HK1200851B

HK1200851B - Etanercept formulations stabilized with amino acids

Info

Publication number: HK1200851B
Application number: HK15101255.1A
Authority: HK
Inventors: 马克．曼宁; 布莱恩．墨菲
Original assignee: 科荣生生物科学公司
Priority date: 2011-10-18
Filing date: 2012-10-18
Publication date: 2018-07-20

Description

Etanercept formulations stabilized with amino acids

Technical Field

The present invention relates to aqueous pharmaceutical compositions stabilized with amino acids for long term storage of etanercept, methods of making the compositions, methods of administering the same, and kits containing the same. The present invention encompasses etanercept formulations that do not require arginine for stabilization.

Background

The polypeptide must generally be stored prior to its use. Polypeptides are generally unstable in solution when stored for a period of time (Manning et al, 1989, pharm. Res.6: 903-918). To extend their shelf life, additional processing steps have been developed, such as drying, e.g., freeze drying. However, lyophilized pharmaceutical compositions are less convenient to use.

Typical practice to improve polypeptide stability can be addressed by varying the concentration of elements of the formulation, or by adding excipients to modify the formulation (see, e.g., U.S. patent nos. 5580856 and 6171586). However, the use of additives may still result in inactivated polypeptides. Alternatively, in the case of freeze-drying, the rehydration step can result in inactivation of the polypeptide by, for example, aggregation or denaturation (Hora et al, 1992, pharm. Res., 9: 33-36; Liu et al, 1991, Biotechnol. Bioeng, 37: 177-184). Aggregation of polypeptides is undesirable because it can lead to immunogenicity (Cleland et al, 1993, Crit. Rev. therapeutic Drug Carrier Systems,10: 307-.

Another approach to improve polypeptide stability is to use L-arginine at specific concentrations (U.S. patent No. 7,648,702).

One polypeptide that is stored for up to two years prior to use is etanercept (b: (b))Immunex corporation) which is a dimeric fusion polypeptide consisting of the extracellular ligand binding portion of human 75 kilodaltons (p75) Tumor Necrosis Factor Receptor (TNFR) linked to the Fc portion of human IgG 1. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons (Physicians Desk Reference, 2002, Medical Economics). The Fc component of etanercept contains heavy chain constant domain 2(CH2), heavy chain constant domain 3(CH3) and the hinge region of human IgG1, but no heavy chain constant domain 1(CH 1). The Fc domain may contain one or all of the domains described above. Etanercept is typically produced by recombinant DNA technology in a Chinese Hamster Ovary (CHO) mammalian cell expression system.

The present invention provides novel stable liquid formulations of etanercept that allow long-term storage thereof.

Disclosure of Invention

The present invention is an aqueous pharmaceutical composition comprising etanercept and a stabilizer to inhibit instability, aggregation, misfolding and/or fragmentation of etanercept, wherein the stabilizer comprises an amino acid compound selected from the group consisting of serine, proline and glutamic acid.

Various technical terms used in the following discussion are defined in the following section entitled "definitions" and throughout the remainder of this specification.

The stabilized etanercept formulations of the present invention induce long term storage stability characterized by at least one of: (1) at M₃Or T₂Or T₄A SEC analysis (as defined herein) of greater than about 90% monomer content; an aggregate content of less than about 3 wt%; and a fragment 3 content of less than about 5 wt%; and (2) at M₃Or T₂Or T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 20 wt%.

In a related aspect the proline, serine or glutamic acid containing formulation elicits long term storage stability characterized by at least one of: (1) at M₃Or T₂Or T₄A SEC analysis of greater than about 90 wt% monomer content; an aggregate content of less than about 3 wt%; and less than about 5 wt% fragment 3; and (2) at M₃Or T₂Or T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 20 wt%.

In the above-mentionedPreferred aspects of the stable formulation, which results in long term storage stability characterized by: at M₃Or T₂Or T₄The HIC analysis of (1), wherein the amount of the component represented by peak 2 of the HIC chromatogram is greater than or equal to about 95 wt%; and wherein, if peak 3 is present in the HIC chromatogram, the amount of the component represented by peak 3 is less than or equal to about 1 wt%.

The stabilized etanercept formulation as summarized above, optionally and preferably, is free of arginine, or substantially free of arginine.

The formulations of the present invention have excellent stability as determined by SEC (exclusion chromatography) and HIC (hydrophobic interaction chromatography) analysis performed after storage for one, two or three months at 5 ℃. These formulations are comparable or superior to commercially available etanercept formulations in which arginine is an essential component. The invention therefore also relates to a formulation of etanercept stabilized with serine, proline or glutamic acid, which is arginine-free or substantially arginine-free, and wherein the composition is at M₃Or T₂Or T₄Results in long term storage stability meeting one or both of the following criteria: (A) comparable or superior to that in trademarksThe stability of commercial etanercept sold under (b) as determined by SEC analysis of the amount of aggregates, monomers and fragments 3 in the composition (as defined in the specification), and (ii) HIC analysis of the amount of material in the composition corresponding to peaks 1,2 and 3 of a HIC chromatogram (as defined in the specification); and (B) a HIC chromatogram in which (i) peak 3 is absent or substantially absent and (ii) peak 2 represents more than about 95 wt% of the composition; a SEC chromatogram substantially free of peaks corresponding to aggregates; and wherein the monomer content represents a SEC chromatogram of at least about 95 wt% of said composition.

In embodiments of the invention wherein the stabilizing agent is glutamic acid, the stable formulation comprises about 25 to about 75mg/ml etanercept; glutamic acid up to about 150 mM; less than about 6 wt% sucrose; optionally up to about 100mM NaCl; about 1 to about 30mM sodium phosphate, wherein the composition has a pH of about 6.0 to 6.6, and wherein, optionally and preferably, the composition is free of arginine or substantially free of arginine.

Another embodiment of the stable formulation of glutamic acid comprises about 50mg/ml etanercept; less than about 150mM glutamic acid; about 0 to 3% sucrose; about 1 to 30mM phosphate buffer, optionally up to about 100mM NaCl, and having a pH of about 6.0 to 6.6.

Another stable formulation of glutamic acid comprises about 50mg/ml etanercept; about 100 to about 120mM glutamic acid; less than about 4 wt% sucrose, and about 10-30, preferably about 25mM phosphate, and having a pH of about 6.3 to 6.5.

Another stable formulation of glutamic acid comprises about 50mg/ml etanercept; about 100mM glutamic acid; less than about 2 wt% sucrose, about 100mM NaCl; about 10-30, preferably about 25mM, phosphate; and has a pH of about 6.3 to 6.5.

Yet another stable glutamic acid formulation of the invention comprises about 50mg/ml etanercept; about 50mM glutamic acid; less than about 2 wt% sucrose, about 100mM NaCl; about 10 to 30, preferably about 25mM, phosphate; and has a pH of about 6.3 to 6.5.

In yet another embodiment wherein the stabilizing agent is serine, the stabilized etanercept composition comprises up to about 150mM serine; about 0.5 to about 3 wt% sucrose; about 1 to about 30mM sodium phosphate, and wherein the composition has a pH of about 6.0 to 6.6; and wherein optionally and preferably arginine is absent or substantially absent.

Another embodiment of a serine stabilized etanercept composition comprises about 50mg/ml etanercept; about 100mM serine; about 1 wt% sucrose, and about 10-30, preferably about 25mM phosphate, and having a pH of about 6.3 to 6.5.

In another embodiment wherein the stabilizing agent is proline, the proline stabilized etanercept composition comprises up to about 150mM proline; about 0.5 to about 3 wt% sucrose; about 1 to about 30mM sodium phosphate, about 15 to about 100mM NaCl; and wherein the composition has a pH of about 6.0 to 6.6; and wherein optionally and preferably, the composition is free of arginine or substantially free of arginine.

Another embodiment of the proline stabilized etanercept compositions of the invention contains about 50mM proline, less than about 4 wt% sucrose, about 25mM NaCl, about 10-30, preferably about 25mM phosphate, and has a pH of about 6.3 to 6.5.

The etanercept compositions of the present invention further afford the ability to provide formulations containing acceptable levels of microscopically visible particles. Thus, the invention also relates to serine, proline or glutamic acid stabilized etanercept formulations at M₃Or T₂Or T₄On average, has no more than about 10000 microscopically visible particles per ml, each having a size greater than 5 μm. Microscopically visible particles can be measured using the known means of FlowCam analysis.

The stabilized etanercept compositions of the present invention are further characterized by: (a) at M₃Or T₂Or T₄A SEC analysis of greater than about 90 wt% monomer content; and an aggregate content of less than about 3 wt%; and (b) at M₃Or T₂Or T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 20 wt%.

The stability of the formulation may be further characterized by the composition as shown below at M₃Or T₂Or T₄The HIC analysis of (A) was characterized by: wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 1%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than about 95 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 3 wt%.

In another embodiment wherein the stabilizing agent is glutamic acidThe stability of the etanercept formulation may be characterized by: at M₃Or T₂Or T₄A SEC analysis of greater than about 97 wt% monomer content and less than about 1 wt% aggregate content; and at M₃Or T₂Or T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than about 82 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 15 wt%. These features are achievable without the need for arginine as a stabilizer.

In another embodiment wherein the stabilizing agent is serine, the stabilized etanercept formulation is characterized by: at M₃Or T₂Or T₄A SEC analysis of greater than about 97 wt% monomer content and less than about 1 wt% aggregate content; at M₃Or T₂Or T₄The HIC analysis of (1), wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 4 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than about 82 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 15 wt%. These properties are achieved without the use of arginine as a stabilizer.

Preferred stable compositions of the present invention exhibit the following at M₃Or T₂Or T₄The HIC assay of (1), wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 2%, preferably less than about 1%; the amount of component represented by peak 2 of the HIC chromatogram is greater than about 95 wt%, preferably greater than about 97%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 1 wt%, preferably 0 to 1%. These properties are achieved without the use of arginine as a stabilizer.

Unlike commercially available etanercept, which is provided in the form of an arginine-containing formulation, we have unexpectedly found that, in view of U.S. patent No. 7648702, the formulation embodiments of etanercept described and exemplified herein do not require arginine for long-term stabilization, although arginine may still be added if desired. To provide an etanercept preparation stabilized without using arginineThe ability of the agent to be administered by providing patients and healthcare providers with a combination of arginine for stabilization of current commercial etanercept formulations (i.e.,) Potentially significant benefits for medical systems are shown compared to storage-stable etanercept replacement formulations available at lower cost.

The term "instability" or similar terms as used herein refers to the tendency of etanercept monomers to undergo various undesirable transformations in storage. Such transformations include the formation of oligomers and high molecular weight aggregates (hereinafter the term "aggregates") in which a plurality of substantially intact etanercept monomers are irreversibly bound to one another by various non-covalent bond attractions (e.g., electrostatic interactions). Undesirable conversion during storage may also include degradation of the etanercept monomer into smaller fragments and/or broken forms. Ideally, the formulation of etanercept should minimize as much as possible the tendency of the formulation to cause aggregate, misfolded proteins, oligomers, and/or fragments of etanercept to form during storage. An important benefit arising from the ability to reduce the formation of unwanted aggregates or fragments is the reduction in potential toxicity and/or immunogenicity of the drug.

The etanercept formulation of the present invention, which is optionally and preferably free or substantially free of arginine. The term "substantially free of arginine" is intended to mean that arginine, if present, does not contribute to the stabilization of the etanercept monomer in the formulation to such an extent that one of skill in the art would judge its presence to be advantageous or necessary from a stability standpoint.

These and other aspects will become apparent from the following description, although variations and modifications therein may be made without departing from the spirit and scope of the novel concepts of the disclosure.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Detailed Description

Various embodiments of the present invention will now be described in detail. As used in the specification and throughout the claims, the meaning of "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Also, as used in the specification and throughout the claims, the meaning of "in … …" includes "in … …" and "above … … (on)" unless the context clearly indicates otherwise. In addition, some terms used in this specification are defined more specifically below.

Definition of

The terms used in this specification generally have their ordinary meanings in the art within the context of the invention, and the specific context in which each term is used. Certain terms used to describe the invention are discussed below or elsewhere in the specification to provide additional guidance to the practitioner regarding the description of the invention. Synonyms for specific terms are provided. Recitation of one or more synonyms does not exclude the use of other synonyms. The use of any positional example in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or any exemplary terms. The present invention is not limited to the various embodiments given in this specification.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present document, including definitions, will control.

"left and right (around)," "about (about)" or "approximately" generally means within 20%, within 10%, within 5, 4, 3, 2 or 1% of a given value or range. A given quantity is approximate, meaning that the terms "left and right", "about" or "approximate" can be inferred if not expressly stated.

The term "etanercept" or "etanercept monomer" or "monomer" withSynonymously. It refers to a polypeptide that is a dimeric fusion polypeptide consisting of the extracellular ligand binding portion of human 75 kilodalton (p75) Tumor Necrosis Factor Receptor (TNFR) linked to the Fc portion of human IgG 1. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons. For the purposes of this application, the term "etanercept" also encompasses etanercept with minor modifications in the amino acid structure (including deletion, addition, and/or substitution of amino acids) that do not significantly affect the function, potency, or affinity of the etanercept. The term "etanercept" embracesIncluding but not limited to concentrated formulations, injectable ready-to-use formulations; reconstituted with water, alcohol, and/or other ingredients, and others.

The term "monomer" as used herein is intended to mean the dimeric etanercept fusion protein referenced above.

The term "serine" refers to an amino acid whose codons are UCU, UCC, UCA, UCG, AGU, and AGC.

The term "proline" refers to the alpha-amino acids whose codons are CCU, CCC, CCA and CCG.

The term "glutamic acid" refers to the deprotonated form or salt of the alpha-amino acid glutamic acid (Glu). For the purposes of this application, the term "glutamic acid" also encompasses glutamic acid itself.

The term "sugar" refers to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, trehalose, dextrose, and others.

The term "polyol" refers to an alcohol containing a plurality of hydroxyl groups. Examples of polyols include, but are not limited to, mannitol, sorbitol, and others.

The term "long term storage" is understood to mean that the pharmaceutical composition may be stored for three months or more, six months or more, and preferably one year or more. Long term storage is also understood to mean that the pharmaceutical composition is stored as a liquid at 2-8℃, or frozen, for example, at-20℃ or lower. It is also contemplated that the composition may be frozen and thawed more than once.

The term "stable" or "stabilized" in relation to long term storage is understood to mean that etanercept contained in the pharmaceutical composition does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to the activity of the composition at the start of storage.

The term "mammal" includes, but is not limited to, humans.

The term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid or excipient of any conventional type. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and are compatible with other ingredients of the formulation.

The term "composition" refers to a mixture that typically contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may comprise a cell culture, wherein the polypeptide or polynucleotide is present within the cell or within the culture medium. For example, compositions for oral administration may be formed into solutions, suspensions, tablets, pills, capsules, sustained release formulations, mouth rinses, or powders.

The terms "pharmaceutical composition" and "formulation" are used interchangeably.

The term "treatment" refers to any administration or use of a therapeutic agent for a disease in a mammal and includes inhibiting the disease, controlling its development, alleviating the disease, e.g., by causing regression, or restoring or repairing lost, absent, or defective function; or stimulate inefficient processes. The term includes any treatment to obtain a desired pharmaceutical and/or physiological effect, covering a pathological condition or discomfort in a mammal. The effect may be prophylactic in terms of completely or partially preventing the discomfort or symptoms thereof, and/or may be therapeutic in terms of partially or completely treating the discomfort and/or adverse effects due to the discomfort. It includes: (1) preventing the occurrence or recurrence of the discomfort in a subject predisposed to the discomfort but not yet symptomatic, (2) inhibiting the discomfort such as controlling its development, (3) halting or terminating the disease or at least its associated symptoms such that the host is not suffering from the discomfort or its symptoms, such as causing regression of the discomfort or its symptoms, for example, by restoring or repairing lost, absent, or defective function, or stimulating an inefficient process, or (4) alleviating, or ameliorating the discomfort or symptoms associated therewith, wherein amelioration is used in a broad sense to refer to a reduction in at least one parameter such as inflammation, pain, and/or tumor size magnitude.

The term "disease" refers to any condition, infection, discomfort or symptom that requires or is desirable for medical intervention. Such medical intervention may include treatment, diagnosis and/or prevention.

The term "therapeutically effective amount" refers to an amount that, when administered to a living subject, achieves a desired effect on the living subject. For example, a therapeutically effective amount of a polypeptide of the invention for administration to a living subject is an amount that prevents and/or treats an integrin α v β 3-mediated disease. The exact amount will depend on the purpose of the treatment and will be determinable by one of skill in the art using known techniques. As is known in the art, modulation of systemic versus local delivery, age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition is necessary and can be determined by one of skill in the art using routine experimentation.

The term "T₁"refers to the point in time at which the etanercept formulation has been stored at 40 ℃ for about one week.

The term "T₂"refers to the point in time at which the etanercept formulation has been stored at 40 ℃ for about two weeks.

The term "T₄"refers to the point in time at which the etanercept formulation has been stored at 40℃ for about four weeks.

The term "M₃"in total refers to three time points, specifically to the analytical results observed for an etanercept formulation after about one, about two, or about three months of storage at a storage temperature of 5 ℃. For example, this document refers to the compound at M₃Reference to an assay being performed is understood to mean that such an assay is performed at a point in time when the etanercept formulation has been stored for a period selected from about one, about two, or about three months. Thus, if the storage duration corresponds to at least one of: the requirement herein is met when the desired value is observed at a time point of approximately one month, approximately two months, or approximately three months of storage at 5 deg.C₃Etanercept formulations elicit specific analytical or measured values.

The terms "peak 1", "peak 2" and "peak 3" when used herein in conjunction with the discussion of HIC chromatography results refer to the same peaks 1,2 and 3 discussed in U.S. patent 7,294,481.

Detailed Description

When containing etanerceptWhen pharmaceutical compositions comprising aqueous and lyophilized formulations of etanercept are stored for extended periods, the activity of etanercept may be lost or diminished by instability of the etanercept monomer, including fragment formation, through aggregation and/or chemical degradation. Accordingly, the present invention provides several aqueous formulations of etanercept that allow stable long-term storage of etanerceptEmbodiments enable etanercept, whether in a liquid or frozen state, to be stable during storage. The provided formulations include, but are not limited to, formulations that do not contain arginine and do not require any additional steps such as rehydration.

These embodiments are explained in more detail below.

Etanercept

All compositions of the present invention comprise etanerceptAs explained in the background section of the present application, etanercept is a dimeric fusion polypeptide consisting of the extracellular ligand binding portion of human 75 kilodalton (p75) tumor necrosis factor receptor linked to the Fc portion of human IgG 1. Etanercept consists of 934 amino acids. The Fc component of etanercept contains the heavy chain constant domain 2(CH2), heavy chain constant domain 3(CH3) and hinge region of human IgG 1. The Fc domain may contain one or all of the domains described above.

Etanercept suitable for storage in the present pharmaceutical compositions of the invention may be produced by a living host cell expressing etanercept, such as a hybridoma in the case of an antibody, or a host cell genetically engineered to produce a polypeptide in the case of a fusion polypeptide or antibody. Methods of genetically engineering cells to produce polypeptides are well known in the art. See, e.g., Ausubel et al, eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods comprise introducing into a living host cell a nucleotide encoding and permitting expression of the polypeptide. These host cells may be bacterial cells, fungal cells or preferably animal cells grown in culture. Bacterial host cells include, but are not limited to, E.coli cells. Examples of suitable E.coli strains include: HB101, DH5.alpha, GM2929, JM109, KW251, NM538, NM539 and any E.coli strain that is not capable of cleaving foreign DNA. Fungal host cells that may be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Aspergillus cells. Some examples of animal cell lines that may be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3 and W138. New animal cell lines can be established using methods well known to those skilled in the art (e.g., by transformation, viral infection, and/or selection). Optionally, etanercept may be secreted by the host cell into the culture medium.

Purification of the expressed etanercept may be carried out by any standard method. When etanercept is produced intracellularly, the particulate fragment is removed, for example, by centrifugation or ultrafiltration. When etanercept is secreted into the culture medium, the supernatant of such an expression system may first be concentrated using a standard polypeptide concentration filter. Protease inhibitors may also be added to inhibit proteolysis, and antibiotics may be included to prevent the growth of microorganisms.

Etanercept may be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and known or yet to be discovered purification techniques including, but not limited to, protein A chromatography, ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparinChromatography, anion or cation exchange resin chromatography (such as polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

Etanercept stabilized with serine, proline or glutamic acid

The present invention provides a stable aqueous pharmaceutical composition comprising etanercept and a stabilizer to inhibit instability, aggregation, misfolding and/or fragmentation of etanercept, wherein the stabilizer comprises a compound selected from the group consisting of serine, proline and glutamic acid. In a preferred embodiment the stabilizer comprises glutamic acid.

Without wishing to be bound by any particular theory of the invention, serine, proline and glutamic acid are believed to act as conformational stabilizers to reduce the tendency of etanercept to aggregate. The reduction in aggregation is believed to last for a long period of time, e.g., two years or more. Serine, proline and glutamic acid are believed to stabilize aqueous pharmaceutical compositions containing etanercept because they are excluded from the surface of the protein, resulting in a net conformational stabilization. The stabilizing effect of serine, proline and/or glutamic acid includes, but is not limited to, benefiting from a reduction of etanercept monomer in a formulation containing said monomer.

The pharmaceutical compositions of the present invention may be prepared by combining purified etanercept and a stabilizer. Further, buffers, tonicity modifying agents and additional excipients and other commonly used inactive ingredients may be added as needed. For simplicity, these will be discussed more fully later in this specification. One of ordinary skill in the art will appreciate that the combination of the various components to be included in the composition can be accomplished in any suitable order. For example, the buffer may be added first, intermediate or last, and the tonicity modifier may be added first, intermediate or last. One of ordinary skill in the art will also appreciate that some of these chemicals may be incompatible in certain combinations and thus easily replaced with different chemicals having similar properties but which are compatible in the relevant mixture.

In a preferred embodiment, the concentration of the serine, proline or glutamate stabilizer in a provided formulation is preferably up to about 150 mM.

Serine, proline and glutamic acid are available from commercial suppliers.

In embodiments wherein the stabilizing agent comprises glutamic acid, the formulation of the invention may comprise about 25 to 50mg/ml of etanercept; glutamic acid up to 150 mM; less than 6 wt% sucrose; optionally up to 100mM NaCl; about 1 to about 30mM sodium phosphate, and wherein the formulation has a ph of between 6.0 to about 7.0 and more preferably about 6.0 to about 6.6, and most preferably about 6.3 to 6.5.

In embodiments where the stabilizer comprises serine, the formulations of the present invention may comprise from about 25 to about 50mg/ml of etanercept; less than about 150mM serine; about 0.5 to about 3 wt% sucrose; about 1 to about 30mM sodium phosphate, and wherein the formulation has a ph of about 6.0 to about ph7.0, and more preferably about 6.0 to about 6.6, and most preferably between about 6.3 to about 6.5.

In embodiments wherein the stabilizing agent comprises proline, the formulation of the present invention may comprise about 25 to about 50mg/ml etanercept; less than about 150mM proline; about 0.5 to about 3 wt% sucrose; about 1 to about 30mM sodium phosphate, about 15 to about 100mM NaCl; and wherein the formulation has a ph of between 6.0 and about ph7.0, more preferably between about 6.0 and about 6.6, and most preferably between about 6.3 and about 6.5.

Etanercept formulations containing serine, proline or glutamic acid according to the invention are preferably characterized by: at T₂A SEC analysis of about 80 to about 95 weight percent monomer content; less than about 4 wt% aggregate content; and a fragment 3 content of less than about 8 wt%.

The present invention contains serine, proline or glutamic acid for use in a stable specific formulation which causes stability characterized by:

(a) at T₄A SEC analysis of greater than about 90, 91, 92, 93, 94, 95, 96, or 97 wt% monomer content; and an aggregate content of less than about 3, 2, or 1 wt%; and

(b) at T₂Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3,

2 or 1 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is more than 80, 81, 82, 83, 84, or 85 wt%; and the amount of a component represented by peak 3 of the HIC chromatogram is less than about 20, 19, 18, 17, 16, 15, 14, or 13 wt%; and

(c) at T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3, 2, or 1 wt%; the amount of component represented by peak 2 of the HIC chromatogram is more than 80, 81, 82, 83, 84 or 85 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16, 15, 14, or 13 wt%.

The terms "SEC", "T₂”“、“T₄”、“M₃"," HIC "monomer content", "aggregate" and "fragment 3", "peak 1", "peak 2" and "peak 3" are defined in the examples below.

In particularly preferred formulations containing serine, proline or glutamic acid for stabilization, it is preferred to have the following at T₄Or T₂The HIC analysis of (A) was characterized by: wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 1%; the amount of component represented by peak 2 of the HIC chromatogram is more than about 95 wt%, and most preferably more than about 99 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 3 wt%.

Another preferred formulation for stabilization of etanercept using serine, proline and/or glutamic acid comprises about 50mg/ml etanercept; serine, proline or glutamic acid of less than about 150mM, and most preferably glutamic acid; about 0 to 3% sucrose; about 1 to 30mM phosphate buffer, and having a pH of about 6.0 to 6.6; and is characterized by: at T₄A monomer content of greater than about 97 wt% and an aggregate content of less than about 1 wt% in SEC analytical sites of (a); at T₂Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than about 82 wt; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 15 wt%; and at T₄Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 2 wt%; the amount of component represented by peak 2 of the HIC chromatogram is greater than about 84 wt%; and the amount of the component represented by peak 3 of the HIC chromatogram is less than about 13 wt%.

In another embodiment of the invention, the stabilized etanercept composition is stabilized using serine, proline or glutamic acid, the formulation is free or substantially free of arginine, and the formulation elicits long term storage stability characterized by at least one of:

at M₃Has a monomer content of greater than about 90% and an aggregate content of less than about3 wt%; hexipian (Chinese character of 'He')

The level of stage 3 is less than about 5 wt%; and

at M₃Wherein the amount of the component represented by peak 1 of the HIC chromatogram is less than about 3 wt%;

the amount of the component represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and peak 3 from HIC chromatogram

The amount of the component represented is less than about 20 wt%.

Preferably the serine, proline or glutamic acid stabilized composition of the invention results in a long term storage stability characterized by: at M₃Wherein the amount of the component represented by peak 2 of the HIC chromatogram is greater than or equal to about 95 wt%; and wherein the amount of the component represented by peak 3 is less than or equal to about 1 wt% if peak 3 is present in the HIC chromatogram.

In another aspect of the invention, the serine, proline or glutamic acid stabilized composition of the invention results in long term storage stability characterized by:

at M₃Or T₄A SEC analysis of greater than about 90 wt% monomer content; less than about 3 wt% aggregates

Content (c); and less than about 5 wt% fragment 3; and

at M₃Or T₂Or T₄The HIC analysis of (1), wherein the amount of the component represented by peak 1 of the HIC chromatogram is small

At about 3 wt%; the amount of the component represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and by HIC

Peak 3 of the chromatogram represents an amount of less than about 20 wt% of the component.

Another preferred glutamate-stabilized etanercept formulation comprises: about 50mg/ml etanercept; about 120mM glutamic acid; about 1% sucrose and about 25mM phosphate; has a pH of about 6.3 to about 6.5, and exhibits the SEC and HIC analysis characteristics described above.

Preferably, the stable etanercept formulation according to the present invention upon storage at 5 ℃ or 25 ℃ for one, two or three months is (a) characterized by an HIC chromatogram in which peak 3 is substantially absent; (b) characterized by a SEC chromatogram comprising no or substantially no peak corresponding to the aggregate; and (c) characterized by a SEC chromatogram of said composition in which said monomer content represents at least about 97 weight percent.

The arginine-free serine, proline or glutamic acid stabilized etanercept formulation was found to result in a stability at 5 ℃ for three months that is comparable or superior to currently available commercial Enbrel formulations containing arginine. The invention therefore also relates to serine, proline or glutamic acid stabilized etanercept formulations that are arginine-free or substantially arginine-free, and wherein the composition is at M₃Or T₂Or T₄Results in long term storage stability meeting one or both of the following criteria:

(A) comparable or superior to that in trademarksThe stability of commercially available etanercept sold under the following, measured by: (i) SEC analysis of the amount of aggregates, monomers and fragments 3 in the composition (as defined in the specification), and (ii) HIC analysis of the amount of material in the composition corresponding to peaks 1,2 and 3 of a HIC chromatogram (as defined in the specification); and

(B) a HIC chromatogram in which (i) peak 3 is absent or substantially absent and (ii) peak 2 represents more than about 95 wt% of the composition; a SEC chromatogram substantially free of peaks corresponding to aggregates; and a SEC chromatogram of said composition in which the monomer content represents at least about 95 wt%.

Although the present invention does not exclude the use of arginine, etanercept formulations comprising serine, proline and/or glutamic acid for stabilization according to the present invention do not require arginine for stabilization and therefore are preferably free or substantially free of arginine.

Other Components of the pharmaceutical compositions provided

The formulations of the present invention may also include buffers, tonicity modifying agents, excipients, pharmaceutically acceptable carriers and other inactive ingredients commonly used in pharmaceutical compositions. For simplicity, these will be discussed more fully later in this specification.

The buffer maintains the pH within the desired range. Suitable buffering agents include histidine, potassium phosphate, citric acid, sodium or potassium maleate, ammonium acetate, tris- (hydroxymethyl) aminomethane (tris), various forms of acetate and diethanolamine. The concentration of the buffer in the formulation is preferably between about 1mM to about 1M, more preferably about 10mM to about 200mM formulation. Buffers are well known in the art and are manufactured by known methods and are available from commercial suppliers.

Examples of suitable buffers are phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris- (hydroxy) -aminomethane (tris), bicarbonate.

In a preferred embodiment, the buffer salt is sodium phosphate.

In a preferred embodiment, the pH of the pharmaceutical composition is at or near physiological levels. Thus, preferably, the pH of the provided compositions is between about 5.8 to about 8.4; even more preferably, between about 6.2 and about 7.4. Those skilled in the art will appreciate that the pH can be adjusted as needed to maximize the stability and solubility of etanercept in a certain formulation. Thus, formulations of etanercept having a pH outside the physiological range, but which are tolerable to the patient, are also outside the ranges described herein.

Tonicity modifiers are molecules that contribute to the osmolality of the solution. The osmolality of the pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and/or to minimize discomfort to the patient upon administration. It is generally preferred that the pharmaceutical composition is isotonic with serum, i.e., has the same or similar osmolality, which can be achieved by the addition of tonicity modifying agents.

In a preferred embodiment, the osmolality of the provided formulation is from about 180 to about 420 mOsM. It is to be understood, however, that the osmolality can be higher or lower as the case may require.

Examples of tonicity modifying agents suitable for modifying osmolality include, but are not limited to, amino acids (excluding arginine) (e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate), and/or sugars (e.g., sucrose, glucose and mannitol).

Preferred tonicity modifiers are glycine, alanine, sodium chloride, potassium chloride and sodium sulfate.

In a preferred embodiment, the concentration of the tonicity modifying agent in the formulation is preferably between about 1mM to about 1M, more preferably about 10mM to about 200 mM. Tonicity modifiers are well known in the art and can be made by known methods and are available from commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, or co-solvents, which stabilize the polypeptide when in solution (and in dried or frozen form) may also be added to the pharmaceutical composition. Excipients are well known in the art and can be manufactured by known methods and are available from commercial suppliers.

Examples of suitable excipients include, but are not limited to, sugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, poly (vinyl alcohol), PVA, Hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), Hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyols, (e.g., PEG and glycerol) and Dimethylformamide (DMF); amino groupAcids such as: proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid; surfactants such as:-80 (polysorbate 80),-20 (polysorbate 20), SDS, polysorbate, poloxamer, and various excipients such as potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (such as zinc, calcium, and magnesium), CHAPS, monolaurate, 2-O- β -mannosylglyceride, or any combination of the foregoing.

Preferred excipients are sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), recombinant albumin, dextran, PVA, Hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), Hydroxyethylcellulose (HEC), polyethylene glycol, ethylene glycol, glycerol, alanine, glycine, lysine hydrochloride, sarcosine, SDS, polysorbate 20, polysorbate 80, poloxamer 188, trimethylamine N-oxide, betaine, zinc ion, calcium ion, magnesium ion, CHAPS, sucrose monolaurate and 2-O- β -mannosylglyceride.

The concentration of one or more excipients in the formulations of the present invention is preferably between about 0.001 to 5 weight percent, more preferably about 0.1 to 2 weight percent.

Method of treatment

In another embodiment, the present invention provides a method of treating a mammal comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the mammal has a disease or disorder that is beneficially treated with etanercept.

In a preferred embodiment, the etanercept is derived from the same species of mammal to be treated with the composition.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that may be treated with the provided compositions include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, wegener's disease (granulomatosis), crohn's disease (or inflammatory bowel disease), Chronic Obstructive Pulmonary Disease (COPD), hepatitis c, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis. Other diseases or disorders that may be treated with the compositions of the present invention include those described in WO00/62790, WO 01/62272, U.S. patent application No. 2001/0021380, and U.S. patent No. 7,648,702B2, the relevant portions of which are incorporated herein by reference.

The provided pharmaceutical compositions can be administered by systemic injection, such as intravenous injection; or by injection or administration to the relevant site, such as by direct injection or administration to the site when the site is exposed to surgery; or by topical administration to a subject in need of treatment.

In one embodiment, the present invention provides a method of treating and/or preventing rheumatoid arthritis comprising administering to a mammal in need thereof a therapeutically effective amount of one of the etanercept compositions provided.

The therapeutically effective amount of etanercept in a provided composition will depend on the condition being treated, the severity of the condition, previous treatments, and the clinical history of the patient and response to the therapeutic agent. The appropriate dosage may be adjusted at the discretion of the attending physician so that it is administered to the patient at one time or over a series of administrations.

In one embodiment, the effective amount of etanercept per adult dose is from about 1 to 500mg/m²Or about 1-200mg/m²Or about 1 to 40mg/m²Or about 5-25mg/m²。

Alternatively, a fixed dose may be administered, which may be in an amount of 2-500 mg/dose, 2-100 mg/dose, or about 10-80 mg/dose.

If the dose is to be administered more than once per week, exemplary dosage ranges are the same or lower than those previously described, and are preferably administered twice or more per week at each dosage range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration by injection contains 80-100mg per dose, or 80mg per dose.

The dose may be administered weekly, biweekly, or every few weeks (e.g., 2 to 8 weeks).

In one embodiment, etanercept is administered by single Subcutaneous (SC) injection at 25 to 75 mg/ml.

In some examples, improvement in the condition of a patient will be obtained by administering a dose of the pharmaceutical composition of up to about 100mg once to three times per week for at least three weeks. Treatment over a longer period of time may be necessary to cause the desired degree of improvement. The therapy may continue indefinitely for an incurable chronic condition. For pediatric patients (ages 4-17), suitable therapy may involve administering etanercept at a dose of 0.4mg/kg to 5mg/kg once or more weekly.

In another embodiment, the pharmaceutical formulations of the present invention may be prepared in bulk formulations and, as such, the components of the pharmaceutical compositions are adjusted to be higher than required for administration and diluted appropriately prior to administration.

The pharmaceutical composition may be administered as a single therapeutic agent or in combination with additional therapies as desired. Thus, in one embodiment, the provided methods of treatment and/or prevention are used in conjunction with the administration of a therapeutically effective amount of another active agent. The additional active agent may be administered before, during or after administration of the pharmaceutical composition of the present invention. The other active agent can be administered as part of the provided composition, or alternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can be achieved in a variety of ways, including parenteral oral, buccal, sublingual, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreal injection, and topical application.

The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, intraperitoneally, intracerobrospinally, intraarticularly, intrasynovially, intravitreally, and/or intrathecally. Parenteral administration can be by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be presented in unit dose form, for example, in ampoules or in multi-dose containers, with an added preservative. In addition, many recent drug delivery methods have been developed and the pharmaceutical compositions described herein are suitable for administration using these new methods, e.g.Injector pens such asAnd needleless devices such asAndthe pharmaceutical compositions may also be adapted for yet to be discovered methods of administration. See also Langer,1990, Science,249: 1527-.

The pharmaceutical compositions provided may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the formulations may be modified using suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example as a sparingly soluble salt.

If desired, the pharmaceutical composition may be presented in vials, packages, or dispensing devices that may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispensing device may comprise a syringe having a single dose of ready-to-use liquid formulation. The syringe may be accompanied by instructions for administration.

In another embodiment, the invention relates to a kit or container containing an aqueous pharmaceutical composition of the invention. The concentration of the polypeptide in the aqueous pharmaceutical composition may vary within a wide range, but is typically in the range of from about 0.05 to about 20000 micrograms per milliliter (μ g/ml) of aqueous formulation. The kit may also be accompanied by instructions for use.

The invention is more particularly described in the following examples, which are intended to be illustrative, since numerous modifications and variations therein will be apparent to those skilled in the art.

Example 1

Etanercept stabilized with serine

Etanercept formulations stabilized with serine, proline or glutamic acid can be prepared using the following procedure:

each solid formulation component is weighed to the amount required for a given volume of formulation buffer. These components are combined into a beaker or vessel capable of holding and measuring the given volume of formulation buffer. Approximately 3/4 volumes of deionized water, equivalent to approximately 3/4 for a given formulation buffer of interest, were added to the beaker and the components were subsequently dissolved. The pH of the buffer was adjusted to the pH of the desired formulation using 1M sodium hydroxide or 1M hydrochloric acid. The final formulation buffer volume was then raised to the target volume by adding deionized water. The etanercept protein solution is placed in a bag of Dialysis material (such as ThermoScientific Slide-a-Lyzer MINI Dialysis unit10,000mwco) which is then placed in contact with the desired formulation buffer for 12 hours at 4 ℃. The ratio of the buffer volume of the formulation to the volume of the protein solution should be no less than 1000: 1. The dialysis bag and the protein solution it contains are then placed in a second identical volume of formulation buffer at 4 ℃ for another 12 hours. The resulting protein solution was removed from the dialysis material bag and the concentration of the protein was determined using ultraviolet spectroscopy. Protein concentration is adjusted to a desired level using centrifugation (such as Amicon ultra10,000mwco centrifugal concentrator) and/or dilution with formulation buffer.

The compositions can be tested for long-term stability, as well as binding and biological activity at different time points by exclusion chromatography (SEC), denaturing SEC (dsec), Hydrophobic Interaction Chromatography (HIC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The biological activity can be measured by any number of well known assays.

For example, exclusion chromatography techniques are described in Hawe et al, pharm. Res.2011,28:2302 and/or van Rarschalkerweerd et al, Eur. J. pharm. Biopharm.2011,78: 213. Similarly, denaturing exclusion chromatography, hydrophobic interaction chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis techniques are well known to those of ordinary skill in the art.

The composition is believed to be stable over a period of two years or more.

(preparation 1:15)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Serine (inactive ingredient)	25mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)
		NaCl (inactive)	100mM

(preparation 1:12)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Serine (inactive ingredient)	25mM
		Sodium phosphate, pH6.4 (inactive)	25mM
Sucrose (inactive)	2.5% (w/v) or 5% (w/v)
		NaCl (inactive)	100mM

(preparation 1:16)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Serine (inactive ingredient)	50mM

Sodium phosphate, pH6.4 (inactive)	25mM
		Sucrose (inactive)	5％(w/v)
NaCl (inactive)	25mM

(preparation 2:4)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Serine (inactive ingredient)	100mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)

(preparation 3:8)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Serine (inactive ingredient)	120mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)

Example 2

Etanercept stabilized with proline

(preparation 1:4)

Composition (I)

Concentration of

Etanercept (active ingredient)	50mg/ml
		Proline (inactive ingredient)	25mM
Sodium phosphate, pH6.3 (inactive)	25mM
		Sucrose (inactive)	2.5％(w/v)
NaCl (inactive)	50mM

(preparation 1:5)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Proline (inactive ingredient)	50mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1.0％(w/v)
		NaCl (inactive)	25mM

(preparation 1:6)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Proline (inactive ingredient)	100mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1.0％(w/v)

Example 3

Etanercept stabilized with glutamic acid

Compositions stabilized with glutamic acid can be prepared and tested using procedures similar to those described in example 1.

A glutamate stabilized etanercept composition, free of arginine, as exemplified below:

(preparation 1:9)

Composition (I)	Concentration of
		CanaXipu (active ingredient)	50mg/ml
Glutamic acid (inactive ingredient)	25mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)
		NaCl (inactive)	100mM

(preparation 2:2)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Glutamic acid (inactive ingredient)	50mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)
		NaCl (inactive)	50mM

(preparation 2:3)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Glutamic acid (inactive ingredient)	100mM
		Sodium phosphate, pH6.3 (inactive)	25mM
Sucrose (inactive)	1％(w/v)

(preparation 3:5)

Composition (I)	Concentration of
		Etanercept (active ingredient)	50mg/ml
Glutamic acid (inactive ingredient)	120mM
		Sodium phosphate, pH6.5 (inactive)	25mM
Sucrose (inactive)	1％(w/v)

The composition can be tested for long term stability and the biological activity can be measured in the same manner as discussed in example 1.

The composition is believed to be stable over a period of two years or more.

Example 4

Preparation of etanercept

Step 1: and (4) expanding the cells.In a manner known in the art, CHO cell clones expressing etanercept fusion protein are used for the expansion of cells necessary to generate an effective number of cells sufficient for seeding of a production bioreactor. The product of this expression process (harvested cell culture fluid) is a mixture of correctly folded etanercept and incorrectly folded and/or aggregated etanercept along with additional impurities. The harvested cell culture fluid containing this protein mixture is subjected to detergent virus inactivation.

Step 2: affinity chromatography. Conventional protein A affinity was used in a well-known manner for the cell culture fluid harvested in step 1 aboveThe column is subjected to affinity chromatography. The product recovery was approximately 85%. The obtained product is a complex protein mixture comprising correctly folded etanercept, incorrectly folded etanercept and/or aggregates of correctly and/or incorrectly folded etanercept, or protein fragments. The product obtained from this protein a affinity column purification step was adjusted to ph3.5 and then subjected to a virus inactivation step. After virus inactivation the product was adjusted to ph5.5 and further purified in a known manner using commercially available capsule filters.

Step 3AMixed mode cation exchange chromatography using 31.8L (45cm diameter × cm bed height) packed bed GE Healthcare Capto MMC chromatography column to purify the product obtained in step 2 above prior to use, the column was equilibrated with 25mM acetate at 2cvph5.5 and sterilized with 2CV of 0.1N NaOH, 1M NaCl, and neutralized with 25mM acetate at 2cvph5.5, 0.7M NaCl, then the column was equilibrated with 25mM acetate at 8-10cvph5.5 until the effluent was ph5.5 and 3.5 mS/cm. the protein a pool (pool) from step 2 above was diluted to ≦ 6mS/cm using WFI mixed mode and applied to the column load of up to 15g/L media per cycle, the column was operated at linear speed of 200cm/h to give a 6 minute residence time after loading, the column was washed with 25mM acetate at 2 ph5.5 with mill 8.5% to collect the product from the column using a gradient of 0.5 mM acetate at 10mM ph7 mM, the maximum absorbance of the column acetate was collected from the column at 0.5 mM ph5.5 mM filter, the column was eluted with 0.5 mM acetate at 10mM ph7 mM of 0.5 mM 1.5 mM and the effluent was collected from the column with a 0.5 mM eluent, the column, the eluent was discarded the effluent was collected with a 0.5 mM acetate at a maximum absorbance of 0.5 mM acetate concentration gradient of 0.5 mM acetate (0.5 mM ph 5.5.5.5.5 mM ph) and the column, the eluent was collected from the column, the eluent was collected at 0.5 mM eluent, the column²) Filtration is carried out. The product obtained from this step showed a recovery of about 70% of the protein a material obtained in step 2.

Step 3BMixed mode anion exchange chromatography A27.0L (45cm diameter × 17cm bed height) packed bed GE Healthcare Capto Adhere column was used to further purify the product obtained in step 3A above, before use, 25mM T of 2CV pH8.0 was usedThe ris equilibrated the column, and using 2CV 0.1N NaOH, 1M NaCl disinfection and use 2CV pH8.0 25mM Tris neutralization and equilibrium. Before product loading, the column was equilibrated with 3CV of 10mM Tris pH 8.0. The Capto MMC pool from step 3A above was adjusted to pH8.1 using 0.045Kg of 1M Tris (pH8.3) per Kg of pool. The product from step 3A above was diluted with WFI in a continuous 1:3.8 dilution to adjust conductivity to 12.0mS/cm and pH 8.0. The material obtained was then applied to a column load of up to 15g/L of medium. The column was operated at a linear speed of 170cm/h to give a residence time of 6 minutes. After loading, the column was washed with 2CV of 25mM Tris pH 8.0. The product was then eluted with a10 CV gradient (20% to 90%) of 25mM Tris, pH8.0 to 10mM Tris,1M NaCl, pH 8.0. Product collection started at 0.15OD (a280,1.0cm path length) and ended at 25% of the maximum peak collected. The volume of the eluent is 4-6 CV. The eluted product is filtered using a commercially available capsule filter, followed by virus inactivation and tangential flow filtration steps in a known manner. The total product recovery from step 3B (including final virus and tangential flow filtration steps) was approximately 68%. The product recovery rate measured before the filtration step was about 75%. A graphical representation of HIC data obtained on the eluted fractions from this step is shown in figure 12.

Analysis of: the final filtered product obtained in this example was found to have greater than about 90 wt% correctly folded etanercept as determined by HIC; less than 5 wt% of incorrectly folded etanercept form as determined by HIC; less than about 3 wt% of a cleavage mass (considered to be an etanercept fragment in which the TNFR moiety is truncated) as determined by HIC analysis, and more than 95 wt% of the combined amount of correctly and incorrectly folded etanercept as determined by exclusion chromatography.

Analysis of etanercept formulations

A. Heat stable storage

After dialysis and concentration, samples of the etanercept formulation exemplified above were sterile filtered in a biosafety cabinet. Samples of etanercept formulation were transferred to the pre-preg using a sterile pipette and high pressure steam treated pipette tipLabeled and autoclaved 1mL lyophilized vials. The vial was stoppered with a sterile butyl plug and clamped with an aluminum cap. All vials were then transferred to a heat stable oven. The samples were subjected to two thermal stability protocols: (1) two weeks at 40 ℃, and (2) four weeks at 25 ℃. Throughout this specification, these two temperature regimes are denoted respectively as "T₂"and" T₄”。

B. Exclusion Chromatography (SEC)

The etanercept formulations disclosed herein are performed using well known techniques of exclusion chromatography (SEC), wherein analytes are analyzed by Size-separated high performance liquid chromatography methods (see Rogner, M. (2000).Protein Liquid ChromatographyM.Kastner.Amsterdam, Elsevier.61: 89-145). To evaluate the thermal stability of the etanercept samples described above, the thermal stress testing was performed by a literature-based (van maarschalkerweerd, a., g.j. wolbink, et al (2011) "company of analytical methods to detect the stability of the experimental work" European Journal of Pharmaceutics and Biopharmaceutics78(2):213- & 221.). the mobile phase buffer was prepared to contain 50mM sodium phosphate monobasic monohydrate and 150mM arginine the pH was adjusted to 6.5 using 1MHCl all separations were performed using a Tosoh TSK-Gel SW × l6mM × 4cm guard column (cat. No.8543) linearly connected to Tosoh TSK-Gel G4000SW × l7.8mm × 30cm (cat. No. 8542). to perform the separations, the column was brought to room temperature (23 ℃) and equilibrated using a mobile phase at a flow rate of 0.5 mL/min.5 microliters of 50mg/mL etanercept formulation was injected into the column using an autosampler needle, the separations were completed over 30 minutes at a flow rate of 0.5 mL/min.the column eluent was detected at a wavelength of 280nm during this period.

C. Integration of exclusion chromatography chromatograms

All integrations were performed using Chromeleon software (Dionex). Prior to integration, SEC chromatograms of buffer without etanercept were subtracted from all chromatograms. All integrations were performed between retention times of 12 minutes and 26 minutes. The peaks are defined using seven parameters. The minimum area of the detected peak was set to 0.05mAu min. The two-dimensional sensitivity of peak detection was set to 0.01mAu and 75 seconds. The peak shoulders were added manually using a manual integration tool. All detected peaks were manually adjusted in two steps. First, the peak baseline (the bottom boundary of the peak) is adjusted to the horizontal. Second, the vertical position of the baseline of the peak is adjusted to the vertical position of the baseline of the chromatogram. The chromatogram is baseline to a signal defined as the absence of analyte. The absence of analyte signal is defined as the absorbance in the 12 minute retention time mAu.

D. SEC fraction of etanercept formulation

In the SEC analysis of the etanercept formulation described above, three SEC chromatogram fractions were identified and studied. The fractions analyzed in the order of elution from the SEC column were: (1) represents the high molecular weight portion of an aggregate (hereinafter "aggregate" or aggregate content) of the intact etanercept TNFR: Fc fusion protein that appears to assemble by non-covalent electrostatic attraction between intact etanercept molecules; (2) a monomeric portion representing the entire etanercept TNFR: Fc fusion protein (hereinafter referred to as "monomer" or "monomer content"); (3) appears to represent a fragment or portions of fragments of the etanercept molecule, where TNFR: the molecular fusion protein has been cleaved from the monomer; the fusion protein loses the arms of the Fab portion at the hinge region of the molecule. The most common fragment or broken form, as measured by SEC, is referred to as fragment 3. In performing the SEC analysis, it will be observed that the aggregate elutes first, followed by the monomer, followed by fragment 3.

The following table shows the relative amounts of aggregate, monomer and fragment 3 as determined by SEC analysis as described above.

TABLE I

SEC analysis of monomers

Note that: the amounts reported in tables I, II and III are in weight percent

T₀The formulations were kept at 5 ℃ and analyzed within 24 hours of formation.

T₁The formulations were stored at 40 ℃ for one week.

T₂The formulations were stored at 40 ℃ for two weeks.

TABLE II

SEC analysis of aggregates

Note that: the amounts reported in tables I, II and III are in weight percent

T₁The formulations were stored at 40 ℃ for one week.

T₂The preparation is stored at 40 ℃ for two weeks

TABLE 3

Analysis of fragment 3

Note that: the amounts reported in tables I, II and III are in weight percent

T₁The formulations were stored at 40 ℃ for one week.

T₂The formulations were stored at 40 ℃ for two weeks.

TABLE IV

SEC analysis of monomer content

(T₄4 weeks/25 ℃)

Table IV below shows that the etanercept formulations prepared in accordance with the present invention when stored at 25 deg.C for four weeks (using the symbol T)₄Expressed) of the monomer (etanercept). In the following table T₀Represents SEC measurements made within 24 hours of preparation at a sample temperature of 5 ℃; and T₄Represents a sample of an etanercept formulation subjected to SEC analysis after 4 weeks storage at 25 ℃.

TABLE V

SEC analysis of aggregate content

(T₄4 week/25 ℃ C.)

Table V below shows the aggregate content of etanercept formulations prepared in accordance with the present invention after four weeks storage at 25 ℃. In the following table T₀Represents SEC measurements made within 24 hours of preparation at a sample temperature of 5 ℃; and T₄Represents a sample of an etanercept formulation that was analyzed by SEC after 4 weeks storage at 25 ℃.

HIC analysis of etanercept formulations

The following tables (tables VI and VII) show the results of hydrophobic interaction chromatography ("HIC chromatography") performed on samples 3:5 and 3: 8. HIC chromatography is performed in a manner known in the art and is generally described in us patent 7,294,481, which is incorporated herein by referenceText. Sample at t₀Evaluation was carried out (within 24 hours of preparation at 5 ℃) and storage at 25 ℃ for two weeks (t)₂) After (see Table VI) or storage at 25 ℃ for four weeks (t)₄) After which (see table VII) it was evaluated again. Peak 1 of the HIC chromatogram is considered to be or includes "fragment 3" as referred to in the discussion of SEC data above, which was identified and quantified using SEC; peak 2 is the etanercept monomer referred to in the discussion of SEC data above; peak 3 includes the "aggregate" referred to in the discussion of SEC data above. It should be further understood that the terms "peak 1", "peak 2" and "peak 3" as used herein also constitute references to the HIC peak 1, peak 2 and peak 3 disclosed and referred to in fig. 4 of U.S. patent 7,294,481, which is incorporated herein by reference.

TABLE IV

HIC data after two weeks of storage at 40 deg.C

TABLE VII

HIC data (T) after four weeks storage at 25 deg.C₄)

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Tables VIII through XVI below contain the results of stability tests conducted on formulation 3:5 (see example 3 above) and etanercept-containing material produced in the manner described in example 4 (preparation of etanercept). The stability of this formulation was evaluated 3:5 based on storage at different temperatures including 5 ℃ for one, two and three months using SEC, HIC and FlowCam analysis for microscopic particles. The methodology used to perform these stability tests is as follows:

bulk etanercept storage. Unformulated bulk etanercept was stored at 2-8 deg.C as describedShown in the package insert.

UV spectroscopy. The protein concentration in the samples of different stabilities was determined using UV spectroscopy. The absorbance of the bulk material (50 mg/mL Enbrel) at 280nm was determined to be 0.625 using a 0.1mm path length unit, resulting in an extinction coefficient of 1.30 mL/mg cm. This value is used for all calculations in this case.

Dialysis and concentration of etanercept formulations. All buffers were prepared in two 1L volumes containing all buffer components. Five minutes after rinsing the dialysis cassette in deionized water, the bulk material was loaded into Slide-a-Lyzer dialysis apparatus (10kD cut off, 1 to 3mL volume). The dialyzed sample was dialyzed at 2-8 ℃ for five hours in 1L buffer followed by a second dialysis overnight at 2-8 ℃ in a second 1L buffer. When dialyzing the etanercept formulation 3:5, two 4L dialysis units were used because the formulation required 24mL of starting protein.

All samples were concentrated above their target values using an Amicon Ultra10K cut-off filter centrifuge (2mL size). The new concentration of the sample was determined using UV, which was then diluted to the appropriate level using formulation buffer.

Thermostable sample incubation. After dialysis and concentration, the heat stable samples were sterile filtered to a biosafety cabinet. Using a sterile pipette and a high pressure steam treated pipette tip, the sample was transferred to a pre-labeled and high pressure steam treated 1mL lyophilized vial. The vial was stoppered with a sterile butyl plug and clamped with an aluminum cap. All vials were then transferred to a heat stable oven.

Exclusion Chromatography (SEC). Exclusion using different methodsChromatography (SEC) in the SEC method referred to herein as "method 2", a mobile phase buffer was prepared to contain 50mM sodium phosphate monobasic monohydrate and 150mM arginine hcl. pH was adjusted to 6.5 using 1m maho. separation was performed using Phenomonex Yarra3micron SEC3000,30cm × 4.6.6 mM. to perform the separation, the column was brought to room temperature (23 ℃) and equilibrated using the mobile phase at a flow rate of 0.5 mL/min. one microliter of 50mg/mL etanercept formulation was injected into the column using an autosampler needle.

In another SEC method, hereinafter referred to as "method 3", NaCl is used as a salt at a concentration of 100mM, ph6.3, in the mobile phase instead of arginine HCl.

In another SEC method, referred to herein as method 1, the SEC analysis is performed as follows: the mobile phase buffer was prepared to contain 50mM sodium phosphate monobasic monohydrate and 150mM arginine. The pH was adjusted to 6.5 using 1M HCl. All separations were performed using a Tosoh TSK-Gel SW × l6mm × 4cm guard column (cat No.8543) connected linearly to a Tosoh TSK-Gel G4000SW × l7.8mm × 30cm (cat No. 8542). For separation, the column was brought to room temperature (23 ℃) and equilibrated with the mobile phase at a flow rate of 0.5 mL/min. 5 microliters of 50mg/mL etanercept formulation was injected into the column using an autosampler needle. The separation was completed at a flow rate of 0.5mL/min for 30 minutes. During this time the column eluate was detected at a wavelength of 280 nm.

Integration of exclusion chromatography chromatograms. All integrations were performed using Chromeleon software (Dionex). Prior to integration, SEC chromatograms of buffer without etanercept were subtracted from all chromatograms. The integration is performed between retention times of 2 minutes and 8 minutes (CHS) or 12 minutes and 26 minutes (Innovator). The peaks are defined using seven parameters. The minimum area of the detected peak was set to 0.05mAu min. The two-dimensional sensitivity of peak detection was set to 0.01mAu and 75 seconds. The peak shoulders were added manually using a manual integration tool. All detected peaks were manually adjusted in two steps. First, the peak baseline (the bottom boundary of the peak) is adjusted to the horizontal. Second, adjust theThe vertical position of the baseline of the peak to the vertical position of the baseline of the chromatogram. The chromatogram baseline value is defined as the signal for the absence of analyte. In this case, the absence of analyte signal is defined as the absorbance in the 2 minute retention time mAu.

Hydrophobic Interaction Chromatography (HIC). Undiluted formulation samples were loaded into HPLC vials prior to injection into the chromatographic column. The samples were separated by HIC according to the parameters listed in the table below.

TABLE 4 HIC method description

For testing formulations 3:5 Description of the method. The method development work was first performed using a Manuel Prime without sample program at 7/10/12 (liquid-liquid interface). Significant mixing effects were seen in the flow cell, so an alternative air gap procedure (Manual Prime with sample) was chosen for sample evaluation.

Frozen 3:5 samples of the baseline (T0) preparation were received and stored at-20 ℃ until thawed in the ambient environment. Once thawed, the formulation was stored at refrigeration temperatures (2-8 ℃). The T0 sample method included a matched buffer pretreatment step to accommodate the flow cell. 0.4mL or more of the formulation buffer is flushed through the system before loading the sample. This pretreatment was determined to be unnecessary and therefore was not used for the t3 test time point.

Samples of formulation 3:5 that had been subjected to thermal stress for three months at 5 ℃ and 25 ℃ were evaluated in the FlowCAM assay. All samples were thawed on the date they were analyzed. Once thawed, it was stored at refrigeration temperatures (2-8 ℃).

Apparatus and device

FlowCAM instrument: model VS1, Serial #551 with Sony SX90 camera and C70 pump with 1mL syringe (fluid imaging technique)

FlowCAM software: DSP Firmware Version: 54, a first electrode; version3.0.3

Flow cell Field of View (FOV FC80) with a depth of 80 μm and a width of 700 μm (fluid imaging technique)

An objective lens: 10 is rich

Background settings (method and setting parameters)

The method comprises the following steps: manual Prime with sample (air gap)

And (3) sample analysis: 0.200mL volume, 0.170mL was analyzed

Flow rate: 0.100ml/min

Automatic imaging speed: 22frames per second

Efficiency: 38.7 percent

Operating time: 1.7 minutes

Distance to Nearest

0 μm of Neighbor

Close Hole:5iterations

Image: 5 collage image boundary padding

Particle segmentation: dark threshold 15.00, light threshold 15.00

An acceptance domain: left 15, right 1255, top 0, bottom 959

A camera: shutter 8

Obtained 57

Automatic imaging speed: 22frames per second

Flash camera latency 100 milliseconds

Duration of flash: 18.5 milliseconds

Diameter (ESD): minimum 2.00, maximum 1000.00 microns

Before running the sample, the flow cell and objective lens were mounted and field and focus optimized. System certification includes running a particle size standard in blank water and multiple replicates. A cleaning procedure is performed prior to running the samples to ensure that the particle count is below an acceptable level of typically 1000 particles/mL between samples, or less than 5% sample particles/mL between replicates. This conventional cleaning procedure used water (millipore direct-Q type1,0.22M filtration, 18.2M) between cleaners and as a final rinse before determining count levels. Once the particle technology reaches an acceptable level of particles per mL, the sample is carefully pipetted into the sample tip and loaded into the stream before starting the sample analysis.

The quality of the runs was determined during and immediately after each run, using a series of diagnostic tools in visual spray sheet including the use of x-y capture maps of various particle characteristics (e.g. size, roundness, length, aspect ratio) (to visualize flow regime dynamics), aspect ratio maps for the diameter size (to identify blocking particles), image detection during the run and image analysis at the end of the run.

Individual particle sizes were determined using a fluid imaging technique software measurement technique known as Equivalent Spherical Diameter (ESD). ESD is the average ferter measurement of particles based on 36 sample measurements (performed every 5 °). The ferlet measurement is the perpendicular distance between two parallel tangent lines touching opposite sides of the particle.

The following data table describes the behavior of formulation 3:5 to withstand three months of thermal stress:

(Note: in tables VIII to XIII, the numbers in parentheses (e.g., 3:6) refer to the formulations tested. the designation "C" is a control sample in which 50mg/ml of etanercept prepared according to example 4 is present in a formulation consisting of 25mM sodium phosphate, 1% sucrose, 100mM sodium chloride and 25mM arginine hydrochloride.

TABLE VIII

One, two and three month stability

SEC data

Monomer content

(formulation 3:5 and control)

¹Sec method 1;²SEC method 2;³SEC method 3

(Note: in tables VIII to XIII, the numbers in parentheses (e.g., 3:5) refer to the formulations. the designation "C" is a control sample in which 50mg/ml of etanercept prepared according to example 4 is present in a formulation consisting of 25mM phosphate buffer, 1% sucrose, 100mM sodium chloride and 25mM arginine hydrochloride.)

TABLE IX

One, two and three month stability

SEC data-fragment 3 "

(formulation 3:5 and control)

²SEC method 2;³SEC method 3.

Table X

Stability in two months

SEC data-aggregates "

(formulation 3:5 and control)

²SEC method 2.

TABLE XI

HIC Peak 1 (broken/fragmented form)

One, two and three months

Storage of

(formulation 3:5 and control)

TABLE XII

HIC Peak 2 (etanercept)

One, two and three months

Storage of

(formulation 3:5 and control)

TABLE XIII

HIC Peak 3 (misfolded/aggregated material)

One, two and three months

Storage of

(formulation 3:5 and control)

The microscopically visible particles of formulation 3:5 were evaluated using the FlowCam flow imaging system. These instruments are designed to measure the level of microscopic visible particles (SVP). It was measured initially (table XIV) and then after three months at 5 ℃ (table XV) and 25 ℃ (table XVI). Consistent with the low level of SEC and HIC data shown above, which symbolize aggregated or misfolded material in formulation 3:5 after three months of thermal stress, formulation 3:5 exhibited a low level of microscopically visible particles (less than 10000 particles greater than 5 μm in size per mL).

In the data presented in tables XIV to XVI, the controls were the same as those shown in tables VIII to XIII above, i.e., a formulation containing 50mg/ml etanercept prepared as in example 4, was provided in a formulation comprising 25mM phosphate buffer, 1% sucrose, 100mM sodium chloride and 25mM arginine hydrochloride.

TABLE XIV

Initial number of particles of different sizes/mL (before thermal stress) for formulation 3:5 as measured by FlowCam

Preparation 3:5

Comparison product

2-5μm	14000±16000	7400±8000
			5-10μm	3100±3400	1900±1800
10-15μm	500±500	290±260
			15-25μm	180±120	100±80
25-40μm	130±130	50±40
			40-50μm	20±20	10±10
>50μm	10±10	10±20
			>2μm	17000±21000	9700±11000
>5μm	3900±4100	2300±2200

TABLE XV

Formulation 3:5 storage at 5 ℃ for three months at different particle size/mL measured by FlowCam

	Preparation 3:5	Comparison product
			2-5μm	38000±18000	7900±4200
5-10μm	8000±3300	1600±700
			10-15μm	820±270	150±30
15-25μm	200±30	40±30
			25-40μm	110±40	10±10
40-50μm	0±0	0±0
			>50μm	10±10	0±0
>2μm	47000±22000	9600±4900
			>5μm	9100±3600	1800±700

TABLE XVI

Formulation 3:5 storage at 25 ℃ for three months at different particle size/mL measured by FlowCam

	Preparation 3:5	Comparison product
			2-5μm	16000±7000	15000±10000
5-10μm	4300±1500	3200±2000
			10-15μm	490±150	310±190
15-25μm	190±60	170±140
			25-40μm	60±40	50±40
40-50μm	10±10	10±10
			>50μm	20±10	10±10
>2μm	22000±8600	19000±13000
			>5μm	5000±1700	3800±2300

The data presented in tables VIII to XVI above show that the amino acid stabilized formulations according to the invention are able to achieve storage stability comparable or superior to the comparative dosage forms comprising arginine as stabilizer.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. An arginine-free aqueous pharmaceutical composition comprising etanercept and a stabilizer, wherein the stabilizer is 100-120mM glutamic acid and wherein the composition has a pH of 6-6.6.

2. The aqueous pharmaceutical composition of claim 1, further comprising one or more additional components selected from the group consisting of buffers, tonicity modifying agents and excipients.

3. The aqueous pharmaceutical composition of claim 2, wherein the formulation consists of: 25 to 75mg/ml etanercept; 100-120mM glutamic acid; less than 6 wt% sucrose; 0 to 100mM NaCl; 1 to 30mM sodium phosphate buffer; optionally a surfactant.

4. The aqueous pharmaceutical composition of claim 1, consisting of: 50mg/ml etanercept; 100-120mM glutamic acid; 0 to 3% sucrose; 1 to 30mM phosphate buffer, and 0 to 100mM NaCl; optionally a surfactant.

5. The aqueous pharmaceutical composition of claim 1, consisting of: 50mg/ml etanercept; 100-120mM glutamic acid; less than 4 wt% sucrose, and 10-30mM phosphate; optionally a surfactant; and has a pH of 6.3 to 6.5.

6. The aqueous pharmaceutical composition of claim 1, consisting of: 50mg/ml etanercept; 100mM glutamic acid; less than 2 wt% sucrose, 100mM NaCl; 10-30mM phosphate; optionally a surfactant; and has a pH of 6.3 to 6.5.