HK1145990A - Peg-interferon-beta formulations - Google Patents

Description

PEG-interferon-beta formulations

Technical Field

The present invention relates to pegylated interferon-beta formulations (PEG-IFN- β).

Background

Interferon-beta is a protein that has been identified as a useful drug and is currently used, for example, in the treatment of Multiple Sclerosis (MS).

Proteins may be modified in sequence or otherwise modified to alter or improve, for example, their activity or stability. One such modification is the introduction of a linkage to a polymer such as polyethylene glycol (PEG).

Interferon-beta linked to polyethylene glycol (PEG-IFN-beta) has been described, for example, in WO99/55377 and EP 0593868A 1.

In the pharmaceutical field, various methods such as freeze-drying have been employed to stabilize proteins, or to prepare them in the form of liquid pharmaceutical compositions. The lyophilized material must be reconstituted into a solution prior to use. Liquid pharmaceutical compositions, which are concentrated or ready-to-use, are easier and of particular interest, since they do not require any further preparation when applied to a patient.

WO95/31213 and WO2004/096263 describe liquid pharmaceutical formulations of interferon-beta.

WO2005/084303 describes coupled polymeric compositions of interferon-. beta.1b-.

US 6,531,122 relates to pegylated interferon variant formulations containing excipients, stabilizers and buffers.

WO2004/060299 provides a method of synthesizing cytokine-coupled polymers comprising interferon-beta and receptor binding antagonists thereof.

US20060051320 a1 relates to a pegylated interferon lyophilized formulation prepared with trehalose as cryoprotectant.

WO99/48535 provides formulations that prevent loss and damage of pegylated interferon-alpha conjugates during and after lyophilization.

WO03002152 relates in particular to stable compositions comprising an interferon polypeptide and a sulfoalkyl ether cyclodextrin derivative.

None of the above references disclose or suggest the liquid formulations of the present invention.

Summary of The Invention

In one aspect, the invention provides a liquid pharmaceutical composition comprising pegylated interferon-beta (PEG-IFN- β), an excipient, a surfactant, and a buffer, wherein the excipient is a polyol, the surfactant is a non-ionic surfactant, and the buffer is a sodium acetate buffer.

In another aspect of the invention, there is provided a method of preparing a liquid pharmaceutical composition, said method comprising adding a calculated amount of excipients and surfactant to a buffer, followed by the addition of PEG-IFN- β.

In another aspect of the invention, there is provided a container sealed under sterile conditions, suitable for storage ready for use, which container comprises a liquid pharmaceutical composition according to the invention.

In another aspect, the invention provides a kit of pharmaceutical compositions comprising a container containing a pharmaceutical composition of the invention.

Brief description of the tables and drawings

I. Formulations of the invention containing 0.044mg/ml PEG-interferon-beta

Table 1 describes various formulations of the present invention containing 0.044mg/ml PEG-interferon-beta.

Table 2 describes the stability test (SE-HPLC) of the formulations of the invention at 40 deg.C, 25 deg.C and 2-8 deg.C, respectively, with a pH of 4.2 throughout the test period.

Table 3 describes the stability tests (RP-HPLC) of the formulations of the invention at 40 deg.C, 25 deg.C and 2-8 deg.C, respectively, with a pH of 4.2 throughout the test period.

Table 4 describes the titre (mcg/mL) of the formulations of the invention as determined by SE-HPLC.

Table 5 depicts the titer of the formulations of the invention (mcg/mL) as determined by SE-HPLC; the% recovery before and after filtration is shown in the figure (FIG. 1:% recovery is expressed as percentage AF and TO recovery%).

Table 6 shows the pH of the formulations of the invention after storage at 25 ℃ and 2-8 ℃ for 4-26 weeks, respectively.

Table 7 describes the results of the bioassay of the formulations of the present invention over time.

Formulations of the invention containing 0.055and 0.110mg/ml PEG-interferon-beta, respectively

The PEG-interferon-beta content of these formulations has been increased to 0.055and 0.110mg/ml PEG-interferon-beta, respectively.

Table 8 describes the SE-HPLC test to check the purity of the formulations of the invention at 40 deg.C, 25 deg.C and 2-8 deg.C storage, respectively, with a pH of 4.2 throughout.

Table 9 describes the SE-HPLC assay to determine the protein content of the formulations of the invention stored at 40 deg.C, 25 deg.C and 2-8 deg.C, respectively, with a pH of 4.2 over the entire period.

Table 10 describes the RP-HPLC tests to determine the purity of the formulations of the invention when stored at 40 deg.C, 25 deg.C and 2-8 deg.C, respectively, at a pH of 4.2 throughout.

Table 11 shows the pH of the formulations of the invention after storage at 25 ℃ and 2-8 ℃ for 4-13 weeks, respectively.

Table 12 shows the bioassay data for the formulations of the present invention over time after storage at 25 ℃ and 2-8 ℃ respectively.

Table 13 describes data on peptide profiles of oxidized forms of PEG-interferon-beta formulations of the invention.

Detailed Description

The present invention relates to a liquid pharmaceutical composition comprising pegylated interferon-beta (PEG-IFN- β), an excipient, a surfactant and a buffer, wherein the excipient is a polyol, the surfactant is a non-ionic surfactant and the buffer is a sodium acetate buffer.

One aspect of the invention relates to a liquid pharmaceutical composition comprising pegylated interferon-beta (PEG-IFN- β) or a variant or active fragment thereof, an excipient, a surfactant and a buffer, wherein the excipient is a polyol, the surfactant is a non-ionic surfactant and the buffer is a sodium acetate buffer.

The following section provides definitions of the various compounds that make up the formulations of the present invention, which are intended to be synonymous throughout the specification and claims, unless the definitions expressly set forth otherwise provide a broader definition.

The pegylated interferon-beta in the formulation is any interferon-beta that has been covalently modified with polyethylene glycol or a modification equivalent thereto. An example of pegylated interferon beta is PEG-interferon beta, which pegylation may be performed using known methods, e.g. as described in WO 99/55377.

In particular, the IFN- β of the invention is covalently bound to a hydrophilic polyethylene glycol (PEG), also known as polyethylene oxide (PEO). PEG can be a linear polymer with hydroxyl groups at both ends:

HOCH₂CH₂O(CH₂CH₂O)nCH₂CH₂OH

methoxy-PEG-OH (m-PEG) may also be used, one end of which is a relatively inert methoxy group and the other end is a hydroxyl group susceptible to chemical modification:

CH₃O(CH₂CH₂O)nCH₂CH₂OH

n in the above formula may be 1 to several hundreds.

In another preferred embodiment, the PEG may also be a branched PEG represented by R (PEG-OH) m, wherein R represents a central core, such as pentaerythritol or glycerol, and m represents the number of branched arms. The number of arms (m) of the branches ranges from 3 to 100 or several hundred. The hydroxyl groups are susceptible to chemical modification.

Another branched form of the invention is described, for example, in WO96/21469, wherein PEG has one end susceptible to chemical modification. This type of PEG can be represented as (CH)₃OPEG) pRX, wherein p is equal to 2 or 3, R represents a central core, such as lysine or glycerol, and X represents a functional group, such as a carboxyl group susceptible to chemical activation.

Another branched form of the invention is referred to as "pituitary PEG" which contains reactive groups, such as carboxyl groups, along the PEG backbone rather than at the ends of the PEG chain.

PEG-interferon- β of the present invention with weak or degradable linkages in the backbone can also be prepared as described in U.S. patent application 06/026,716. Thus, PEG containing ester linkages in the polymer backbone that are susceptible to hydrolysis can be prepared. Hydrolysis can result in the polymer being cleaved to form low molecular weight fragments according to the following reaction equation:

-PEG-CO₂-PEG+H₂O→-PEG-CO₂H+HO-PEG-

copolymers of ethylene oxide and propylene oxide are closely related chemically to PEG and may be used in place of PEG in accordance with the present invention.

Various methods of PEGylating proteins have been developed. Electrophilically activated PEG derivatives are commonly used to allow PEG to bind to reactive groups of proteins. One method utilizes the alpha-or epsilon-amino group and the N-terminus of lysine residues to produce conjugates comprising a product mixture.

The conjugate preferably comprises a population of molecules of 1 to several PEG molecules conjugated to each protein molecule in an amount of 1 to the number of amino acids in the protein.

It may be desirable to introduce a site for pegylation in interferon- β, such as Woghiren et al bioconjugugatechem, 4 (5): 314-318, 1991, thiol-selective PEG derivatives were synthesized.

In one embodiment, the IFN is specifically pegylated. Specific PEGylation at the N-terminal residue may be carried out, for example, with mPEG-N-propionaldehyde (proprionaldehyde) according to the method of EP 675201. Particularly preferred are compositions such as Woghiren et al Bioconjugate chem, 4 (5): 314-318, 1991.

One embodiment of the invention is a composition comprising a covalent binding to Cys as described in WO99/55377¹⁷Mono-pegylated interferon beta of (a). The PEG moiety can be linear or branched methoxy PEG, one or more polyols and copolymers of hydrolyzable or enzymatically degradable PEG and PEG, and PLGA (polylactic/glycolic acid).

According to one embodiment of the invention, the thiol group of cysteine may be reacted with a thiol-reactive PEGylation reagent. Or PEG with functional group such as dithio-pyridine, vinyl sulfone, maleimide, and iodoacetamide. Preferred thiol-reactive PEGylation agents are dithio-pyridine (OPSS) derivatives of PEG. PEGylation agents can be used in mono-methylated form, where only one end is available for coupling, or in bifunctional form, where both ends are available for coupling.

In a preferred reaction embodiment, IFN- β -Cys is used¹⁷The reaction was carried out according to the reaction scheme described in the present application. The reaction is directed against Cys¹⁷Because the other two cysteine residues (positions 31 and 141) in IFN- β have formed disulfide bonds and are not PEGylated. The effective molecular size of PEG-interferon-beta corresponds to the molecular size of proteins with molecular weight of 50-110kDa (preferably about 70 kDa). In this particular site of interferon- β modification, the PEG molecule preferentially binds to proteins with a molecular weight greater than 20 kDa. In one embodiment, the PEG molecule is conjugated to Cys of a 2x20kDa protein via a spacer arm¹⁷。

This spacer forms IFN-PEG 2kDa-OPSS with IFN according to the following synthetic scheme.

After pegylation, the solution is purified to separate PEG-interferon-beta from free spacer and/or PEG. This purification step is preferably carried out by ultrafiltration. The ultrafiltration is carried out at a temperature below 10 ℃, preferably 5+/-3 ℃, more preferably 4 ℃. The purification step preferably employs 0.1M NaOH. The resulting PEG-interferon-beta solution contains less than 0.5EU/ml endotoxin, more preferably less than 0.25EU/ml endotoxin.

In one embodiment, the PEG is introduced into interferon- β according to the following scheme:

in one embodiment, a low molecular weight PEG is linked to interferon-beta.

The low molecular weight PEG has the following structural formula:

W-CH₂CH₂O(CH₂CH₂O)_nCH₂CH₂-X

w and X in this formula are groups that can independently react with amine, thiol, carboxyl, or hydroxyl functional groups, allowing low molecular weight PEG to bind to interferon-beta. W and X are preferably independently selected from: dithio-pyridine, maleimide, vinyl sulfone, iodoacetamide, amine, thiol, carboxyl, active ester, phenylpropyl triazole carbonate, p-nitrophenol carbonate, isocyanate and biotin.

The low molecular weight PEG is preferably PEG with a molecular weight of 100-. In a preferred embodiment, the PEG molecular weight is 1000-.

The preferred molecular weight range for the low molecular weight monofunctional or bifunctional PEG containing a free end to which interferon-beta can be bound is 100-200 Da. Preferably methoxy PEG, branched PEG, hydrolysable or enzymatically degradable PEG, pendant PEG or dendritic PEG.

The monofunctional and bifunctional PEGs further have the following structural formula:

Y-CH₂CH₂O(CH₂CH₂O)mCH₂CH₂-Z

in this formula, Y is a terminal reactive group on the free end of the low molecular weight PEG moiety capable of binding to interferon-beta; z is-OCH₃Or a reactive group capable of forming a bifunctional conjugate.

Thus, PEG-interferon- β for use in the compositions of the invention and as a medical drug can be produced in a stepwise manner using 2 or more PEG molecules.

Advantageously, the disulfide bond between PEG and IFN- β is stable in the blood circulation and can be cleaved once inside the cell.

The PEG-interferon-beta used in the formulations of the present invention has about the same or higher interferon-beta activity as natural interferon-beta.

The excipient may be any polyol that, together with the other components of the formulation, produces a stable PEG-interferon-beta formulation. An example of a polyol is mannitol (PEARLITOL)) Sorbitol (NEOSORB)) Maltitol (maltorb), xylitol (xylosorb) and maltitol (LYCASIN). The preferred polyol excipient is mannitol.

Nonionic surfactants are employed in accordance with the present invention. Nonionic surfactants include polyol derivatives, polyoxyethylene esters and ethers, poloxamers (poloxamer 188), nonylphenyl ether, polyvinyl alcohol, propylene glycol diacetate, alkanolamides. The nonionic surfactant may preferably be poloxamer, and poloxamer 188 is particularly preferred.

Any buffer that can maintain the pH at the selected level can be used. The buffer may be sodium acetate buffer or PBS. Sodium acetate buffer is particularly preferred.

In one embodiment, the invention is a liquid pharmaceutical composition comprising PEG-interferon-beta, an excipient, a surfactant, and a buffer, wherein the excipient is mannitol, the surfactant is poloxamer 188, and the buffer is a sodium acetate buffer.

Interferon- β may be native human interferon- β or recombinantly produced interferon- β subjected to pegylation of the present invention. Also, interferon- β (IFN- β) of the present invention refers to a glycoprotein produced in the body in response to viral infection.

Interferon units or international units of interferon (U or IU) are reported as a measure of interferon activity, defined as the amount necessary to protect 50% of cells from viral damage. A reported test for measuring biological activity is the cytopathic effect inhibition test (Rubinstein et al, 1981; Famililetti, P.C. et al, 1981). In this antiviral assay, approximately 1 unit/ml of interferon is the amount of interferon required to produce 50% of the cytopathic effect. The unit amount can be measured by using human-interferon-beta international reference standard (Pestka, S.1986) provided by national institutes of health.

Interferon-beta is also known as a Biological Response Modifier (BRM) because it plays a role in the recognition of an organism's response to tumors, infection, through immunomodulation.

Human fibroblast interferon (IFN-beta) has antiviral activity, and can stimulate natural killer cells to resist tumor cells. It is a polypeptide of about 20,000Da that is induced by viruses and double-stranded RNA. The nucleotide sequence of the fibroblast interferon gene has been cloned by recombinant DNA techniques (Derynk et al, 1980) and the complete amino acid sequence of the protein has been deduced. It is 166 amino acids in length.

Interferon Rebif for treating Multiple Sclerosis (MS)(Merck Serono-recombinant human interferon-. beta.) is Interferon (IFN) -1a produced from a mammalian cell line. Its recommended international non-patent drug name (INN) is "interferon-beta-1 a".

As used herein, "interferon- β" or "IFN- β" is meant to include any molecule so defined in the literature, including, for example, any type of IFN- β mentioned in the above section. IFN- β s suitable for use in the invention are commercially available, e.g., Rebif(Merck Serono)、Avonex(Biogen Idec) as long as it contains a dedicated attachment site for PEGylation. The present invention also preferably employs human-derived interferon. The term interferon-beta as used in this specification includes salts, functional derivatives, variants, muteins, congeners and active fragments thereof.

The term "interferon-beta (IFN- β)" as used herein, is intended to include fibroblast interferons, particularly interferons of human origin, such as those isolated from biological fluids and obtainable from eukaryotic host cells using recombinant DNA techniques, and salts, functional derivatives, variants, analogs and active fragments thereof. Preferred interferon-beta means interferon-beta-1 a.

The term "mutein" as used herein refers to analogs of IFN- β and PEG-IFN- β having one or more amino acid residues of the native IFN- β substituted or deleted by different amino acid residues or having one or more amino acid residues added to the native sequence of IFN- β, but which produce a product that has no significant change in activity compared to the wild-type IFN- β. These muteins can be prepared by known synthetic techniques or site-directed mutagenesis techniques, or any other suitable technique known. Preferred muteins include, for example, those described by Shepar et al (1981) or Mark et al (1984). In particular, it is possible to describe,the mutant protein molecule of the invention contains Cys¹⁷Including any of the variations described above.

Any such mutein preferably has an amino acid sequence that substantially replicates the sequence of IFN- β, e.g., has substantially similar or even better activity than IFN- β. Those skilled in the art are familiar with the biological function of interferons, whose biological standards have been established from the national institute of health and control center (http://immunology.org/links/NIBSC) And (4) obtaining the product.

Biological assays for determining IFN- β/PEG-IFN- β activity have been reported. The interferon assay can be performed, for example, as described by Rubinstein, 1981 et al. Thus, routine experimentation can be used to determine whether any given mutein has substantially similar or even better activity than IFN- β.

IFN- β muteins and PEG-IFN- β or nucleic acids encoding them useful in the present invention comprise a limited set of substantially corresponding sequences, such as substituted peptides or polynucleotides, which are routinely obtained by one of ordinary skill in the art without undue experimentation, based on the instructions and guidance provided herein.

Preferred variations of the muteins of the present invention are substitutions referred to as "conservative". Conservative amino acid substitutions for polypeptides or proteins of the invention may include amino acid substitutions within the synonymous groups that have substantially similar physicochemical properties, with substitutions between members of the group retaining the biological function of the molecule. It is clear that amino acid insertions or deletions can be made in the above-mentioned sequences without altering their function, in particular if these insertions or deletions involve only a few amino acids, such as less than 30, preferably less than 10, and do not move or replace amino acids which are critical for the functional conformation, such as cysteine residues. Proteins and muteins produced by such deletions and/or insertions fall within the scope of the present invention.

Amino acid substitutions may be made in the protein which may be used to obtain the PEG-IFN- β muteins used in the present invention, examples of which include any known method steps, see for example, U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462 to Mark et al, 5,116,943 to Koths et al, 4,965,195 to Namen et al, 4,879,111 to Chong et al and 5,017,691 to Lee et al. Lysine substituted proteins are described in U.S. Pat. No. 4,904,584(Shaw et al). The characteristic muteins of IFN- β have been described, for example, in Mark et al, 1984.

"functional derivatives" as used herein include PEG-IFN- β derivatives and muteins and fusion proteins thereof, which can be prepared from functional groups or N-or C-terminal groups of the side chains of amino acid residues by methods known in the art. These proteins are included in the present invention as long as they remain pharmaceutically acceptable, i.e., the activity of the protein is not disrupted substantially similar to that of IFN- β, and does not cause toxicity to the composition in which it is included. These derivatives may include, for example, aliphatic esters with carboxyl groups, amides resulting from the reaction of carboxyl groups with ammonia or primary or secondary amines, N-acyl derivatives of the free amino group of the amino acid residue with acyl groups, such as alkanoyl or carbocyclic aroyl groups, or O-acyl derivatives of the free hydroxyl group, for example of seryl or threonyl residues, with acyl groups.

As "active portions" of PEG-IFN- β, or muteins and fusion proteins, the present invention includes any fragment or precursor of the polypeptide chain of the protein molecule, whether alone or in combination with a molecule or residue attached thereto (e.g., a sugar residue or a phosphate residue), or aggregates of the protein molecule or sugar residue itself, so long as the activity of the portion is not significantly less than that of the corresponding IFN.

The term "salt" is used herein to refer to a carboxylic acid salt or an amino acid addition salt of the above-mentioned protein or its congener. Salts of the carboxylic groups may be formed by methods known in the art, including salts of inorganic acids, such as sodium, calcium, ammonium, iron, or zinc salts, and the like; those salts formed with organic bases, for example amines such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with inorganic acids such as hydrochloric acid or sulfuric acid; salts with organic acids, such as acetic acid or oxalic acid. Of course, any of these salts must retain the biological activity of the protein (IFN) of interest of the invention, i.e.the ability to bind to the corresponding receptor and initiate receptor signaling.

In yet another embodiment, the fusion protein comprised by the composition of the invention comprises an Ig fusion. Such fusions may be direct or via a short linker peptide, which may be 1-3 amino acid residues in length or longer, e.g., 13 amino acid residues in length. The linker may be, for example, a tripeptide sequence E-F-M (Glu-Phe-Met) introduced between the IFN sequence and the immunoglobulin sequence, or a linker sequence of 13 amino acids Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met. The resulting fusion protein may have improved properties such as increased residence time in body fluids (half-life), increased specific activity, increased expression levels or facilitated purification of the fusion protein.

The concentration of PEG-IFN- β in the composition is suitably from about 0.1mg/ml to about 0.01mg/m, preferably from about 0.06mg/ml to about 0.03mg/ml, and even more preferably about 0.044 mg/ml.

In another embodiment, the PEG-IFN- β concentration in the formulation is 0.05-0.150mg/ml, preferably 0.055 or 0.110 mg/ml.

The dose used in the composition or the dose which can be applied to an individual varies depending on various factors, including pharmacokinetic properties, administration route, condition and characteristics of a patient (sex, age, body weight, health condition and size of head), degree of symptoms, concurrent treatments, frequency of treatment and effect to be achieved.

The standard dose range for human IFN- β/PEG-IFN- β is 80000IU/kg-200000IU/kg per day or 6MIU (million International units) -12MIU per person per day, or equivalent to 22-44 μ g (micrograms) of IFN- β per person. In accordance with the present invention, the amount of PEG-IFN- β in the compositions of the invention is preferably from about 1 to about 50 μ g, more preferably from about 10 to about 30 μ g, or from about 10 to about 20 μ g per person per day, of IFN- β.

The active ingredients of the invention can be administered by intravenous, intramuscular, subcutaneous routes. Preferred routes of administration for the compositions of the invention are subcutaneous and intramuscular routes.

The PEG-IFN- β compositions of the invention may also be administered daily, every other day, or infrequently. Preferably, the PEG-IFN- β composition is administered once, twice, or three times a week. Preferably once every two weeks.

A preferred route of administration is subcutaneous, e.g. three times a week. Another preferred route of administration is intramuscular, e.g. once a week.

Preferably, the PEG-IFN- β compositions of the invention are administered three times a week subcutaneously, corresponding to 22-44 μ g or 6MIU-12MIU of IFN- β.

The PEG-IFN- β composition is administered subcutaneously at a dose of 25-30 μ g or 8MIU-9.6MIU every other day. A PEG-IFN- β composition corresponding to 30 μ g or 6MIU IFN- β can also be administered intramuscularly once a week.

The term "stability" refers to the physical, chemical and conformational stability (including maintenance of biological effects) of the interferon formulations of the present invention. Destabilization of protein preparations is caused by chemical degradation or aggregation of protein molecules to form higher order polymers, deglycosylation, glycosylation modification, oxidation, or any other structural modification that results in a reduction in at least one biological activity of the interferon polypeptide of the present invention.

A "stable" composition, solution or formulation is one in which degradation, modification, degree of aggregation, loss of biological activity, etc., of the protein is acceptably controlled and does not increase to unacceptable levels over time. Preferably, at least or about 60%, more preferably at least or about 70%, and most preferably at least or about 80% of the activity of the labeled interferon is retained for 12-24 months. Preferred PEG-IFN- β compositions of the invention preferably have a half-life of at least about 6 months, 12 months, 18 months, more preferably at least 20 months, even more preferably at least about 22, and most preferably at least about 24 months when stored at 2-8 ℃.

Methods of monitoring the stability of a liquid PEG-IFN- β pharmaceutical composition of the present invention are available in the art, including those described herein. Thus, by measuring the change in soluble PEG-IFN- β in solution over time, the formation of PEG-IFN- β aggregates during storage of the liquid pharmaceutical compositions of the invention can be readily determined. The amount of soluble polypeptide in solution can be determined using various analytical assays suitable for detecting particulate PEG-IFN- β. Such tests include, for example, Reverse Phase (RP) -HPLC and UV absorption spectrophotometry as described in the examples below

Detection of soluble and insoluble aggregates in a liquid composition during storage can be accomplished, for example, by analytical ultracentrifugation as set forth in the examples below, to distinguish between the portion of soluble polypeptide present as soluble aggregates and the portion present as non-aggregated bioactive molecules.

The term "buffer" or "physiologically acceptable buffer" refers to a solution of a compound contained in a formulation that is safe for medical or veterinary use and effective to maintain or control the pH of the formulation within the desired pH range of the formulation. Acceptable buffers to control the pH at mildly acidic pH include, but are not limited to, buffers such as phosphate, acetate, citrate, arginine, TRIS, and histidine. "TRIS" refers to 2-amino-2-hydroxymethyl-1, 3-propanediol and pharmaceutically acceptable salts thereof. Preferred buffers are acetate buffers containing saline or acceptable salts.

The excipient is preferably present at a concentration of about 30mg/ml to 50mg/ml, more preferably at a concentration of about 40mg/ml to 50mg/ml, and even more preferably at a concentration of about 45 mg/ml.

The surfactant is suitably present in a concentration of from about 0.1mg/ml to about 1mg/ml, preferably in a concentration of from about 0.4mg/ml to about 0.7mg/ml, and even more preferably in a concentration of about 0.5 mg/ml.

The term "surfactant" refers to a soluble compound that reduces the surface tension of a liquid or reduces the interfacial tension between two liquids or a liquid and a solid, the surface tension being the force acting on the surface of a liquid that tends to minimize the surface area. Surfactants are sometimes used in pharmaceutical formulations, including the delivery of low molecular weight drugs anda polypeptide to improve absorption or delivery of a drug to a target tissue. Well known surfactants include polysorbates (polyoxyethylene derivatives of sorbitol fatty esters, Tween)) And poloxamers, such as Pluronic, BASF, GermanyOr Lutrol

According to a preferred embodiment of the invention, it has been found that the surfactant employed in formulating the PEG-IFN- β is selected from the group consisting of PluronicF77、PluronicF8、PluronicF88 and PluronicF68, Pluronic being particularly preferredF68(BASF，PluronicF68 also known as poloxamer 188), the resulting stable formulation minimizes loss of active ingredient due to adsorption to the surface of the vial and/or delivery device (e.g., needle, pump, cannula, etc.). It has also been found that the use of a compound selected from PluronicF77、PluronicF87、PluronicF88 and PluronicF68, Pluronic being particularly preferredF68(BASF，PluronicF68 also known as poloxamer 188) is used to formulate PEG-IFN- β, the resulting stable composition is more resistant to oxidation and formation of protein aggregates.

PluronicThe surfactant is a block copolymer of Ethylene Oxide (EO) and Propylene Oxide (PO). The propylene oxide block (PO) is sandwiched between two Ethylene Oxide (EO) blocks.

In PluronicIn F77, the polyoxyethylene (hydrophilic) percentage is 70%, and the hydrophobic (polyoxypropylene) molecular weight is about 2,306 Da.

In PluronicIn F87, the polyoxyethylene (hydrophilic) percentage is 70%, and the hydrophobic (polyoxypropylene) molecular weight is about 2,644 Da.

In PluronicIn F88, the polyoxyethylene (hydrophilic) percentage is 80% and the hydrophobic (polyoxypropylene) molecular weight is about 2,644 Da.

In PluronicIn F68, the polyoxyethylene (hydrophilic) percentage is 80% and the hydrophobic (polyoxypropylene) molecular weight is about 1,967 Da.

Other polymers having properties similar to the Pluronic (Pluronic) series may also be employed in the compositions of the present invention. A preferred surfactant is PluronicF68 and surfactants having similar properties.

Pluronic, in particular PluronicF68, preferably present at a concentration sufficient to maintain PEG-IFN- β stability throughout storage (e.g., 12-24 months), and also at a concentration sufficient to prevent loss of protein by adsorption to a surface (e.g., vial, ampoule or cannula, syringe surface).

Pluronic in liquid formulationIn particular PluronicPreferred concentrations of F68 are or about 0.01mg/ml to 10mg/ml, more preferably or about 0.05mg/ml to 5mg/ml, and still more particularlyPreferably at or about 0.1mg/ml to 2mg/ml, most preferably at or about 1 mg/ml.

In another preferred embodiment, methionine, in particular L-methionine, is used. Specifically, a concentration of 0.1mg/ml to 0.5mg/ml is used, with a preferred concentration of about 0.1mg/ml to 0.3mg/ml, more preferably about 0.25mg/ml or about 0.12 mg/ml.

The addition of methionine was found to facilitate further stabilization of the formulation and reduce protein oxidation.

The various compounds contained or contained in the formulations of the present invention may be varied as described above, yet still achieve the positive effects of methionine. In one embodiment, the formulation comprises or contains 0.055mg/ml PEG-IFN- β, 10mM sodium acetate buffer (pH 3.5-4.5, preferably pH4.2), 45mg/ml mannitol and 0.5mg/ml poloxamer 188, or may comprise 0.110mg/ml PEG-IFN- β.

The compositions of the present invention contain a sufficient amount of buffer to maintain the pH of the composition at ± 0.5 units of a particular pH. This particular pH is about 3.0-5.0, preferably said pH is about 3.5-4.5, even more preferably said pH is about 4.2 ± 0.2.

The buffer concentration in the compositions of the invention is from about 5mM to 500mM, preferably at a concentration of about 10 mM.

The composition is preferably an aqueous solution.

The present invention includes liquid compositions. The preferred solvent is water for injection.

Adjusting the pH of the PEG-IFN- β composition to about pH3-5, preferably to about 3.5-4.5, more preferably to about 4.2+/-0.2, can be shown to provide a stable formulation.

The composition of the invention achieves a positive effect on the PEG-IFN- β degradation process which is negatively effected in a liquid composition. This effect can be detected by SE-HPLC at, for example, 40 ℃ and 25 ℃ respectively. No significant reduction in the PEG-IFN- β content (SE-HPLC) was observed for the compositions of the invention, particularly no reduction in the PEG-IFN- β content for two weeks at 40 ℃ and six weeks at 25 ℃. The compositions of the invention show good stability characteristics, in particular a reduced shelf-life protein aggregation is achieved.

A positive effect was particularly achieved with a composition comprising 0.044mg/ml PEG-IFN- β, 10mM sodium acetate buffer at pH about 4.2, 45mg/ml mannitol and 0.5mg/ml poloxamer 188 prepared in a glass vial (preferably 3ml) or glass needle tubing (preferably 1 ml).

The purity is measured by using SE-HPLC and RP-HPLC, and the protein content and the biological activity are measured by using SE-HPLC, so that the composition has good stability.

In another aspect, the invention relates to a method of preparing the above liquid pharmaceutical composition, wherein the method comprises adding a calculated amount of excipient and surfactant to the buffer, followed by the addition of PEG-IFN- β.

In yet another aspect, the present invention relates to a container suitable for storage ready for use, sealed under sterile conditions, comprising a liquid pharmaceutical composition of the present invention.

Any container suitable for medical use may be used. The container is preferably a pre-filled syringe, vial or cartridge for an automatic injector.

The formulations of the present invention may be administered using approved devices. Examples of systems containing these single vials include auto-injectors or injection pen devices that deliver solutions, such as Rebiject

The product claimed in this application comprises a packaging material. The packaging material provides the conditions under which the product is used in addition to the information required to be provided by the regulatory agency. The packaging material of the present invention provides instructions to the patient for use and, if desired, instructions for preparing two vials of the final solution of the dry/wet product and using the final solution within 24 hours or more. For a single vial of solution product, the label indicates that the solution can be used for 24 hours or more. The product claimed in this application is a pharmaceutical product for human use. The composition provided to the patient is a clear solution.

In accordance with the present invention, PEG-IFN- β can be administered to a patient by a variety of delivery methods, including SC or IM injection, transdermal, pulmonary, transmucosal, implantation, osmotic pump, syringe cartridge, micropump, oral, or other means well known in the art and recognized by a skilled artisan.

In another aspect, the invention relates to a kit of pharmaceutical compositions comprising a container filled with a pharmaceutical composition of the invention.

The container preferably acts as a needle for the delivery device.

All references cited in this specification, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, approved U.S. or foreign patents, or other references, are incorporated herein by reference in their entirety, including all data, tables, figures, and text presented in the cited references. In addition, the references cited in this specification are also incorporated by reference in their entirety.

The invention is illustrated below by means of examples, which are not to be construed as limiting the scope of the invention.

Examples

The following examples will describe preferred embodiments of the present invention.

Preparation of PEG-IFN- β conjugates

The following scheme illustrates an example of the preparation of PEG-IFN- β:

PEG-IFN synthesis and purification scheme：

PEG 2k OPSS ═ (2-pyridyldithiol)₂-PEG-2K

PEG(20K)₂HS-cystamide-lysine- (PEG-20K)₂

PEG-IFN synthesis and purification scheme:

2. preparation

PEG-IFN- β compositions are formulated with PEG-IFN- β 1 a. The solution was filtered through a 0.22um membrane (Durapore) and filtered into the final container (1ml vial or syringe).

The test composition contained 0.044mg/ml PEG-IFN-. beta.1 a, 10mM sodium acetate buffer (pH4.2), 45mg/ml mannitol and 0.5mg/ml poloxamer 188. Alternatively, 0.055 or 0.110mg/ml PEG-IFN- β 1a and methionine may be added.

Samples of the test compositions were stored at 40 deg.C, 25 deg.C and 2-8 deg.C and tested, purity was measured by SE-HPLC or RP-HPLC, protein content was measured by SE-HPLC, biological activity was measured by an antiviral assay based on IFN- β induced cytoprotection, and pH was measured as a function of time, and oxidized forms of the peptides were measured by peptide mapping/UPLC analysis.

3. Stability testing and other experiments

3.1. Purity and SE-HPLC determination

Purity was assessed by SE-HPLC on Shodex columns (aquous SE Corp., No. KW-803); PBS 10x (Gibco BRL, number 70013-; detection was performed with ultraviolet light UV at 214 nm.

Samples of IFN-PEG formulation were injected using the following injection volumes: 200mcL (for 44 and 55mcg/mL samples); 100mcL (for 110mcg/mL samples).

The assay uses a control reference standard PS200-01 (single point assay) for quantitative protein determination.

RP-HPLC determination of purity

Purity measurements were carried out by RP-HPLC at a constant temperature of 35 ℃ on a C4 column (Symmetry 300C4, 5m size4.6X250mm, Waters). The wavelength was set at 214nm and elution was carried out at 1 mL/split flow rate using the following conditions:

mobile phase:a ═ 0.1% TFA/water; b-0.1% TFA/acetonitrile

Gradient:30% → 70% B, 40 min

Time (minutes)	Flow rate (mL/min)	％A	％B
				0	1.0	70	30
3	1.0	70	30
				40	1.0	30	70
45	1.0	70	30

Samples of IFN-PEG formulation were injected using the following injection volumes: 160mcL (for 44mcg/mL samples); 200mcL (for 55mcg/mL samples); 100mcL (for 110mcg/mL samples).

3.3. Biological Activity (in vitro assay)

The biological activity was determined using the current method for detecting IFN- β, an antiviral assay based on the cytopathic effect of IFN- β inducible cells (WISH cells, human amniotic cells) to protect against the virus (vesicular stomatitis virus).

pH determination

The pH measurements were carried out using a calibrated pH meter (Mettler-Toledo, model 713) according to standard protocols.

3.5. Analysis of oxidized forms with peptide mapping/UPLC

The method for quantitative determination of oxidized methionine residues (Met 1, Met 117, Met 36) allows the prediction of proteolytic digestion of IFN-PEG samples, digestion with endoproteinase Lys-C followed by gradient UPLC analysis; pretreatment of the formulation samples prior to proteolysis removes interference from matrix components.

Separating the proteolytic mixture on an Acquity UPLC analytical column (BEH C181, 7 μm 2.1x50mm cod.1860002350, Waters corporation); elution was performed with a gradient of 0.1% TFA/water (A1) and 0.1% TFA/acetonitrile (B1).

The following instrument settings and analysis parameters can be used:

UV detector wavelength	214nm
		Temperature of automatic sampler	+5℃±3℃
Column temperature	+40℃±5℃
		Flow rate of column	0.6 mL/min
Analysis time	23 minutes
		Delay time for next injection	10 minutes

TABLE 1PEG-IFN- β formulation composition (mg/mL)

	PEG-IFN	Buffer solution	Mannitol	L-methionine	Poloxamer 188	Container with a lid
							PEG-IFN Man/F68-Small bottle	0.044	10mM sodium acetate pH4.2 (*)	45.0	-	0.5	DIN2R vial
PEG-IFN Man/Met-Vial	0.044	10mM sodium acetate pH4.2 (*)	45.0	0.12	-	DIN2R vial
							PEG-IFNMan/F68/Met-Small bottle	0.044	10mM sodium acetate pH4.2 (*)	45.0	0.12	0.5	DIN2R vial
PEG-IFNMan/F68/Met needle tube	0.044	10mM sodium acetate pH4.2 (*)	45.0	0.12	0.5	1mL needle tube

(^*) PBS buffer also remained in the bulk composition (due to the 5-fold dilution of PEG-IFN drug).

TABLE 2 purity by SE-HPLC%

TABLE 3 purity by RP-HPLC%

	At 0 time	1 week +40 deg.C	2 weeks +40 deg.C	4 weeks +40 deg.C
								PEG-IFN Man/F68-Small bottle	96.0	94.1	91.7	87.4
PEG-IFN Man/Met-Vial	96.3	93.9	90.8	88.5
								PEG-IFN Man/F68/Met-Vial	95.9	93.7	92.3	90.0
PEG-IFN Man/F68/Met syringe	96.4	93.4	92.5	88.8
	At 0 time	2 weeks +25 deg.C	4 weeks +25 deg.C	6 weeks +25 deg.C	8 weeks +25 deg.C	12 weeks +25 deg.C	26 weeks +25 deg.C
								PEG-IFN Man/F68-Small bottle	96.0	95.0	94.5	93.3	93.5	93.9	90.6
PEG-IFN Man/Met-Vial	96.3	94.3	95.0	93.4	93.3	94.1	90.9
								PEG-IFN Man/F68/Met-Vial	95.9	94.5	94.4	93.4	93.1	94.3	90.8
PEG-IFN Man/F68/Met syringe	96.4	94.3	94.3	93.3	93.4	93.1	90.6

	At 0 time	1 week +40 deg.C	2 weeks +40 deg.C	4 weeks +40 deg.C
									At 0 time	4 weeks +2-8 deg.C	6 weeks +2-8 deg.C	8 weeks +2-8 deg.C	12 weeks +2-8 deg.C	26 weeks +2-8 deg.C
PEG-IFN Man/F68-Small bottle	96.0	94.5	94.9	94.4	95.7	96.0
								PEG-IFN Man/Met-Vial	96.3	95.2	93.9	94.0	95.1	95.8
PEG-IFN Man/F68/Met-Vial	95.9	94.9	94.5	93.9	95.3	95.7
								PEG-IFN Man/F68/Met syringe	96.4	94.7	94.1	94.6	94.7	96.1

TABLE 4 titer determined by SE-HPLC (mcg/mL)

	At 0 time	1 week +40 deg.C	2 weeks +40 deg.C	4 weeks +40 deg.C
								PEG-IFN Man/F68-Small bottle	41.2	44.4	44.3	43.7
PEG-IFN Man/Met-Vial	40.5	43.1	43.3	41.5
								PEG-IFN Man/F68/Met-Vial	43.0	46.3	45.3	44.9
PEG-IFN Man/F68/Met syringe	43.4	46.7	46.0	45.7
	At 0 time	2 weeks +25 deg.C	4 weeks +25 deg.C	6 weeks +25 deg.C	8 weeks +25 deg.C	12 weeks +25 deg.C	26 weeks +25 deg.C
								PEG-IFN Man/F68-Small bottle	41.2	43.6	42.7	41.9	44.5	41.8	41.4
PEG-IFN Man/Met-Vial	40.5	42.0	41.2	40.4	42.7	40.6	38.4
								PEG-IFN Man/F68/Met-Vial	43.0	45.1	44.2	43.3	45.7	43.1	42.5
PEG-IFN Man/F68/Met syringe	43.4	45.6	44.6	43.7	46.2	43.8	43.4

	At 0 time	1 week +40 deg.C	2 weeks +40 deg.C	4 weeks +40 deg.C
									At 0 time	4 weeks +2-8 deg.C	6 weeks +2-8 deg.C	8 weeks +2-8 deg.C	12 weeks +2-8 deg.C	26 weeks +2-8 deg.C
PEG-IFN Man/F68-Small bottle	41.2	42.7	42.0	44.4	42.2	41.9
								PEG-IFN Man/Met-Vial	40.5	41.7	40.4	43.3	41.1	39.9
PEG-IFN Man/F68/Met-Vial	43.0	44.4	43.3	45.7	43.6	43.3
								PEG-IFN Man/F68/Met syringe	43.4	44.7	44.1	46.4	44.3	44.2

TABLE 5 titer determined by SE-HPLC (mcg/mL)

	Before filtration (mcg/mL)	After filtration (mcg/mL)	T0(mcg/mL)	AF recovery (%)	T0 recovery (%)
						PEG-IFN Man/F68-Small bottle	43.4	41.7	41.2	96.1	94.9
PEG-IFN Man/Met-Vial	43.4	42.7	40.2	98.3	92.5
						PEG-IFN Man/F68/Met-Vial	43.5	42.0	43.0	96.7	98.9
PEG-IFN Man/F68/Met syringe	43.5	42.0	43.4	96.7	99.9

TABLE 6 determination of pH

	At 0 time	4 weeks +25 deg.C	8 weeks +25 deg.C	12 weeks +25 deg.C	26 weeks +25 deg.C
						PEG-IFN Man/F68-Small bottle	4.22	4.20	4.16	4.19	4.22
PEG-IFN Man/Met-Vial	4.23	4.20	4.17	4.21	4.20
						PEG-IFN Man/F68/Met-Vial	4.22	4.21	4.19	4.23	4.22
PEG-IFN Man/F68/Met syringe	4.22	4.20	4.19	4.22	4.21
							At 0 time	4 weeks +2-8 deg.C	8 weeks +2-8 deg.C	12 weeks +2-8 deg.C	26 weeks +2-8 deg.C
PEG-IFN Man/F68-Small bottle	4.22	4.22	4.17	4.21	4.17
						PEG-IFN Man/Met-Vial	4.23	4.23	4.17	4.19	4.18
PEG-IFN Man/F68/Met-Vial	4.22	4.25	4.19	4.23	4.20
						PEG-IFN Man/F68/Met syringe	4.22	4.26	4.18	4.24	4.22

TABLE 7 biological assays (MIU/mL)

TABLE 8 purity (%) -by SE-HPLC

TABLE 9 protein content by SE-HPLC (mcg/mL)

TABLE 10 purity by RP-HPLC (%)

TABLE 11pH values

TABLE 12 biological assays (U/mL)

TABLE 13 peptide map/oxidized form (%) -displayed by UPLC

And (4) prompting: in all tables, mcg stands for microgram, HMW for high molecular weight species, DIM for dimer, and Mo for month.

Reference to the literature

Derynk R. et al, Nature 1980; 285, 542-547.

Familletti, p.c., Rubinstein, S. and Pestka, S; 1981 "convenient Rapid cytopathic Effect inhibition assay for Interferon", Methods in Enzymology, Vol.78 (S.Pestka, eds.), Academic Press, New York, 387-394.

Mark D.F et al, Proc. Natl. Acad. Sci. U.S.A., 81(18)5662-5666 (1984).

Pestka, S. (1986) "standards and common abbreviations for interferon", Methods in enzymology (S. Pestka, eds.), Academic Press, New York 119, 14-23.

Rubinstein, s., Familletti, P.C and Pestka, S; "simple assays for interferons", j.virol 1981; 37, 755-758.

Shepard H.M et al, Nature 1981; 294, 563-565.

Woghiren et al, Bioconjugate chem, 4 (5): 314-318, 1993.

Claims (26)

1. A liquid pharmaceutical composition comprising pegylated interferon-beta (PEG-IFN- β) or mutein or active fragment thereof, an excipient, a surfactant and a buffer, wherein the excipient is a polyol, the surfactant is a non-ionic surfactant and the buffer is a sodium acetate buffer.

2. The composition of claim 1, comprising PEG-IFN- β, an excipient, a surfactant, and a buffer, wherein the excipient is mannitol, the surfactant is poloxamer 188, and the buffer is sodium acetate buffer

3. The composition of claim 1 or 2, wherein the PEG-IFN- β is at a concentration of about 0.01mg/ml to about 0.1 mg/ml.

4. The composition of claim 3, wherein the PEG-IFN- β is at a concentration of about 0.044mg/ml, 0.055mg/ml, or 0.110 mg/ml.

5. The composition according to any preceding claim, wherein the PEG-IFN- β comprises a linear or branched PEG, preferably a branched PEG.

6. The composition of claim 5, wherein the PEG has a molecular weight of at least 20kDa, preferably at least 40kDa, more preferably 40 kDa.

7. The composition of claim 6, wherein the PEG-IFN- β comprises a spacer arm.

8. The composition of claim 7, wherein the spacer has a molecular weight of 100-.

9. The composition of claim 1 or 2, wherein the excipient is at a concentration of about 30mg/ml to 50 mg/ml.

10. The composition of claim 1 or 2, wherein the excipient is at a concentration of about 40mg/ml to 50 mg/ml.

11. The composition of claim 1 or 2, wherein the excipient is at a concentration of about 45 mg/ml.

12. The composition of claim 1 or 2, wherein the surfactant is at a concentration of about 0.1mg/m to 1 mg/ml.

13. The composition of claim 1 or 2, wherein the surfactant is at a concentration of about 0.4mg/ml to 0.7 mg/ml.

14. The composition of claim 1 or 2, wherein the surfactant is at a concentration of about 0.5 mg/ml.

15. The composition of claim 1 or 2, wherein the amount of buffer is sufficient to maintain the pH of the composition at ± 0.5 units of a specific pH, the specific pH being about 3.0-5.0.

16. The composition of claim 15, wherein the pH is about 3.5-4.5.

17. The composition of claim 15, wherein the pH is about 4.2 ± 0.2.

18. The composition of claim 1 or 2, wherein the buffer is at a concentration of about 5mM to 500 mM.

19. The composition of claim 16, wherein the buffer is at a concentration of about 10 mM.

20. The composition of any of the preceding claims, wherein said composition is an aqueous solution.

21. The composition according to any of the preceding claims, wherein the composition further comprises methionine.

22. A composition according to claim 21, wherein the concentration of methionine is 0.10-0.50mg/ml, preferably 0.20-0.40mg/ml, more preferably 0.12 or 0.25 mg/ml.

23. A method of preparing a liquid pharmaceutical composition according to any one of claims 1 to 22, comprising adding to said buffer calculated amounts of excipient and surfactant, followed by addition of PEG-IFN- β.

24. A container suitable for storage ready for use, sealed under sterile conditions, comprising a liquid pharmaceutical formulation according to any one of claims 1 to 22.

25. The container of claim 24, wherein the container is a prefilled syringe or a vial for an automatic syringe.

26. A kit of pharmaceutical compositions comprising a container filled with a pharmaceutical composition according to any one of claims 1 to 22.