HK1144178A - Antibody-containing solution pharmaceuticals - Google Patents

Description

Solution formulations comprising antibodies

This application is a divisional application of the invention patent application having application number 03805052.8.

Technical Field

The present invention relates to stable antibody-containing solution formulations.

Background

With the development of genetic engineering techniques, it has become possible to use antibodies such as immunoglobulins, monoclonal antibodies and humanized antibodies as pharmaceutical products. In order to provide an antibody drug product in a stable amount, it is necessary to determine formulation conditions and storage conditions under which the structure and activity of the antibody can be maintained.

When proteins are stored in the form of highly concentrated solutions, they often deteriorate, for example the formation of insoluble aggregates, which must be avoided. In particular, antibody formulations have a disadvantage in that they tend to form polymers, resulting in insoluble aggregates during solution storage.

For example, we have found that an anti-IL-6 receptor antibody has a therapeutic effect on immature myeloma cells (JPA HEI 8-99902), and have succeeded in mass-producing a reconstituted humanized antibody, hPM-1 antibody as an anti-IL-6 receptor antibody, and we have attempted to prepare such a purified anti-IL-6 receptor antibody into a pharmaceutical product. Humanized anti-IL-6 receptor antibodies are labile proteins susceptible to physical or chemical changes, such as cross-linking or aggregation under pressure of filtration, concentration, heat and light, during purification procedures to remove viruses and other microorganisms.

When the antibody is obtained by genetic engineering techniques, antibody-producing cells are cultured in large quantities and purified to produce a solution containing the antibody, and then the solution is cryopreserved and thawed before preparation. However, the content of the remaining antibody in the solution may be reduced due to the formation of antibody dimers or insoluble microparticles during repeated freeze/thaw cycles, or the degradation of the antibody to form degradation products during long-term storage.

Many efforts have been made to provide a method for storing proteins in a solution, in which a stabilizing effect is obtained by preventing chemical or physical changes by adding a polymer including proteins such as human serum albumin or purified gelatin, or oligomers such as polyols, amino acids and surfactants as a stabilizer. However, the addition of biopolymers such as proteins as stabilizers is inconvenient, for example, it requires a very complicated step of removing contaminants such as viruses and prions. If oligomers are to be added, they should preferably be minimized.

Lyophilized antibody formulations stabilized with a sugar or amino sugar, an amino acid and a surfactant have also been reported (JPA HEI 2001-503781).

However, due to the great demand for convenient to use solution formulations (which may not be solubilized and reduced prior to use), stable antibody-containing solution formulations have been sought.

Disclosure of the invention

It is an object of the present invention to provide an antibody-containing solution preparation in which the content of an antibody is high, which is stable even after long-term storage by suppressing the formation of insoluble microparticles and polymers during the preparation or storage of the antibody-containing solution preparation, and further suppressing the formation of degradation products.

As a result of intensive studies to achieve the above object, we have found that the formation of dimers during the freeze/thaw cycle or the formation of polymers and degradation products during long-term storage can be suppressed by the addition of a sugar, and the formation of insoluble microparticles during the freeze/thaw cycle can be significantly suppressed by the addition of a surfactant, and have completed the present invention based on this.

Accordingly, the present invention provides:

(1) a solution formulation comprising an antibody, which comprises a sugar as a stabilizer;

(2) the solution formulation as defined in (1), further comprising a surfactant as a stabilizer;

(3) the solution formulation as defined in (1) or (2), wherein the sugar is a sugar alcohol or a non-reducing oligosaccharide;

(4) the solution formulation as defined in (1) or (2), wherein the saccharide is a non-reducing oligosaccharide;

(5) the solution formulation as defined in (1) or (2), wherein the sugar is mannose, sucrose, trehalose or raffinose;

(6) the solution formulation as defined in (1) or (2), wherein the sugar is sucrose, trehalose or raffinose;

(7) the solution formulation as defined in (1) or (2), wherein the sugar is sucrose or trehalose;

(8) the solution formulation as defined in (1) or (2), wherein the sugar is sucrose;

(9) the solution formulation as defined in any one of (2) to (8), wherein the surfactant is polysorbate 80 or 20;

(10) the solution formulation as defined in any one of (1) to (9), wherein the antibody is a recombinant antibody;

(11) the solution formulation as defined in (10), wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody;

(12) the solution formulation as defined in any one of (1) to (11), wherein the antibody is an IgG class antibody;

(13) the solution formulation as defined in (12), wherein the IgG class antibody is an IgG1 class antibody;

(14) the solution formulation as defined in any one of (1) to (13), wherein the antibody is an anti-interleukin-6 receptor antibody or an anti-HM 1.24 antibody;

(15) a method of inhibiting the formation of antibody multimer molecules in a solution formulation comprising an antibody, comprising adding a sugar to the solution;

(16) a method of inhibiting the formation of antibody multimer molecules during freeze/thaw cycling of a solution comprising an antibody, comprising adding a non-reducing oligosaccharide to the solution;

(17) a method of inhibiting the formation of antibody multimer molecules during freeze/thaw cycling of a solution comprising an antibody, comprising adding a non-reducing disaccharide or a non-reducing trisaccharide to the solution;

(18) a method of inhibiting the formation of insoluble microparticles during freeze/thaw cycling of a solution comprising an antibody, comprising adding a surfactant;

(19) a method of stabilizing an antibody during freeze/thaw cycling of a solution comprising the antibody, comprising adding a non-reducing sugar and a surfactant.

Most preferred embodiments of the invention

As used herein, "antibody-containing solution preparation" refers to a solution preparation containing an antibody as an active ingredient, for administration to an animal such as a human, preferably a solution preparation which does not contain a lyophilization step in the preparation process.

As used herein, an "antibody-containing solution" may be a solution containing any antibody (whether biologically derived or recombinant), preferably a culture medium in which antibody-containing mammalian cells such as CHO cells have been cultured, or a solution obtained by subjecting such a culture medium to, for example, local purification (bulk solution), or a solution preparation as defined above for administration to an animal such as a human.

The term "insoluble microparticle" as used herein refers to an insoluble particulate matter of 10 μm or more as defined in the chapter "test of insoluble particulate matter in injection" in the section "general test, method and apparatus" in the pharmaceutical formulation of Japan. Insoluble particles can be measured using a microscope, insoluble particle collection filter, analytical membrane filter or conveniently using an automated light blocking particle counter.

As used herein, "insoluble matter" refers to an easily detectable insoluble matter, and as defined in the chapter "heterogeneous insoluble matter test of injection" in the general test, method and apparatus section of the Japanese pharmacopoeia, when the container is inspected with the naked eye under an incandescent light intensity of about 1000 lux, the injection must be clear and free of insoluble matter.

As used herein, "polymer" and "degradation product" refer to a polymer and a degradation product, respectively, of an antibody molecule constituting an active ingredient of a preparation, and their contents can be measured by a peak area percentage method (peak area percent method) based on gel permeation chromatography, which will be described later.

The antibodies used in the solution formulation of the present invention are not particularly limited as long as they bind to a specific antigen, and mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, chimeric antibodies, humanized antibodies, human antibodies, and the like are suitable antibodies. The antibody may be polyclonal or monoclonal, but is preferably monoclonal because a homogeneous antibody can be stably produced. Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art.

Hybridomas producing monoclonal antibodies can be basically constructed by the following known techniques. The specific antigen or cells expressing the specific antigen are used as the immunizing host cell for the immunizing antigen (according to standard immunization techniques), the resulting immune cells are fused with known parent cells (using standard cell fusion techniques), and the fused cells are then screened using standard screening methods to find cells producing monoclonal antibodies (hybridomas). Construction of hybridomas can be carried out, for example, by the method of Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). If the antigen has low immunogenicity, it can be conjugated to an immunogenic macromolecule such as albumin for immunization.

Recombinant ANTIBODIES can also be used, which are produced by genetic engineering techniques by transfecting a host with a suitable vector containing the antibody genes cloned from the hybridoma (see, e.g., Carl, A.K. Borrebaeck, James, W.Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, MACMILLIAN PUBLISHERS LTD, published in UK, 1990). In particular, the cDNA sequence of the antibody variable region (V region) is synthesized from the mRNA of the hybridoma using reverse transcriptase. Once the DNA sequences encoding the V regions of the antibody of interest are obtained, they are ligated to the DNA sequences encoding the constant regions (C regions) of the antibody of interest and integrated into an expression vector. Alternatively, the DNA sequence encoding the V region of the antibody may be incorporated into an expression vector comprising the DNA sequence of the C region of the antibody. They are incorporated into expression vectors by means of expression under the control of regulatory regions such as enhancers and promoters. Host cells are then transfected with this expression vector to express the antibody.

In the present invention, recombinant antibodies, that is, antibodies artificially modified to reduce antigenicity to humans or achieve other purposes, such as chimeric antibodies and humanized antibodies, can be used. These modified antibodies can be prepared by known methods. The chimeric antibody, which is composed of the heavy and light chain variable regions of an antibody derived from a non-human animal such as a mouse and the heavy and light chain constant regions of a human antibody, can be obtained by ligating a DNA sequence encoding the variable region of a mouse antibody to a DNA sequence encoding the constant region of a human antibody and transfecting a host with an expression vector containing the ligated sequence to produce the chimeric antibody.

Humanized antibodies, also called reshaped human antibodies, are obtained by grafting Complementarity Determining Regions (CDRs) of an antibody derived from a non-human animal such as a mouse onto complementarity determining regions of a human antibody, and typical genetic recombination techniques for preparing them are known. In particular, DNA sequences that attempt to link the CDRs of a mouse antibody to Framework Regions (FRs) of a human antibody can be synthesized by PCR using several prepared oligonucleotides with terminal overlapping regions. The resulting DNA sequence is ligated to a DNA sequence encoding a human antibody constant region and then integrated into an expression vector, which is transfected into a host to produce a reconstituted antibody (see European patent publication No. EP 239400, International publication No. WO 96/02576). FRs of human antibodies linked to the CDRs are selected by forming appropriate antigen binding sites using the CDRs. If desired, the reshaped humanized antibody may have some amino acid changes in the framework regions of the variable regions so that the CDRs form the appropriate antigen binding site (Sato, K. et al, Cancer Res. (1993)53, 851-.

Methods for obtaining human antibodies are also known. For example, a specific human antibody having a binding activity to a specific antigen can be obtained by immunizing human lymphocytes in vitro with the specific antigen or with cells expressing the specific antigen and fusing the immune lymphocytes with human myeloma cells such as U266 (see JPB No. HEI 1-59878). Specific human antibodies can also be obtained by immunizing transgenic animals with the entire human antibody gene bank with antigens (see international publication nos. WO 93/12227, WO92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). Methods for obtaining human antibodies by panning using human antibody gene libraries are also known. For example, phage binding to antigen can be selected by expressing the variable region of a human antibody as a single-chain antibody fragment (scFv) on the phage surface using phage surface display technology. The DNA sequence encoding the variable region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phage. The complete human antibody can be obtained by preparing an appropriate expression vector based on the determined DNA sequence of the scFv fragment that binds to the antigen. These processes are known from WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388.

When an antibody is prepared by transfecting a previously isolated antibody gene into an appropriate host, the appropriate host may be used in combination with an expression vector. Suitable eukaryotic cells for use as hosts include animal cells, plant cells, and fungal cells. Known animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (hamster kidney), HeLa and Vero cells; (2) amphibian cells such as xenopus oocytes; or (3) insect cells such as sf9, sf21 and Tn 5. Known plant cells include cells of tobacco, such as Nicotiana tabacum, which can be used for callus culture. Known fungal cells include yeasts such as Saccharomyces spp, e.g.Saccharomyces serevisiae, and filamentous fungi such as Aspergillus spp, e.g.Aspergillus niger. Prokaryotic cells can be used as production systems using bacterial cells. Known bacterial cells include e.coli and Bacillus subtilis. The antibody can be obtained by transfecting the cells with the antibody gene of interest and culturing the transfected cells in vitro.

The antibody contained in the stabilized preparation of the present invention includes, but is not limited to, an anti-IL-6 receptor antibody, an anti-HM 1.24 antigen monoclonal antibody, an anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody), and the like.

Preferred reshaped humanized antibodies to be used in the present invention include a humanized anti-IL-6 receptor antibody (hPM-1) (see International publication No. WO 92-19759), a humanized anti-HM 1.24 antigen monoclonal antibody (see International publication No. WO 98-14580), and a humanized anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody) (see International publication No. WO 98-13388).

The antibody contained in the solution formulation of the present invention may belong to any immunoglobulin class, preferably IgG such as IgG1, IgG2, IgG3 and IgG4, more preferably IgG 1.

The antibody-containing solution formulations of the present invention preferably have no increase in multimers and contain 50 or less than 50 insoluble microparticles per ml after freeze/thaw cycling.

In the antibody-containing solution or solution formulation of the present invention, dimer formation during freeze/thaw cycling can be inhibited by the addition of a sugar. Sugars which may be used include non-reducing oligosaccharides, such as non-reducing disaccharides such as sucrose and trehalose, or non-reducing trisaccharides such as raffinose, with non-reducing oligosaccharides being particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

In the antibody-containing solution or solution formulation of the present invention, the formation of polymers and degradation products during long-term storage can be inhibited by the addition of a sugar. Sugars which may be used include sugar alcohols such as mannitol and sorbitol, non-reducing oligosaccharides such as non-reducing disaccharides such as sucrose and trehalose or non-reducing trisaccharides such as raffinose, with non-reducing oligosaccharides being particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

The sugar should be added at a concentration of 0.1-500mg/mL, preferably 10-300mg/mL, more preferably 25-100 mg/mL.

In the present invention, the formation of insoluble microparticles during freeze/thaw cycles of the antibody-containing solution formulation can be significantly inhibited by the addition of a surfactant. Typical examples of the surfactant include:

nonionic surfactants, such as sorbitan fatty acid esters, such as sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerol fatty acid esters such as glycerol monocaprylate, glycerol monomyristate, glycerol monostearate; polyglycerin fatty acid esters such as decaglycerol monostearate, decaglycerol distearate, decaglycerol monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glycerin monostearate; polyoxyethylene glycol fatty acid esters such as polyoxyethylene glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives, such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides, such as polyoxyethylene stearamides having an HLB of 6 to 18.

Anionic surfactants such as alkyl sulfates having a C10-18 alkyl group such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates having an average EO mole number of 2 to 4 and a C10-18 alkyl group, such as sodium polyoxyethylene lauryl sulfate; alkyl sulfosuccinates having C8-18 alkyl groups, such as sodium lauryl sulfosuccinate; and

natural surfactants, such as lecithin; a glycerophospholipid; sphingomyelin, such as sphingomyelin; sucrose fatty acid ester of C12-18 fatty acid. The formulations of the present invention may also comprise one or more such surfactants. Preferred surfactants for use in the solution formulations of the present invention are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60 or 80, preferably polysorbate 20 and 80. Polyoxyethylene polyoxypropylene glycols, e.g. poloxamers (e.g. poloxamers)F-68) is also preferred.

The amount of surfactant added varies depending on the type of the particular surfactant used, and in the case of polysorbate 20 or polysorbate 80 is typically 0.001-100mg/mL, preferably 0.003-50mg/mL, more preferably 0.005-2 mg/mL.

Preferably, the antibody-containing solution formulation of the present invention does not substantially contain a protein as a stabilizer, such as human serum albumin or purified gelatin.

The antibody preparation of the invention preferably has a pH of 4 to 8, more preferably 5 to 7, most preferably 6 to 6.5. However, the pH depends on the antibody to be contained, and is not limited to these values.

The formulations of the present invention may also contain isotonic agents, such as polyethylene glycol; and sugars such as dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

The antibody-containing solution preparation of the present invention may further contain diluents, solubilizers, excipients, pH adjusters, tranquilizers, buffers, sulfur-containing reducing agents, antioxidants, and the like, if necessary. For example, sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and mercapto-containing compounds, such as thioalkanoic acids having 1-7 carbon atoms. Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, alpha-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium disulfite, sodium sulfite, tripentyl gallate, propyl gallate, or chelating agents such as disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate. Other usual additives such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate, and organic salts such as sodium citrate, potassium citrate and sodium acetate may also be contained.

The formulations of the present invention may be prepared by dissolving these components in aqueous buffers known in the art of solution formulation, such as phosphate buffer (preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate system) and/or citrate buffer (preferably sodium citrate buffer) and/or acetate buffer. The concentration of the buffer is typically 1-500mM, preferably 5-100mM, more preferably 10-20 mM.

The antibody-containing solution preparations of the present invention are usually administered by parenteral routes, such as injection (e.g., subcutaneous, intravenous, intramuscular or intraperitoneal injection) or transdermal, mucosal, nasal or pulmonary administration, but may also be administered orally.

The antibody-containing solution formulations of the invention are typically packaged in a fixed, sealed, sterile plastic or glass container, such as a vial, ampoule or syringe, or in a bulk container such as a bottle. The convention prefers pre-filled bottles.

The amount of antibody included in the formulation of the present invention is typically 0.1-200mg/mL, preferably 1-120mg/mL, more preferably 2-22.5mg/mL, depending on the type of disease to be treated, the severity of the disease, the age of the patient and other factors.

In the solution formulation of the present invention, the formation of insoluble particles, particularly during freeze/thaw cycles, can be significantly suppressed by the addition of a surfactant, and the formation of insoluble matter during long-term storage can also be significantly suppressed by the addition of a surfactant, as shown in the following examples. It was also found that by adding a saccharide, the formation of polymers such as dimers and the formation of degradation products can be significantly inhibited, while the content of residual antibody monomers can be increased.

The following examples further illustrate the invention but do not limit the scope of the invention. Based on the description of the present invention, those skilled in the art can make various changes and modifications, which are also included in the scope of the present invention.

Examples

Antibody sample

The hPM-1 antibody was used as a humanized anti-IL-6 receptor antibody. The hPM-1 antibody is a humanized hPM-1 antibody prepared as described in comparative example 2 of JPA HEI 8-99902 using the human elongation factor I.alpha.promoter as described in example 10 of International patent publication No. WO 92/19759.

An antibody (hereinafter referred to as anti-HM 1.24 antibody) prepared according to the method described in comparative example 2 of International patent publication No. WO98-35698 was used as the humanized anti-HM 1.24 antigen monoclonal antibody.

The hPM-1 antibody and anti-HM 1.24 antibody used in the following examples are both IgG1 class antibodies.

Test method

(A) Assays relating to hPM-1 antibodies

(1) Gel Permeation Chromatography (GPC)

Each sample was diluted with mobile phase to a hPM-1 content of approximately 1mg per 1mL, and 30-60. mu.L was tested under the following HPLC conditions.

Column: TSK gel G3000 SW_XL(TOSOH)

Pre-column: TSK pre-column SW_XL(TOSOH)

Column temperature: constant at about 25 deg.C

Mobile phase: 50mM phosphate buffer (pH 7.0) -300mM sodium chloride

Flow rate: about 1.0 mL/min

Detection wavelength: 280 nm.

The peak area was measured with an automatic integrator, the hPM-1 content was calculated from the peak area of the standard hPM-1 product, and the percentage of remaining hPM-1 was calculated from the initial calculation using the following equation:

hPM-1 content (mg/mL) — (concentration of standard hPM-1 product. times. peak area of test sample) ÷ peak area of standard hPM-1 product

Percent (%) remaining hPM-1 (hPM-1 content after thermal acceleration and freeze/thaw cycles ÷ initial hPM-1 content) × 100.

The percentage of dimers, other polymers and degradation products was calculated by area percentage method using the following equation:

dimer (or other polymer or degradation product) (%) [ peak area of dimer (or other polymer or degradation product) ÷ total peak area ] × 100

(2) Estimation of the number of insoluble particles by means of a light blocking automatic particle counter (HIAC)

The evaluation was made according to the method of an automatic light-blocking particle counter described in the chapter "test of insoluble particulate matter in injection solution" in the section "general test, method and apparatus" in the prescription of the pharmaceutical agency in Japan.

(3) Automatic visual inspection (automated visual inspection)

The automatic visual inspection was carried out according to the method described in the chapter "test of foreign insoluble substance in injection" in the section "general test, method and apparatus" in the prescription of the pharmaceutical agency in Japan.

A vision inspection system: e422 type (Eisai).

(B) Assays relating to anti-HM 1.24 antibodies

(1) Gel Permeation Chromatography (GPC); measured as N-3 to evaluate the percentage (%) remaining with respect to the initial content, and the percentage of polymer and degradation products.

Column: TSK gel G3000 SW_XL(TOSOH)

Pre-column: TSK pre-column SW_XL(TOSOH)

Column temperature: constant at about 25 deg.C

Mobile phase: 50mM phosphate buffer (pH 7.0) -300mM sodium chloride

Flow rate: about 0.5 mL/min

Detection wavelength: 280 nm.

Method for calculating concentration

anti-HM 1.24 antibody content (mg/mL) ═ amount of standard (standard concentration × anti-HM 1.24 antibody peak area × standard used) ÷ (total standard peak area × amount of test sample used)

Percent (%) of remaining anti-HM 1.24 antibody (content of anti-HM 1.24 antibody after thermal acceleration ÷ content of original anti-HM 1.24 antibody) × 100

The percentage of polymer and degradation products was calculated by area percentage method.

Polymer (or degradation product) (%) (peak area of polymer (or degradation product) ÷ total peak area) × 100

Example 1: effect of surfactant addition (1)

The effect of the surfactant (polysorbate 80) on thermal stability and freeze/thaw stability was tested. Samples containing various concentrations of polysorbate 80 as shown in table 1 were prepared and tested as follows.

(1) Stability to thermal acceleration (50 ℃ -2W) was assessed by the percentage of hPM-1 remaining and formation of polymers and degradation products as measured by Gel Permeation Chromatography (GPC). The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

(2) Stability to freeze/thaw cycles (three days at-20 ℃ followed by one day at 5 ℃ for 3 cycles) was evaluated by Gel Permeation Chromatography (GPC) for remaining hPM-1 percent and formation of polymers and degradation products. The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

The results obtained are shown in table 1.

TABLE 1

It has been found that the formation of insoluble particulates can be significantly inhibited by the addition of polysorbate 80 during the freeze/thaw cycle. There was no significant change in stability with varying concentrations of polysorbate 80.

Example 2: effect of surfactant addition (2)

The effect of the surfactant (polysorbate 80) on the stability of freeze/thaw cycles and vibration was tested. Samples containing various concentrations of polysorbate 80 as shown in table 2 were prepared and tested as follows.

The stability to freeze/thaw cycles (8 hours storage at-20 ℃ followed by 8 hours at 5 ℃ for 2 cycles) was evaluated by the number of insoluble microparticles per ml measured by an automatic light blocking particle counter (HIAC). The presence or absence of insoluble material was assessed by automated visual inspection.

The results obtained are shown in table 2.

TABLE 2

(test sample and results)

It has been found that the formation of insoluble particulates and insoluble materials can be significantly inhibited by the addition of polysorbate 80 during the freeze/thaw cycle. The effect on the formation of insoluble material depends on the concentration of polysorbate 80.

Example 3: influence of sugar addition

The effect of sugar addition on freeze/thaw cycles was tested. Samples containing various sugars (sucrose, mannose, trehalose) shown in table 3 were prepared and the stability to freeze/thaw cycles (storage at-20 ℃ for 2 hours, then 5 ℃ for 2 hours, repeated for 22 cycles) was evaluated with the amount of dimer formed as measured from Gel Permeation Chromatography (GPC).

TABLE 3

(test sample and results)

It has been found that dimer formation can be inhibited by the addition of sucrose and trehalose.

Example 4: effect of sucrose on Heat stability and Freeze/thaw stability

Sucrose was tested for its effect on thermal stability and freeze/thaw stability. Samples containing various concentrations of sucrose shown in table 4 were prepared and tested as follows.

(1) Stability to thermal acceleration (50 ℃ -2W) was assessed by the percentage of hPM-1 remaining and formation of polymers and degradation products as measured by Gel Permeation Chromatography (GPC). The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

(2) Stability to freeze/thaw cycles (three days at-20 ℃ followed by one day at 5 ℃ for 3 cycles) was evaluated by Gel Permeation Chromatography (GPC) for remaining hPM-1 percent and formation of polymers and degradation products. The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

The results obtained are shown in table 4.

TABLE 4

(test sample and results)

It has been found that dimer formation during freeze/thaw cycles can be significantly inhibited by the addition of sucrose. There was no significant change in stability with changes in sucrose concentration.

Example 5: effect of antibody concentration

The effect of hPM-1 concentration on thermostability was tested. Samples containing various concentrations of hPM-1 as shown in Table 5 were prepared and subjected to the following tests.

Stability to thermal acceleration (50 ℃ -2W) was assessed by the percentage of hPM-1 remaining and formation of polymers and degradation products as measured by Gel Permeation Chromatography (GPC). The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

The results are shown in Table 5.

TABLE 5

(test sample and results)

The hPM-1 concentration was found to be unchanged with respect to stability.

Example 6: effect of phosphate buffer concentration

The effect of the concentration of phosphate buffer on the thermal stability was tested. Samples containing various concentrations of phosphate buffer solution shown in table 6 were prepared and subjected to the following tests.

Stability to thermal acceleration (50 ℃ -2W) was assessed by the percentage of hPM-1 remaining and formation of polymers and degradation products as measured by Gel Permeation Chromatography (GPC). The number of insoluble particles per ml was measured with an automatic light blocking particle counter (HIAC).

The results are shown in Table 6.

TABLE 6

(test sample and results)

The phosphate concentration was found to be unchanged with respect to stability.

Example 7: influence of sugar addition

A thermostability test was conducted to evaluate the effect of adding sugar (sucrose or mannitol) when the concentration of anti-HM 1.24 antibody was 2.5-10 mg/mL. The remaining percentage (%) of samples containing various concentrations of sugars, polymers (%) and degradation products (%) in small-volume and large-volume anti-HM 1.24 antibody formulations (1mL/5mL ampoules) were measured under various storage conditions (60 ℃ -1W, 50 ℃ -3M, 5 ℃ -6M, start).

The low concentration formulations were tested and the results are shown in tables 7 and 8, while the high concentration formulations were tested and the results are shown in tables 9 and 10.

TABLE 7

	Sample 26	Sample 27	Sample 28	Sample 29	Sample 30	Sample 31	Sample 32
								anti-H.M 1.24 antibody (mg/mL)	2.5	2.5	2.5	2.5	2.5	2.5	2.5
Sucrose (mg/mL)	10	50	100	-	-	-	-

	Sample 26	Sample 27	Sample 28	Sample 29	Sample 30	Sample 31	Sample 32
								Mannitol (mg/mL)	-	-	-	10	50	100	-
Sodium chloride (mM)	100	100	100	100	100	100	100
								pH	6.0	6.0	6.0	6.0	6.0	6.0	6.0

TABLE 8

60℃-1W	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 26	90.9％	5.06％	1.99％
Sample 27	91.1％	4.60％	1.98％
				Sample 28	90.0％	4.14％	2.05％
Sample 29	85.5％	5.04％	2.20％
				Sample 30	90.3％	4.99％	1.99％
Sample 31	86.6％	5.57％	2.63％
				Sample 32	88.9％	5.39％	2.09％
50℃-3M	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 26	77.0％	14.0％	6.98％
Sample 27	81.5％	13.7％	6.46％
				Sample 28	84.9％	12.9％	4.83％
Sample 29	78.9％	14.3％	7.31％
				Sample 30	75.2％	13.2％	6.72％
Sample 31	76.1％	12.7％	6.24％
				Sample 32	76.8％	15.5％	7.62％

5℃-6M	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 26	103.8％	3.82％	0.00％
Sample 27	104.0％	3.44％	0.00％
				Sample 28	104.2％	3.43％	0.00％
Sample 29	103.8％	3.49％	0.00％
				Sample 30	104.3％	3.46％	0.00％
Sample 31	104.3％	3.45％	0.00％
				Sample 32	103.5％	3.49％	0.00％

Start of	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 26	100.0％	3.73％	0.00％
Sample 27	100.0％	3.34％	0.00％
				Sample 28	100.0％	3.34％	0.00％
Sample 29	100.0％	3.38％	0.00％
				Sample 30	100.0％	3.36％	0.00％
Sample 31	100.0％	3.36％	0.00％
				Sample 32	100.0％	3.38％	0.00％

After 50℃ -3M thermal acceleration, the samples showed an increase in the percentage of residual antibody monomer, and a decrease in the formation of polymers and degradation products, depending on the concentration of sucrose added. The samples also showed a decrease in the amount of polymer formed after acceleration of 60 deg.C-1W. The effect of the sugar additive on the percentage of remaining antibody under 50-3M thermal acceleration conditions was that sucrose was more pronounced than mannitol. The effect of mannitol, a sugar additive, on the inhibition of cross-linking was also found.

TABLE 9

	Sample 33	Sample 34	Sample 35	Sample 36	Sample 37	Sample 38
							anti-H.M 1.24 antibody (mg/mL)	2.5	5.0	5.0	10	10	10
Polysorbate 80 (%)	0.025	0.025	0.025	0.025	0.025	0.025

	Sample 33	Sample 34	Sample 35	Sample 36	Sample 37	Sample 38
							Acetate (mM)	20	20	20	20	20	20
NaCl(mM)	100	100	100	100	100	100
							pH	6.0	6.0	6.0	6.0	6.0	6.0
Sucrose (mg/mL)	10	10	20	10	40	0

Watch 10

60℃-1W	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 33	96.6％	4.78％	2.16％
Sample 34	96.1％	6.47％	1.84％
				Sample 35	96.1％	6.33％	1.84％
Sample 36	96.1％	6.66％	1.76％
				Sample 37	97.0％	5.96％	1.75％
Sample 38	95.3％	7.11％	1.82％

50℃-1M	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 33	94.6％	5.01％	2.12％
Sample 34	95.9％	5.62％	2.06％
				Sample 35	95.9％	5.27％	2.09％
Sample 36	96.7％	5.37％	1.97％
				Sample 37	97.1％	4.95％	1.96％
Sample 38	95.5％	5.69％	2.02％
				5℃-6M	Remaining percentage (%)	Polymer (%)	Degradation product (%)
Sample 33	107.8％	3.50％	0.00％

50℃-1M	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 34	106.1％	3.52％	0.00％
Sample 35	106.1％	3.51％	0.00％
				Sample 36	104.0％	3.59％	0.00％
Sample 37	104.1％	3.57％	0.00％
				Sample 38	103.7％	3.61％	0.00％

Start of	Remaining percentage (%)	Polymer (%)	Degradation product (%)
				Sample 33	100.0％	3.40％	0.00％
Sample 34	100.0％	3.36％	0.00％
				Sample 35	100.0％	3.36％	0.00％
Sample 36	100.0％	3.38％	0.00％
				Sample 37	100.0％	3.37％	0.00％
Sample 38	100.0％	3.39％	0.00％

Comparison of the amounts of the polymers formed after thermal acceleration shows that, when the concentration of the anti-HM 1.24 antibody is the same, the concentration of sucrose added increases and crosslinking is suppressed more significantly. Sucrose was also found to have an inhibitory effect on cross-linking of high concentrations of anti-HM 1.24 antibody preparation.

Example 8: influence of sugar additives

The effect of various levels of sucrose additives was further tested. Samples shown in Table 11 were prepared and stored at 50 ℃ to 1M, after which the percentage of residual monomer antibody and the amount of polymer were measured by GPC. The results are shown in Table 12.

TABLE 11

	Sample 39	Sample 40	Sample 41	Sample 42
					anti-H.M 1.24 antibody (mg/mL)	10	10	10	10
Polysorbate 80 (%)	0.05	0.05	0.05	0.05
					Acetate (mmol/L)	10	10	10	10

	Sample 39	Sample 40	Sample 41	Sample 42
					NaCl(mmol/L)	100	100	100	100
pH	6.0	6.0	6.0	6.0
					Sugarcane (mg/mL)	0	25	50	75

TABLE 12

Sucrose has been found to be effective in inhibiting the formation of anti-HM 1.24 antibody multimers.

Example 9: influence of sugar addition (Freeze/thaw test)

The effect of the addition of sugars (non-reducing disaccharide and non-reducing trisaccharide) on the freeze/thaw stability was tested. Sugar-containing samples shown in table 13 were prepared, and freeze/thaw tests were performed under the following conditions.

Stability to freeze/thaw cycles was evaluated by the formation of dimers (multimers) as measured by Gel Permeation Chromatography (GPC).

(test conditions)

Unfreezing: -20 ℃→ 5 ℃ (1 hour) maintaining: 5 deg.C (6 hours)

Freezing: 5 ℃→ -20 ℃ (1 hour): -20 ℃ (16 hours)

The above temperature cycle was repeated 3, 7 and 21 times.

Watch 13

(test sample and results)

These results show that the formation of hPM-1 antibody dimers during the freeze/thaw cycles can be significantly inhibited by the addition of non-reducing disaccharides (sucrose, trehalose).

Example 10: influence of sugar addition (thermal stress test)

The effect of the addition of sugars (non-reducing disaccharide and non-reducing trisaccharide) on the stability during thermal loading was tested. Sugar-containing samples shown in tables 14 and 15 were prepared, and a thermal stress test was performed under the following conditions.

Stability during heat loading was evaluated by the formation of dimers and multimers as measured by Gel Permeation Chromatography (GPC).

TABLE 14

(test sample and results)

These results show that the total amount of dimers and the formation of other multimers in the hPM-1 antibody preparation can be significantly inhibited by the addition of non-reducing disaccharides (sucrose, trehalose).

Watch 15

It has been shown that the anti-HM 1.24 antibody preparation is similar to the hPM-1 antibody preparation in that the formation of total polymer and other polymers can be significantly inhibited by the addition of non-reducing disaccharides (sucrose, trehalose).

Example 11: influence of sugar addition (light acceleration test)

The effect of the addition of sugars (non-reducing disaccharide and non-reducing trisaccharide) on the stability during photo-acceleration was tested. Sugar-containing samples shown in tables 16 and 17 were prepared, and a photo acceleration test was performed under the following conditions.

The stability during light acceleration was evaluated by the formation of dimers and multimers as measured by Gel Permeation Chromatography (GPC).

TABLE 16

(test sample and results)

It has been shown that the light-induced dimerization of hPM-1 antibodies can be significantly inhibited by the addition of sucrose.

TABLE 17

It has been shown that light-induced cross-linking of anti-HM 1.24 antibody can be significantly inhibited by the addition of sucrose.

Example 12: influence of the kind of surfactant added

The effect of surfactant type on freeze/thaw stability was tested. Surfactant-containing samples shown in table 18 were prepared and the following tests were performed.

The stability to freeze/thaw cycles (freeze at-25 deg.c/thaw at 4 deg.c for 3 cycles) was evaluated by the number of microparticles per ml measured using an automatic light blocking particle counter (HIAC).

Watch 18

(test sample and results)

It has been found that the formation of insoluble microparticles during freeze/thaw cycles can be significantly inhibited by the addition of surfactant species (polysorbate 80, polysorbate 20, poloxamer 188).

Claims (19)

1. A solution formulation comprising an antibody, wherein a sugar is included as a stabilizer.

2. The solution formulation of claim 1, further comprising a surfactant as a stabilizer.

3. The solution formulation of claim 1 or 2, wherein the sugar is a sugar alcohol or a non-reducing oligosaccharide.

4. The solution formulation of claim 1 or 2, wherein the saccharide is a non-reducing oligosaccharide.

5. The solution formulation of claim 1 or 2, wherein the sugar is mannose, sucrose, trehalose, or raffinose.

6. The solution formulation of claim 1 or 2, wherein the sugar is sucrose, trehalose or raffinose.

7. The solution formulation of claim 1 or 2, wherein the sugar is sucrose or trehalose.

8. The solution formulation of claim 1 or 2, wherein the sugar is sucrose.

9. The solution formulation of any one of claims 2 to 8, wherein the surfactant is polysorbate 80 or 20.

10. The solution formulation of any one of claims 1 to 9, wherein the antibody is a recombinant antibody.

11. The solution formulation of claim 10, wherein the antibody is a chimeric, humanized or human antibody.

12. The solution formulation of any one of claims 1 to 11, wherein the antibody is an IgG class antibody.

13. The solution formulation of claim 12, wherein the IgG class antibody is an IgG1 class antibody.

14. The solution formulation of any one of claims 1 to 13, wherein the antibody is anti-interleukin 6 receptor antibody or anti-HM 1.24 antibody.

15. A method of inhibiting the formation of antibody multimer molecules in a solution formulation comprising an antibody, comprising adding a sugar to the solution.

16. A method of inhibiting the formation of antibody multimer molecules during freeze/thaw cycling of a solution comprising an antibody, comprising adding a non-reducing oligosaccharide to the solution.

17. A method of inhibiting the formation of antibody multimer molecules during freeze/thaw cycling of a solution comprising an antibody, comprising adding a non-reducing disaccharide or a non-reducing trisaccharide to the solution.

18. A method of inhibiting the formation of insoluble microparticles during freeze/thaw cycling of a solution comprising an antibody comprising adding a surfactant.

19. A method of stabilizing an antibody during freeze/thaw cycling of a solution comprising the antibody, comprising adding a non-reducing sugar and a surfactant.