation exchange resins (e.g. DEAE-cellulose), dialysis, chromatofocusing, use of protein aColumn (e.g. column)G-75) gel filtration to remove impurities, metal chelating columns bound to epitope tag forms, and ultrafiltration/diafiltration (non-limiting examples include centrifugal filtration and tangential flow filtrationFiltration (TFF)).

The viscosity reducing agent introduced at a concentration that reduces viscosity, such as 0.010M to 1.0M, preferably 0.050M to 0.50M, most preferably 0.10M to 0.30M, allows for purification and/or concentration of solutions of pharmaceutically active monoclonal antibodies to higher monoclonal antibody concentrations using conventional methods known to those skilled in the art, including but not limited to tangential flow filtration, centrifugal concentration, and dialysis.

In some embodiments, lyophilized formulations of proteins are provided and/or used in the preparation and manufacture of concentrated, low viscosity protein formulations. In some embodiments, the pre-lyophilized protein in powder form is reconstituted by dissolution in an aqueous solution. In this embodiment, the liquid formulation is filled into a specific dosage unit container such as a vial or pre-filled hybrid syringe, lyophilized, optionally with lyoprotectants, preservatives, antioxidants, and other typical pharmaceutical excipients, and then stored under aseptic storage conditions until just prior to use, at which time it is reconstituted with a defined volume of diluent to bring the liquid to the desired concentration and viscosity.

The formulations described herein may be stored by any suitable method known to those skilled in the art. Non-limiting examples of methods for preparing a protein formulation for storage include freezing, lyophilizing, and spray drying a liquid protein formulation. In some cases, the lyophilized formulation is stored frozen at sub-zero temperatures, such as at about-80 ℃ or in liquid nitrogen. In some cases, the lyophilized or aqueous formulation is stored at 2-8 ℃.

Non-limiting examples of diluents that may be used to reconstitute a lyophilized formulation prior to injection include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, ringer's solution, dextrose solution, or aqueous solutions of salts and/or buffers. In some cases, the formulations are spray dried and then stored.

Administering to a subject in need thereof

Protein formulations, including but not limited to reconstituted formulations, are administered to a human in need thereof by intramuscular, intraperitoneal (i.e., injection into a body cavity), intracerobrospinal or subcutaneous injection using an 18-32 gauge needle (optionally a thin walled needle) in a volume of less than about 5mL, less than about 3mL, preferably less than about 2mL, more preferably less than about 1 mL.

The appropriate dosage of a protein, such as a monoclonal antibody ("therapeutically effective amount") will depend on the condition to be treated, the severity and course of the disease or condition, whether the protein is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the judgment of the attending physician. The protein is suitably administered in a single injection or in multiple injections, either as monotherapy or in combination with other drugs or therapies over a range of treatments.

The dosage formulation is designed so that the injection does not cause significant signs of irritation at the injection site, for example where the primary irritation index is less than 3 when evaluated using the Draize scoring system. In an alternative embodiment, injection causes a visually similar level of irritation when compared to injection of an equal volume of saline solution. In another embodiment, the bioavailability of the protein is higher when compared to an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering agents. In another embodiment, the formulation is pharmaceutically effective at least approximately as much as about the same dose of protein administered by intravenous infusion.

In a preferred embodiment, the formulation is injected to give an increased level of therapeutic protein. For example, the AUC value may be at least 10%, preferably at least 20%, higher than the AUC value calculated for an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering agents.

Viscosity lowering agents can also affect bioavailability. For example, the percent bioavailability of a protein may be at least 1.1-fold, preferably at least 1.2-fold, the percent bioavailability of an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering agents.

Viscosity reducing agents may also affect pharmacokinetics. E.g. C after subcutaneous or intramuscular injection_MAXComparable to a substantially equivalent pharmaceutically effective veinInternal administration dosage of C_MAXAt least 10%, preferably at least 20% lower.

In some embodiments, the protein is administered at a higher dose and with a lower frequency than an otherwise identical formulation that does not contain one or more viscosity-lowering agents.

Formulations with lower viscosities require less injection force. For example, the injection force may be at least 10%, preferably at least 20% less than the injection force required for an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering agents. In one embodiment, the injection is performed with a27 gauge needle and the injection force is less than 30N. In most cases, the formulations will be administered using a very small gauge needle, for example 27 to31 gauge, usually 27, 29 or 31 gauge.

The viscosity-lowering agent may be used in the preparation of dosage unit formulations suitable for reconstitution to form liquid pharmaceutical formulations for subcutaneous or intramuscular injection. The dosage unit may contain a dry powder of one or more proteins; one or more viscosity reducing agents; and other excipients. The protein is present in the dosage unit such that the resulting formulation, after reconstitution in a pharmaceutically acceptable solvent, has a protein concentration (mg/mL) of about 100mg to about 2,000mg/1 mL. Such reconstituted formulations may have an absolute viscosity of about 1cP to about 50cP at 25 ℃.

The low viscosity formulation may be provided in the form of a solution or dosage unit, wherein the protein is lyophilized in one vial with or without the viscosity-lowering agent and other excipients, and the solvent with or without the viscosity-lowering agent and other excipients is provided in a second vial. In this embodiment, a solvent is added to the protein immediately prior to or at the time of injection to ensure uniform mixing and dissolution.

The one or more viscosity-lowering agents are present in the formulation at a concentration that does not cause significant signs of toxicity and/or signs of irreversible toxicity when administered via subcutaneous, intramuscular, or other injection types. As used herein, "significant signs of toxicity" include poisoning, lethargy, behavioral changes such as those that occur when the central nervous system is compromised, infertility, severe signs of cardiotoxicity such as cardiac arrhythmia, cardiomyopathy, myocardial infarction, and cardiac or congestive heart failure, renal failure, liver failure, dyspnea, and death.

In preferred embodiments, the formulation does not cause significant irritation when administered no more than twice daily, once daily, twice weekly, once weekly, or once monthly. The administration of the protein formulation may not cause significant signs of irritation at the injection site, which is measured as follows: the primary irritation index is less than 3, less than 2, or less than 1 when evaluated using the Draize scoring system. As used herein, "significant signs of irritation" include erythema, redness and/or swelling at the injection site greater than 10cm, greater than 5cm, or greater than 2.5cm in diameter, necrosis at the injection site, exfoliative dermatitis at the injection site, and severe pain that interferes with daily activities and/or requires hospitalization or hospitalization. In some embodiments, injection of the protein formulation causes a visually similar level of irritation when compared to injection of an equal volume of saline solution.

When administered via subcutaneous or intramuscular injection, the protein formulation may exhibit improved bioavailability compared to an otherwise identical formulation without the one or more viscosity-lowering agents. "bioavailability" refers to the extent and rate at which a biologically active substance, such as a monoclonal antibody, enters the circulation or reaches the site of action. The overall bioavailability of subcutaneous or intramuscular injection is improved compared to an otherwise identical formulation without the one or more viscosity-lowering agents. "percent bioavailability" refers to the fraction of a administered dose of a biologically active substance that enters the circulation, as determined relative to an intravenously administered dose. One way to measure bioavailability is by comparing the "area under the curve" (AUC) of the plasma concentration versus time curve. The AUC may be calculated, for example, using a linear trapezoidal rule. "AUC" as used herein_∞"refers to the area under the plasma concentration curve from time zero to the time when the plasma concentration returns to the baseline level. "AUC" as used herein_0-t"refers to the area under the plasma concentration curve from time zero to a later time t, e.g., to the time at which baseline is reached. Time will typically be measured in days, although hours may also be used when context is evident. For example,the AUC can increase by more than 10%, 20%, 30%, 40%, or 50% as compared to an otherwise identical formulation administered in the same manner but without the one or more viscosity-lowering agents.

"t" as used herein_max"refers to the time at which the plasma concentration reaches a maximum after administration.

"C" as used herein_max"refers to the maximum plasma concentration after a given dose and before the administration of a subsequent dose.

"C" as used herein_min"or" C_Grain"refers to the minimum plasma concentration after a given dose and before the administration of a subsequent dose.

C after subcutaneous or intramuscular injection_maxComparable to intravenous doses of C_maxSmall, e.g., at least 10%, more preferably at least 20%. C_maxThis reduction in (b) may also result in reduced toxicity.

Pharmacokinetic and pharmacodynamic parameters can be approximated across species using measures known to those skilled in the art. The pharmacokinetics and pharmacodynamics of antibody therapeutics can vary significantly based on the particular antibody. Approved murine monoclonal antibodies will exhibit a half-life of about 1 day in humans, whereas the half-life of human monoclonal antibodies will typically be about 25 days (Waldmann et al, int. Immunol.,2001,13: 1551-1559). The pharmacokinetics and pharmacodynamics of antibody therapeutics can be significantly different based on the route of administration. The time to reach maximum plasma concentration after intramuscular or subcutaneous injection of IgG is typically 2 to 8 days, although shorter or longer times may occur (Wang et al, Clin. pharm. Ther.,2008,84(5): 548-558). The pharmacokinetics and pharmacodynamics of antibody therapeutics can be significantly different based on formulation.

Low viscosity protein formulations may allow greater flexibility in administration and reduce the frequency of administration compared to those protein formulations that do not contain one or more viscosity reducing agents. For example, by increasing the dose administered per injection by a multiple of times, the frequency of administration can be reduced in some embodiments from once every 2 weeks to once every 6 weeks.

Protein formulations, including but not limited to reconstituted formulations, can be administered using heated and/or self-mixing syringes or autoinjectors. The protein formulation may also be preheated in a separate warming unit prior to filling the syringe.

i. Heated syringe

The heated syringe may be a standard syringe that is preheated using a syringe warmer. The syringe warming device will typically have one or more openings each capable of receiving a syringe containing the protein formulation and means for heating and maintaining the syringe at a particular temperature (typically above ambient) prior to use. Which will be referred to herein as a preheated syringe. Suitable warming devices for heated syringes include those available from Vista Dental Products and Inter-Med. The warming device is capable of accommodating syringes of various sizes and heating to any temperature up to about 130 ℃ (typically varying within 1 ℃). In some embodiments, the syringe is preheated in a heating bath, such as a water bath maintained at a desired temperature.

The heated syringe may be a self-heating syringe, i.e., capable of heating and maintaining the liquid formulation in the syringe at a particular temperature. The self-heating injector may also be a standard medical injector to which the heating device has been attached. Suitable heating devices that can be attached to the syringe include syringe heaters or syringe heating tapes available from Watlow Electric Manufacturing Co., St.Louis, Mo, and syringe heating modules, stage heaters, and in-line pre-fill heaters such as SW-61 syringe warmers available from Warner Instruments, Hamden, CT. The heaters may be controlled by a central controller, such as a model TC-324B or TC-344B heater controller available from Warner Instruments.

The heated syringe maintains the liquid protein formulation at a specific temperature or a specific temperature that varies within 1 ℃,2 ℃, or 5 ℃. The heated syringe may hold the protein formulation at any temperature from room temperature up to about 80 ℃, up to about 60 ℃, up to about 50 ℃ or up to about 45 ℃, so long as the protein formulation is sufficiently stable at that temperature. The heated syringe can maintain the protein formulation at a temperature of 20 ℃ to 60 ℃,21 ℃ to 45 ℃,22 ℃ to 40 ℃,25 ℃ to 40 ℃, or 25 ℃ to 37 ℃. By maintaining the protein formulation at an elevated temperature during injection, the viscosity of the liquid formulation is reduced, the solubility of the protein in the formulation is increased, or both.

Self-mixing syringe

The injector may be self-mixing or may have an attached mixer. The mixer may be a static mixer or a dynamic mixer. Examples of static mixers include those disclosed in U.S. patents 5,819,988, 6,065,645, 6,394,314, 6,564,972, and 6,698,622. Some examples of dynamic mixers may include those disclosed in U.S. patents 6,443,612 and 6,457,609 and U.S. patent application publication US 2002/0190082. The syringe may include a plurality of barrels for mixing the components of the liquid protein formulation. Us patent 5,819,998 describes a syringe having two barrels and a mixing tip for mixing two-component viscous substances.

Auto-and pre-filled syringes for protein formulations

Liquid protein formulations may be administered using pre-filled syringe autoinjectors or needleless injection devices. The auto-injector comprises a hand-holdable, generally pen-shaped, cartridge grip for gripping the replaceable pre-filled cartridge and a spring-based or similar mechanism for injecting a dose of liquid drug subcutaneously or intramuscularly from the pre-filled cartridge. Autoinjectors are typically designed for self-administration or administration by untrained personnel. The auto-injector may be used to dispense a single dose or multiple doses from a pre-filled cartridge. Autoinjectors enable different user settings including, inter alia, injection depth, injection speed, etc. Other injection systems may include those described in U.S. patent 8,500,681.

The lyophilized protein formulation may be provided in a pre-filled or unit dose syringe. Us patents 3,682,174, 4,171,698 and 5,569,193 describe sterile syringes containing two chambers that can be prefilled with a dry formulation and a liquid capable of mixing immediately prior to injection. US patent 5,779,668 describes use in medicineA syringe system for lyophilization, reconstitution and administration of a pharmaceutical composition. In some embodiments, the protein formulation is provided in a pre-filled or unit dose syringe in lyophilized form, reconstituted in the syringe prior to administration and administered by a single subcutaneous or intramuscular injection. An autoinjector for delivery of unit dose lyophilized drugs is described in WO 2012/010,832. Autoinjectors such as Safe Click Lyo^TM(marketed by Future Injection Technologies, ltd., Oxford, u.k.) may be used to administer unit dose protein formulations, wherein the formulation is stored in lyophilized form and reconstituted immediately prior to administration. In some embodiments, the protein formulation is provided in a unit dose cartridge (sometimes referred to as a Vetter cartridge) for lyophilizing the drug. Examples of suitable cartridges may include those described in U.S. patents 5,334,162 and 5,454,786.

V. method of purification and concentration

Viscosity reducing agents may also be used to aid in protein purification and concentration. One or more viscosity reducing agents and excipients are added to the protein in an amount effective to reduce the viscosity of the protein solution. For example, the viscosity reducing agent is added to a concentration of about 0.01M to about 1.0M, preferably about 0.01M to about 0.50M and most preferably about 0.01M to about 0.25M.

The protein-containing viscosity-reducing agent solution is then purified or concentrated using a method selected from the group consisting of ultrafiltration/diafiltration, tangential flow filtration, centrifugal concentration, and dialysis.

Examples

The foregoing will be further understood by the following non-limiting examples.

After 5 minutes equilibration at 25 ℃, the viscosities of all well-mixed aqueous monoclonal antibody solutions were measured using an mVROC microfluidic viscometer (RheoSense) or a DV2T cone and plate viscometer (Brookfield; "C)&P ") (unless otherwise specified). The mVROC viscometer is equipped with an "a" or "B" chip, each fabricated with 50 micron channels. Typically, 0.10mL of the protein solution is loaded into an air-tight micro-laboratory instrument syringe (Hamilton; 100. mu.L) at the back, immobilized on a chip and measured at multiple flow rates (about 20%, 40%, and 60% of the maximum pressure per chip). For example, a sample of about 50cPAt about 10,20 and 30 μ L/min (about 180, 350 and 530s, respectively, on an "A" chip)^-1) The measurement is carried out until the viscosity has stabilized, usually after at least 30 seconds. The mean absolute viscosity and standard deviation were then calculated from such at least three measurements. C&The P viscometer was equipped with CPE40 or CPE52 spindle (cone angles of 0.8 ° and 3.0 °, respectively), and 0.50mL samples from 2 to 400s^-1Is measured at a plurality of shear rates. Specifically, the sample is mixed with 22.58, 24.38, 26.25, 28.13, 30, 31.88, 45, 67.5, 90, 112.5, 135, 157.5, 180, 202.5, 247, 270, 292.5, 315, 337.5, 360, 382, 400s^-1Each measurement was taken for 30 seconds, starting with a shear rate that achieved at least 10% torque and continuing until the instrument torque reached 100%. The extrapolated zero shear viscosity is then determined from a plot of dynamic viscosity versus shear rate for the samples measured on the DV2T cone-plate viscometer. The extrapolated zero shear viscosity reported is the mean and standard deviation of at least three measurements.

Example 1: viscosity reducing agent camphorsulfonic acid lysine (CSAL) to biological analogsBy the viscosity of the solution

Materials and methods

For commercial biosimids containing pharmaceutical excipients (polysorbate 80, phosphate buffer and NaCl))(100-400mg) was purified. First useTWEEN Medi Columns (G-Biosciences) were used to remove polysorbate 80. The resulting solution was then buffer exchanged for 20mM sodium phosphate buffer (PB; pH 7.0) or20 mM CSAL (pH 7.0) and concentrated on a Jumbosep centrifugal concentrator (Pall Corp.) to a final volume of less than 10 mL. The collected protein solution was freeze-dried. The dried protein cake containing protein and buffer salts or reagents is reconstituted to 0.15-1.Final volume of 3 mL. These samples were reconstituted with additional PB (pH 7.0) or CSAL (pH 7.0) sufficient to give a final concentration of PB or CSAL of 0.25M. The final concentration of monoclonal antibody in solution was determined by light absorption at 280 nm. The reported protein concentrations represent the range of individual protein samples encompassed by each table or each figure. Specifically, the reported values are the median plus or minus one half of the range. The extrapolated zero shear and reported viscosity using an experimentally determined extinction coefficient, i.e., 1.4L/g-cm, were measured on a DV2T cone and plate viscometer.

Results

The data in FIG. 1 show CSAL versus bioanalogsThe viscosity reducing effect of the aqueous solution of (a). Biological analogsThe viscosity of the solution in Phosphate Buffer (PB) increases exponentially with increasing concentration of monoclonal antibody. Bioanalogue was observedThe viscosity of the solution of (a) increases exponentially with increasing monoclonal antibody concentration in the presence of CSAL, but to a lesser extent than the formulation in PB, i.e. the viscosity gradient is reduced. The data in figure 1 show that the higher the concentration of monoclonal antibody, the greater the viscosity reducing effect. The magnitude of the viscosity-lowering effect achieved by replacing PB with CSAL was 1.1-fold for the 100 + -5 mg/mL monoclonal antibody to 10.3-fold for the 227 + -5 mg/mL monoclonal antibody.

Example 2: viscosity reducing action of viscosity reducing agent camphorsulfonic acid lysine (CSAL) as biosimilarAs a function of concentration of

Materials and methods

Biosimids commercially available and containing pharmaceutical excipients (polysorbate 20, phosphate buffer, citrate buffer, mannitol and NaCl) as described in example 1 (extinction coefficient of 1.7L/g-cm at 280nm)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. The protein was formulated to contain 0.25M phosphate buffer or 0.25M CSAL.

Results

Figure 2 depicts the viscosity of an aqueous solution of monoclonal antibody as a function of the concentration of monoclonal antibody in an aqueous buffered solution with CSAL. Biological analogsViscosity in aqueous phosphate buffer in the presence of CSAL increases exponentially with increasing concentration; however, as biosimilarsThat way, the increase is very less pronounced with CSAL-containing formulations, i.e., the viscosity gradient is reduced. Generally, the higher the concentration of monoclonal antibody, the greater the viscosity reduction observed. The magnitude of the viscosity-lowering effect achieved by replacing PB with CSAL was 2.1-fold for the 80mg/mL monoclonal antibody to 3.7-fold for the 230. + -.5 mg/mL monoclonal antibody.

Example 3: biological analogsThe viscosity reduction of the aqueous solution of (a) as a function of the CSAL concentration

Materials and methods

Samples were purified, buffer exchanged, concentrated, dried, reconstituted and analyzed similarly to example 1 above. The final concentration of CSAL in the aqueous CSAL solution after reconstitution was 0.25M to 0.20M.

Results

Table 1 shows the biological analogs of CSAL formulated in 0.25M phosphate buffer (without CSAL as a control) and having different concentrations of CSALThe viscosity of the solution of (a). The viscosity lowering effect of CSAL was observed to increase from 8.4-fold to 12.1-fold with increasing concentration of viscosity-lowering agent. The data in Table 1 show that the higher the concentration of CSAL, the greater the viscosity reduction effect, at least over the range of agent concentrations tested.

TABLE 1 biological analogs(155. + -. 5mg/mL, pH 7.0) viscosity of aqueous solutions at 25 ℃ in the presence of CSAL at various concentrations

Example 4: biological analogsAs a function of temperature in the presence of various viscosity-reducing agents

Materials and methods

For biosimids containing various viscosity-lowering agents as described in example 1The aqueous solution of (a) is prepared. Specifically, a 20mM solution of the viscosity-lowering agent of interest was used for buffer exchange and the lyophilized cake was reconstituted to 0.25M of each viscosity-lowering agent. For samples containing CSA-APMI, for the biological analogsThe expansion buffer exchange to 2mM PB (pH 7.0) was performed and concentrated on Jumbosep centrifugal concentrator (Pall Corp.) to a final volume of less than 10 mL. Firstly, to the sampleAnd (4) carrying out equal division. An appropriate amount of CSAAPMI solution (pH 7.0) was then added to each aliquot such that the final excipient concentration was 0.25M after reconstitution with water. The protein solution was then freeze dried. The dried protein cake containing the protein and viscosity reducing agent (and negligible amounts of buffer salt) was reconstituted to a final volume of about 0.10mL and viscosity reducing agent concentration as previously described.

Results

Table 2 shows the biological analogsViscosity data in the presence of six viscosity reducing agents, namely, lysine Camphorsulfonate (CSAL), arginine Camphorsulfonate (CSAA), lysine Benzenesulfonate (BSAL), arginine Benzenesulfonate (BSAA), arginine Naphthalenesulfonate (NSAA), and 1- (3-aminopropyl) -2-methyl-1H-imidazole Camphorsulfonate (CSAAPMI). The data in Table 2 show that under otherwise identical conditions, the biological analogsAll six viscosity reducing agents reduced the viscosity by at least about 9-fold compared to solutions in phosphate buffer. The most effective viscosity reducing agent, CSAAPMI, reduces viscosity>40 times.

In addition, the data in Table 3 show that bioanalogs prepared with 0.25M CSAA at various temperatures from 20 ℃ to 30 ℃Has the lowest viscosity of the five viscosity reducing agents. Thus, the viscosity trends observed at 25 ℃ seem to be predictive of those at temperatures of at least 20 ℃ and 30 ℃.

TABLE 2 Bioanalogues formulated with various 0.25M viscosity-lowering agentsHas a viscosity reduction at 25 ℃ compared to the viscosity in a 0.25M sodium Phosphate Buffer (PB) of an aqueous solution (226. + -.6 mg/mL, pH 7.0)

TABLE 3 Bioanalogs formulated with various 0.25M viscosity-lowering agents(225. + -. 5mg/mL, pH 7.0) viscosity of the aqueous solution of (b)

Example 5: temperature vs. biological analogs formulated with various viscosity reducing agentsBy the viscosity of the aqueous solution of

Materials and methods

For biosimids containing different viscosity-lowering agents as described in example 1 aboveThe solution of (3) is prepared. Specifically, a 20mM solution of the viscosity-lowering agent of interest was used for buffer exchange and the lyophilized cake was reconstituted to 0.15 or 0.25M viscosity-lowering agent.

Results

As can be seen from Table 4, the 0.25M CSAL produced bioanalogs at all three temperatures between 20 and 30 deg.CThe viscosity of the 230 + -5 mg/mL solution was reduced. Furthermore, 0.15M CSAL reduced the viscosity to about the same absolute value as 0.25M CSAL at 20 and 25 ℃ and was equivalent at 30 ℃.

The data in Table 5 compares the effect of CSAL and BSAL at a concentration of 0.15M. CSAL is an excellent viscosity reducer compared to BSAL at all three temperatures.

TABLE 4 Bioanalogs formulated with 0.25 and 0.15M CSAL(230. + -. 5mg/mL, pH 7.0) viscosity at different temperatures

TABLE 5 Bioanalogues formulated with 0.15M CSAL and BSAL(230. + -. 5mg/mL, pH 7.0) viscosity at different temperatures

Example 6: removal of CSAL reversed the viscosity lowering effect in the monoclonal antibody solution

Materials and methods

Preparation of biological analogsAnd biological analogsEach triplicate sample. The polysorbate was first removed from the commercial monoclonal antibody solution. The resulting solution (i) with the remaining pharmaceutical excipients was concentrated on a centrifugation device (Pall Corp.) with a molecular weight cut-off (MWCO) of 100kDa as a control sample(original excipients); (ii) buffer exchange to 0.25M CSAL was performed as described in example 1; or (iii) buffer exchanged to 0.25M CSAL, reconstituted and then further exchanged to 0.25M PB as described in example 1. In this third case, the exchange to 0.25M phosphate buffer was first carried out by overnight dialysis against 20mM PB (50kDa MWCO, Spectrum Labs). The partially dialyzed sample was then diluted to 60mL in 0.25M PB and concentrated by centrifugation (30 kDa MWCO Jumbosep (Pall Corp.) followed by 100kDa MWCO Macrosep device (Pall Corp.). The viscosities of these three aqueous solutions were determined as described above in example 1.

Results

Biological analogsAnd biological analogsThe viscosity of the aqueous solution of (a) was reduced by a factor of 2.7 and a factor of 1.5 in the presence of CSAL, respectively, but then increased when the CSAL was removed (see tables 6 and 7). Furthermore, after CSAL was removed, the viscosity of the monoclonal antibody solution returned to about the same level as the original solution, suggesting that CSAL did not destroy the protein and suggesting that it is necessary for the observed viscosity reduction.

TABLE 6 biological analogs(80. + -. 5mg/mL, pH 7.0) viscosity at 25 ℃

TABLE 7 biological analogs(101. + -.5 mg/mL, pH 7.0) at 25 ℃.

Example 7: viscosity reducing agent containing camphorsulfonic acidAnd biological analogsThe viscosity of the aqueous solution of (2) is greatly reduced

Materials and methods

For commercially available and pharmaceutical excipients as described aboveAnd biological analogs(Trehalose, sodium phosphate buffer and polysorbate 20; biological analogsPolysorbate 20, phosphate buffer, citrate buffer, mannitol and NaCl), purification, buffer exchange, concentration, freeze-drying and reconstitution. The samples in Table 8 were prepared as described above in example 1 (using a protein extinction coefficient of 1.7L/g-cm at 280nm) and tested at C&Measured on a P viscometer. Samples with reduced viscosity in table 9 were prepared as described in example 4 above, but the monoclonal antibody was buffer exchanged for 2mM PB. An appropriate amount of viscosity reducing agent is then added to bring the final viscosity reducing agent concentration after reconstitution to 0.15-0.35M. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data in tables 8 and 9 show different viscositiesReducing agent to biological analogueThe viscosity reducing effect of the aqueous solution of (a). For biological analogsUp to a 2.5-fold reduction in viscosity (compared to the monoclonal antibody solution in PB) was observed in the presence of CSA-containing viscosity reducing agents.

TABLE 8 Biologs with various viscosity-lowering agents(200. + -. 5mg/mL, pH 7.0) viscosity at 25 ℃

Contains equimolar NaCl; CSA ═ camphorsulfonic acid, BSA ═ benzenesulfonic acid, MSA ═ methanesulfonic acid, PB ═ phosphate buffer

TABLE 9 biological analogs with 0.15M viscosity-lowering agent (unless otherwise stated)At a viscosity of 25 ℃ of an aqueous solution (pH 7.0)

As shown in FIG. 3, the biosimilar with CSAA will beThe viscosity of the 200. + -.9 mg/mL aqueous solution was measured as a function of pH. Biological analogs when pH is increasedThe magnitude of the viscosity-lowering effect achieved by the presence of CSAA also increases, reaching a minimum viscosity and a maximum viscosity-lowering effect around pH 7. For two different concentrations of biological analogsThe viscosity reduction by CSAA was compared as a function of pH. FIG. 4 shows the 0.25M CSAA along with (i) the biological analogsAnd (ii) an increase in pH, resulting in a greater decrease in viscosity.

Table 10 comparison of bioanalogs with and without CSALViscosity reduction and brandingThe viscosity of (3) is reduced. In the absence of said agent, the brandHas a much higher viscosity than the solution of the bio-analog monoclonal antibody. However, the presence of 0.25M CSAL led to the bioanalogs and brandsThe viscosity of (a) is reduced by 1.8 times and 3.3 times respectively; the biological analogues and brands were observed in the presence of 0.25M CSALThe viscosities of (a) and (b) are similar.

TABLE 10 Bioanalogue containing 205 + -5 mg/mLOr brand nameWith or without 0.25M CSAL, at 25 ℃ and pH 7.0

CSAL ═ Camphoresulfonic acid lysine

As described in Table 11, for 210mg/mL biosimilarCSA 1- (3-aminopropyl) -2-methyl-1H-imidazole with HCl (CSAAPMI) provided superior viscosity reduction over CSAL, reducing viscosity by more than 5-fold compared to the PB control.

TABLE 11 Bioanalogs with various viscosity-lowering agentsAt 25 ℃ and pH 7.0

APMI ═ 1- (3-aminopropyl) -2-methyl-1H-imidazole

For biological analogs containing about 230mg/mLTable 12 shows that the viscosity reducing agent with sulfosalicylic acid reduced the viscosity by about 5-fold and the viscosity reducing effect of CSAAPMI and CSA thiamine.

TABLE 12 Bioanalogue containing 228 + -5 mg/mLWith a viscosity reducing agent at 25 ℃ and pH 7.0

APMI ═ 1- (3-aminopropyl) -2-methyl-1H-imidazole; CSA ═ camphorsulfonic acid

Example 8: pair of viscosity reducing agentsAnd biological analogsBy the action of an aqueous solution of

Materials and methods

Biometrics and brands containing various viscosity reducing agents as described in example 1The aqueous solution of (a) is prepared. Specifically, a 20mM solution of the salt of interest was used for buffer exchange and the lyophilized cake was reconstituted to contain 0.25M of each reagent. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip or a DV2T cone and plate viscometer.

Results

Table 13 shows the biological analogs(222. + -. 5mg/mL) in the presence of five viscosity reducing agents, namely CSAA, CSAL, BSAA, BSAL and NSAA. Table 14 compares the biological analogs using CSAA and CSALViscosity reduction of solutions and use of biosimids of arginine or lysine aloneThe viscosity of the solution decreases.

TABLE 13. havingBioanalogs of 0.25M viscosity lowering agents(222. + -. 5mg/mL, pH 7.0) viscosity at 25 ℃

TABLE 14 biological analogs with 0.25M viscosity lowering agent(222. + -. 5mg/mL, pH 7.0) viscosity at 25 ℃

The data in Table 13 show that under otherwise identical conditions, the biological analogsAll five viscosity reducing agents reduced the viscosity by at least a factor of 9.0 compared to an aqueous solution in phosphate buffer. The most effective viscosity reducing agents, CSAA and BSAA, reduce the solution viscosity by about 21-fold. Biosimids containing 0.25M CSAAThe viscosity of the aqueous solutions of (a) was compared as a function of pH at various protein concentrations. Figure 5 shows that for all protein concentrations, a viscosity minimum is observed around pH 7.0. For higher protein concentrations (253 mg/mL in the examples), the effect of pH on viscosity was most pronounced.

Biological analogs and brands as shown in Table 15Has a similar viscosity in the presence of 0.25M arginine salt BSAA.

TABLE 15 biological analogs with or without 0.25M BSAAOr brand name224 + -4 mg/mL aqueous solution at 25 deg.C and pH 7.0

Against biological analoguesThe effect of the viscosity reducing agent on the formation of irreversible protein aggregates was examined. Preparation of (i) a biological analogAnd (ii) a biological analogue containing 0.25M CSALThe aqueous liquid preparation of (1). These solutions were stored at 4 ℃ and pH 5.4 and 7.0, respectively, for 90 days. The stored samples were examined using size exclusion chromatography (column: Tosoh TSKgel UltraSWAggregate; mobile phase: 0.1M potassium phosphate/0.1M sodium sulfate, pH 6.8, 0.8 mL/min; injection: 20. mu.L of 5mg/mL monoclonal antibody solution). The data in table 16 shows that there is no significant aggregate formation in either the commercially available drug or the high concentration formulation with reduced viscosity.

TABLE 16 for containing biosimids with or without 0.25M CSALIs measured by size exclusion chromatographyPercentage of protein aggregate formation after 90 days of storage at 4 ℃ of

Example 9: pair of viscosity reducing agentsBy the action of an aqueous solution of

Materials and methods

Commercially available formulations containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer)Prepared as described in the prescription information sheet. The aqueous drug product was then purified, buffer exchanged, concentrated, dried, reconstituted and analyzed as described in example 1 above (using an extinction coefficient of 1.4L/g cm at 280 nm). Viscosity was measured using a RheoSensemVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

In Table 17 aboutThe data for aqueous solutions show that (i) viscosity reducing agents containing bulky cyclic groups provide a viscosity reduction greater than 15 fold; and (ii) CSAA, CSAAPMI, and sulfosalicylic acid diarginine (SSA dir) provide about 29-fold maximum viscosity reduction. The solution viscosity in the presence of ArgHCl alone is significantly higher than with bulky cyclic groups.

TABLE 17 viscosity reducer 0.25MAt 25 ℃ and pH 7.0

To is directed atThe aqueous solution of (a) in the presence of CSAA was examined for the dependence of the viscosity-reducing effect on the concentration of the agent. The results shown in Table 18 indicate that the viscosity lowering effect increases with increasing reagent concentration. For example, the viscosity lowering effect of the 0.35M reagent is more than two times higher (viscosity less than half) than that of the 0.20M reagent.

TABLE 18 in the Presence of CSAA at various concentrations(215. + -.5 mg/mL) viscosity at 25 ℃ and pH 7.0

For 90 days, the formulation with 0.25M CSAAThe biophysical properties of the solution of (a) were evaluated. Prepared as described in example 1 above for 0.25M CSAAThe sample of (2) was prepared. As shown in Table 19 and FIG. 6, it was determined by size exclusion chromatography (Tosoh TSKgel UltraSW Aggregate column; 0.1M potassium phosphate/0.1M sodium sulfate buffer, pH 6.8, 0.8 mL/min; injection of about 4.5mg/mL of 20. mu.L solution)Monomer content of concentrated solution in 0.25M CSAA, which is similar to the drug product at all time points and is not observed after 100 days of storage at 4 ℃To detectable aggregation. The viscosity measured using a microfluidic viscometer was shown to remain stable after 30 days of storage at 4 ℃ (table 20). In addition, the processedAntigen binding of proteinsNo decrease in binding was observed from day 0 to day 100 as measured by the specific ELISA assay (table 20). In a similar manner to that described above,the monomer content (table 21) and antigen binding (normalized to the antigen binding of the drug product) (table 22) of the concentrated solution in 0.25M CSAA were comparable to the drug product after 1 week of storage at room temperature. Finally, table 23 shows that when the samples were reconstituted, the CSAA-containing lyophilized cakes were stored at 4 ℃ for 75 days without negatively affecting the solution viscosity or the degree of protein aggregation. The results in tables 19-23 and FIG. 6 show formulations with CSAABiophysical stability before storage and after storage at 4 ℃ for at least 100 days.

TABLE 19 after formulation with 0.25M CSAA and storage at 4 ℃No increased aggregation (compared to the pharmaceutical product) was observed in the aqueous solution of (227mg/mL, pH 7)

TABLE 20 after formulation with 0.25M CSAA and storage at 4 ℃Maintaining a reduced viscosity and antigen binding over time in an aqueous solution of (227mg/mL, pH 7)

TABLE 21 after formulation with 0.25M CSAA and storage at room temperatureNo increased aggregation (compared to the pharmaceutical product) was observed in the aqueous solution of (219mg/mL, pH 7)

TABLE 22 after formulation with 0.25M CSAA and storage at room temperatureIn an aqueous solution (219mg/mL, pH 7) to maintain antigen binding

TABLE 23 storage in lyophilized powder formMaintaining low viscosity and monomer content after reconstitution after 75 days storage at 4 ℃

Example 10: pair of viscosity reducing agentsBy the action of an aqueous solution of

Materials and methods

Commercially available pharmaceutical excipients (histidine buffer, trehalose, polysorbate 80) are includedPrepared as described in the prescription information sheet. The aqueous drug product was then purified, buffer exchanged, concentrated, dried, reconstituted and analyzed as described in example 1 above (using an extinction coefficient of 1.5L/g cm at 280 nm). Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data shown in Table 24 indicate that the compounds containing PBContaining a viscosity reducing agentThe viscosity of the aqueous solution of (a) is lowest in the presence of CSAA. At higher protein concentrations (i.e.>250mg/mL), arginine HCl alone significantly reduced the viscosity and CSA further increased the effect.

TABLE 24 containing 0.25M saltAt 25 ℃ and pH 7.0

PB ═ phosphate buffer; ArgHCl ═ arginine HCl; n.d. ═ undetermined

Example 11: pair of viscosity reducing agentsBy the action of an aqueous solution of

Materials and methods

Pharmaceutical excipients (sodium phosphate buffer, sodium chloride, polysorbate) were included as described in example 1 above (extinction coefficient of 1.5L/g cm at 280nm)80) Is commercially availablePurification, buffer exchange, concentration, drying, reconstitution and analysis were performed. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data shown in Table 25 indicate that the viscosity-lowering agent is includedThe viscosity of the aqueous solution (2) was reduced by about 2.5 times (compared with a PB-containing solution) when the protein concentration was about 276 mg/mL.

TABLE 25 viscosity reducer containing 0.25MAt 25 ℃ and pH 7.0

PB ═ phosphate buffer; ArgHCl ═ arginine HCl; n.d. ═ undetermined

Example 12: viscosity reducing agent to biological analogsBy the action of an aqueous solution of

Materials and methods

Pharmaceutical excipients (citrate buffer, sodium chloride and sodium chloride) were dosed as described in example 1 above (extinction coefficient of 1.7L/g cm at 280nm)80) Of (a) commercially available biological analogsPurifying, exchanging buffer solution, and concentratingShrinking, drying, redissolving and analyzing. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data shown in table 26 indicate that the biological analogs containing the viscosity-lowering agent compared to the monoclonal antibody formulated in PBThe viscosity of the aqueous solution of (a) is reduced by more than 13 times in the case of a protein concentration of about 213mg/mL and by more than 5 times in the case of a protein concentration of about 202 mg/mL.

TABLE 26 Bioanalogs with viscosity-lowering agentsAt 25 ℃ and pH 7.0

*Is 220mg/mL

DMP ═ dimethyl piperazine

Example 13: pair of viscosity reducing agentsBy the action of an aqueous solution of

Materials and methods

Commercial vehicle containing pharmaceutical excipients as described in example 1 above (using an extinction coefficient of 1.25L/g cm at 280nm)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data shown in Table 27 indicate that the solutions containing viscosity reducing agents compared to solutions formulated with PB instead of viscosity reducing agentsThe viscosity of the aqueous solution of (a) is reduced by about 2 times at a concentration of 291mg/mL and by about 3 times at a concentration of 252 mg/mL.

TABLE 27 with 0.25M viscosity reducerAt 25 ℃ and pH 7.0

Example 14: pair of viscosity reducing agentsBy the action of an aqueous solution of

Materials and methods

Commercial vehicle containing pharmaceutical excipients as described in example 1 above (using an extinction coefficient of 1.5L/g cm at 280nm)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. Viscosity was measured using a RheoSense mVROC microfluidic viscometer equipped with an "a" or "B" chip.

Results

The data shown in Table 28 show that solutions containing viscosity reducing agents compared to solutions formulated with PB instead of viscosity reducing agentsThe viscosity of the aqueous solution of (a) is reduced by a factor of about 3 at a concentration of 274mg/mL and at a concentration of 245mg/mLA reduction of about 2 times.

TABLE 28 with 0.25M viscosity reducerAt 25 ℃ and pH 7.0

Example 15: comparison of different methods for measuring viscosity

Materials and methods

As described in example 1 above for a composition containing 220mg/mLAnd 0.25M CSAA in water. The viscosities at 25 ℃ and pH 7.0 are reported in table 29 as extrapolated zero shear viscosity from cone and plate viscometer measurements and absolute viscosity measured with a microfluidic viscometer. Cone plate measurements have been performed using a DV2T cone plate viscometer (Brookfield) equipped with a CPE40 or CPE52 spindle for 2 to 400s^-1Are measured. The extrapolated zero shear viscosity is determined from a plot of absolute viscosity versus shear rate. Microfluidic viscometer measurements were performed using a RheoSense mVROC microfluidic viscometer equipped with either an "a" or "B" chip at multiple flow rates (approximately 20%, 40%, and 60% of the maximum pressure per chip).

Results

The data in table 29 indicate that the absolute viscosity from the microfluidic viscometer can be directly compared to the extrapolated zero shear viscosity determined by the cone and plate viscometer.

TABLE 29 with 0.25M CSAAViscosity at 25 ℃ and pH 7.0 measured on two different viscometers

To compare a broader range of viscosity and protein concentration, aqueous solutions of a model antibody, bovine gamma globulin, with and without 0.25M CSAL were prepared. As described above, the viscosity was measured at a protein concentration of 110mg/mL to 310 mg/mL. The data shown in table 30 indicates that the absolute viscosity from the microfluidic viscometer can be directly compared to the extrapolated zero shear viscosity for low and high viscosity protein solutions.

TABLE 30 viscosities of aqueous gamma globulin solutions with and without 0.25M CSAL measured at 25 deg.C and pH 7.0 on two different viscometers

Example 16: the viscosity-lowering agent showed no signs of toxicity when injected subcutaneously

Materials and methods

Sprague-Dawley rats, 30 at 11 weeks of age, were divided into 6 groups of 5 rats each (3 saline control groups and 3 CSAA groups). Rats were injected subcutaneously with 0.5mL endotoxin-free phosphate buffered saline or endotoxin-free 0.25M CSAA according to the following schedule: one set of injections for each condition was given on day 1 and then sacrificed after 1 hour; one injection for each condition on day 1, one on day 2, and then sacrificed 24 hours after the second injection; and one injection for each condition on day 1, one on day 2, one on day 3, and then sacrificed 24 hours after the third injection.

Clinical observations regarding any pharmacological toxicological signs were recorded prior to dosing, immediately after dosing, at 1 and 4 hours (± 15 minutes) after dosing, and daily thereafter. Stimulation (if present) at the injection site was scored using Draize scores before dosing, immediately after dosing, 1 hour (+ -15 minutes) after dosing, and before sacrifice.

Results

Overall, the results observed with the saline and CSAA injections throughout the study were macroscopically similar. Both caused no to slight irritation at each time point with an edema score of 0-2. Microscopic examination of the injection sites indicated that CSAA caused very little clinically insignificant stimulatory effect, which was no longer evident by day 4.

Example 17: formulated with viscosity-lowering agentsExhibit low syringe extrusion force and high monomer content when discharged through various gauge needles

Materials and methods

Commercially available formulations containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer)Prepared as described in the prescription information sheet. The aqueous drug product was then purified, buffer exchanged, concentrated, dried, reconstituted and analyzed as described in example 1 above (using an extinction coefficient of 1.4L/g cm at 280 nm). A 20mM solution of phosphate buffer, CSAAPMI or CSAA was used for buffer exchange and the lyophilized cake was reconstituted to a concentration of 0.25M for each viscosity-lowering agent. After reconstitution, the viscosity of each solution was measured using a microfluidic viscometer as described in the examples above. The solution was then returned to a 1mL BD insulin syringe with a27, 29 or 31 gauge fixed needle. Then concentrated by extrusionThe force required for the solution was measured using an Instron at a displacement rate equivalent to a fluid flow rate of 3 mL/min. The solution expelled from the syringe was collected and analyzed by size exclusion chromatography.

As a result, the

All containing viscosity-lowering agentsThe solution was able to be expelled through the syringe with relatively low squeeze force (table 31). Solutions containing phosphate buffer cannot be discharged due to high viscosity. As shown in Table 31, both solutions containing viscosity reducing agents maintained high monomer content after extrusion regardless of needle gauge.

Table 31.The concentrated aqueous solution is extruded through the injectability of needles of various specifications

Example 18: viscosity reducing agents for biosimidsThe viscosity of the concentrated aqueous solution is reduced

Materials and methods

For commercial biosimids containing pharmaceutical excipients (polysorbate 20, phosphate and citrate buffers, mannitol and NaCl)And (5) purifying. First useTWEEN Medi Columns (G-Biosciences) to remove the polysorbate 20. The resulting solution is then expandedThe buffer was exchanged to 20mM sodium Phosphate Buffer (PB) for the PB sample and 2mM PB for the viscosity reducer sample and concentrated on a Jumbosep centrifugal concentrator (Pall Corp.) to a final volume of less than 10 mL. The viscosity reducing agent was then added to the 2mM PB sample as described in example 4 above. One or more viscosity reducing agents are added in an amount sufficient to give the concentrations specified below after reconstitution. In the case of the combined reagents, the concentration of each component was 0.15M. The protein solution was then freeze dried. The dried protein cake was reconstituted in phosphate buffer (for PB samples) or water (for samples containing viscosity reducing agents) to a final volume of about 0.10 mL. Final concentration of monoclonal antibodies in solution by Coomassie protein quantification by mixing samples of unknown concentration with biosimilarsIs compared or, where possible, determined by using a280 with an extinction coefficient of 1.7L/g cm. The reported viscosities were measured on a RheoSense mVROC microfluidic viscometer. The results are reported in table 32.

Results

Various GRAS, IIG and API compounds are capable of reducing the concentration of bioanalogs compared to phosphate buffered samplesThe viscosity of the solution. Of those compounds included in table 32, local anesthetics such as procaine and lidocaine and GRAS substances such as biotin are the most effective viscosity-lowering excipients.

TABLE 32 viscosity reducers vs. biosimilarsBy the action of the solution of

Average of two biological replicates

CSA ═ camphorsulfonic acid

Example 19: the viscosity-lowering effect is an agent concentration-dependent effect

Materials and methods

For commercial biosimids as described in example 4The aqueous solution of (a) is prepared. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of about 0.10mL and a final concentration of 1- (3-aminopropyl) -2-methyl-1H-imidazole dihydrochloride (APMI x 2HCl) of 0.10M or 0.25M. Final concentration of monoclonal antibody in solution by Coomassie protein quantitation assay by combining samples of unknown concentration with biological analogsIs compared to determine. The reported viscosities were measured on a RheoSense mVROC microfluidic viscometer.

Results

As shown in fig. 7, the viscosity lowering effect increased as the concentration of APMI x 2HCl increased.

Example 20: the single viscosity reducing agent reduces the viscosity of a plurality of therapeutically relevant monoclonal antibodies

Materials and methods

For commercial biosimids as described in example 4The aqueous solution of (a) is prepared. The dried protein cake was reconstituted in phosphate buffer or water to a final volume of about 0.10mL and a final concentration of thiamine HCl of 0.10M or 0.25M. Final concentration of monoclonal antibody in solution by Coomassie protein quantitation assay by combining samples of unknown concentration with biological analogsIs compared to determine the standard curve.

In the same manner for commercially available formulations containing pharmaceutically acceptable excipients (sodium phosphate buffer, NaCl, polysorbate 80)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. In the same manner for commercially available formulations containing pharmaceutically acceptable excipients (sodium phosphate buffer, NaCl, polysorbate 80)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. In the same manner for commercially available biosimids containing pharmaceutically acceptable excipients (polysorbate 80, phosphate buffer and NaCl)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. Commercially available excipients (sucrose, polysorbate 80, sodium phosphate buffer) are includedPrepared as described in the prescription information sheet. The aqueous drug product is then purified, buffer exchanged, concentrated, dried, reconstituted and analyzed in the same manner. The reported viscosities were measured on a RheoSense mVROC microfluidic viscometer.

Results

The data in table 33 show that thiamine HCl can reduce the viscosity of concentrated aqueous solutions of various therapeutically relevant monoclonal antibodies. Thiamine HCl produced a greater than 4-fold viscosity reduction for each monoclonal antibody.

TABLE 33 Effect of Thiamine HCl on solution viscosity

Examples 21 to 24: the viscosity-lowering agent reduces the viscosity of aqueous solutions of a plurality of therapeutically relevant monoclonal antibodies

Materials and methods

For commercial biosimids as described in examples 18 and 19Biological analogsAndthe aqueous solution of (a) is prepared. Tables 34-38 show that viscosity-lowering agents can be advantageously used with a variety of different monoclonal antibodies.

Results

TABLE 34 biological analogsIn the presence of a 0.15M viscosity reducing agent

Average of two biological replicates

Table 35.In the presence of 0.15M viscosity reducing agent (unless otherwise stated)

Table 36.In the presence of 0.15M viscosity reducing agent (unless otherwise stated)

Table 37.In the presence of 0.15M viscosity reducing agent (unless otherwise stated)

Table 38.In the presence of 0.15M viscosity reducing agent (unless otherwise stated)

Example 25: viscosity lowering effects of TPP and TPPAPIMI as biosimilarsAs a function of concentration of

For commercial biosimids as described in example 1 aboveThe aqueous solution of (a) is prepared. The protein was formulated to contain 0.25M phosphate buffer, 0.10M thiamine pyrophosphate (TPP), or 0.10M TPP-1- (3-aminopropyl) -2-methyl-1H-imidazole (TPPAPIM).

FIG. 8 depicts biological analogsViscosity of the aqueous solution as a function of the concentration of monoclonal antibody in the presence of phosphate buffer, TPP or tppamil. Biological analogs in phosphate bufferIncreases exponentially over the range of protein concentrations tested. In the presence of the TPP containing excipient, the increase in viscosity is reduced, i.e. the viscosity gradient is reduced.

Example 26: viscosity lowering effect of thiamine HCl as viscosity lowering agent SIMPONI as bio-analogAs a function of concentration of

Materials and methods

Commercial SIMPONI containing pharmaceutical excipients (histidine, sorbitol, polysorbate 80) as described above in example 1 (extinction coefficient of 1.4L/g-cm at 280nm)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. The protein was formulated to contain 0.15M phosphate buffer or 0.15M thiamine HCl.

Results

FIG. 9 depicts SIMPONIThe viscosity of the aqueous solution as a function of the concentration of monoclonal antibody in the presence of phosphate buffer or thiamine HCl. SIMPONI in phosphate bufferIncreases exponentially over the range of protein concentrations tested. In the presence of thiamine HCl, the increase in viscosity is reduced, i.e. the viscosity gradient is reduced.

Example 27: viscosity lowering effect of thiamine HCl asAs a function of concentration of

Materials and methods

For commercially available pharmaceutical excipients (mannitol, sucrose, tromethamine) as described in example 1 above (0.96L/g-cm extinction coefficient at 280nm)Purification, buffer exchange, concentration, drying, reconstitution and analysis were performed. The protein was formulated to contain 0.15M phosphate buffer or 0.15M thiamine HCl。

Results

Table 39 depicts the results with phosphate buffer or thiamine HClViscosity of the aqueous solution. Addition of thiamine HClIs reduced by up to about 2 times.

Table 39.In the presence of 0.15M PB or thiamine HCl

Example 28: the isotonic solution of the excipient with reduced viscosity is reducedViscosity of the concentrated solution of (2)

Materials and methods

Commercially available formulations containing pharmaceutical excipients (sucrose, polysorbate 80, sodium phosphate buffer)Prepared as described in the prescription information sheet. The aqueous drug product was then purified, buffer exchanged, concentrated, dried, reconstituted and analyzed as described above in example 1, in addition to the addition of an equal tensor of charged hydrophobic compound.

Results

As shown in Table 40, the CSAA and CSAAPMI of equal tensor can be reducedViscosity of the concentrated solution of (2)And in some cases, by as much as about 10 times.

Table 40.In the presence of an isotonic (0.3M) viscosity-reducing excipient

Unless otherwise explicitly defined above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.

Claims (27)

1. A liquid pharmaceutical formulation for injection comprising:

(i) at least 100mg/ml of an antibody;

(ii) cimetidine or a pharmaceutically acceptable salt thereof; and

(iii) an aqueous pharmaceutical solvent;

wherein the liquid drug formulation has an absolute viscosity of 1cP to 100cP at 25 ℃ when in a volume suitable for injection, measured using a cone and plate viscometer or a microfluidic viscometer; and the absolute viscosity of the liquid pharmaceutical formulation is less than the absolute viscosity of a control composition comprising the antibody and the pharmaceutically acceptable solvent but not the cimetidine or pharmaceutically acceptable salt thereof;

wherein the absolute viscosity is an extrapolated zero shear viscosity.

2. The liquid pharmaceutical formulation of claim 1, wherein the antibody has a molecular weight of 120kDa to 250 kDa.

3. The liquid pharmaceutical formulation of claim 1, wherein the antibody is a monoclonal antibody.

4. The liquid pharmaceutical formulation of claim 1, comprising from 183mg/ml to 215mg/ml of the antibody.

5. The liquid pharmaceutical formulation according to claim 1, wherein the cimetidine or pharmaceutically acceptable salt thereof is present at a concentration of 0.15M.

6. The liquid pharmaceutical formulation of claim 1, further comprising one or more pharmaceutically acceptable excipients.

7. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutically acceptable excipients comprise a diluent or carrier.

8. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutically acceptable excipients comprise a sugar, a sugar alcohol, a buffer, a natural polymer, a synthetic polymer, a surfactant, a bulking agent, or any combination thereof.

9. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutically acceptable excipients comprise a stabilizer.

10. The liquid pharmaceutical formulation of claim 9, wherein the stabilizer comprises a preservative, an antioxidant, a chelating agent, a cryoprotectant, a lyoprotectant, or any combination thereof.

11. The liquid pharmaceutical formulation of claim 6, wherein the one or more pharmaceutically acceptable excipients comprise a polysorbate, poloxamer 188, sodium lauryl sulfate, or a polyol.

12. The liquid pharmaceutical formulation of claim 11, wherein the polyol is poly (ethylene glycol), glycerol, propylene glycol, or poly (vinyl alcohol).

13. The liquid pharmaceutical formulation of claim 8, wherein the sugar alcohol is sorbitol or mannitol.

14. The liquid pharmaceutical formulation of claim 1 in the form of a single dose vial, a multi-dose vial, a cartridge, or a pre-filled syringe.

15. The liquid pharmaceutical formulation of claim 1, wherein the liquid pharmaceutical formulation is reconstituted from a lyophilized composition.

16. The liquid pharmaceutical formulation of claim 1, wherein the liquid pharmaceutical formulation is isotonic with human serum.

17. The liquid pharmaceutical formulation of claim 1, wherein the viscosity is at least 0.5s when measured using a cone and plate viscometer^-1The absolute viscosity is measured at the shear rate of (2).

18. The liquid pharmaceutical formulation of claim 1, wherein the amount of the surfactant is at least 1.0s when measured using a microfluidic viscometer^-1The absolute viscosity is measured as the shear rate.

19. Use of cimetidine or a pharmaceutically acceptable salt thereof in the manufacture of a liquid pharmaceutical formulation according to any one of claims 1-18, wherein the liquid pharmaceutical formulation is prepared for subcutaneous or intramuscular injection.

20. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared for subcutaneous or intramuscular injection with a syringe, wherein the syringe is a heated syringe, a self-mixing syringe, an autoinjector, a pre-filled syringe, or a combination thereof.

21. The use of claim 20, wherein the syringe is a heated syringe and the liquid pharmaceutical formulation is prepared to have a temperature of 25 ℃ to 40 ℃.

22. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared to produce a primary irritation index of less than 3 when evaluated using the Draize scoring system.

23. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared to have an injection force that is at least 10% less than the injection force of a control composition comprising the antibody and the pharmaceutically acceptable solvent, but not the cimetidine or pharmaceutically acceptable salt thereof.

24. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared to have an injection force that is at least 20% less than the injection force of a control composition comprising the antibody and the pharmaceutically acceptable solvent, but not the cimetidine or pharmaceutically acceptable salt thereof.

25. The use of claim 19, wherein the liquid pharmaceutical formulation is prepared for injection with a needle 27 to31 gauge in diameter and the liquid pharmaceutical formulation is prepared to have an injection force with a27 gauge needle of less than 30N.

26. A process for preparing a liquid pharmaceutical formulation according to any one of claims 1-18 comprising the step of combining the antibody, the pharmaceutically acceptable solvent and cimetidine or a pharmaceutically acceptable salt thereof.

27. A lyophilized composition comprising:

(i) an antibody;

(ii) cimetidine or a pharmaceutically acceptable salt thereof; and

(iii) a pharmaceutically acceptable excipient;

wherein the lyophilized composition is reconstituted with an aqueous pharmaceutical solvent to have an absolute viscosity of at least 100mg/ml of the antibody and 1cP to 100cP at 25 ℃ measured using a cone and plate viscometer or a microfluidic viscometer, and the absolute viscosity after reconstitution is less than the absolute viscosity of a control composition comprising the antibody and the pharmaceutical solvent but not the cimetidine or a pharmaceutically acceptable salt thereof,

wherein the absolute viscosity is an extrapolated zero shear viscosity.