Reducing polysorbate degradation in protein formulations with low amounts of citric acid

Abstract

The present invention relates to methods for reducing polysorbate degradation with low amounts of citric acid in protein formulations, and to protein formulations with low amounts of citric acid that have reduced polysorbate degradation.

Description

[Technical field]

[0001] The present invention relates to methods for reducing polysorbate degradation with low amounts of citric acid in protein formulations, and to protein formulations with low amounts of citric acid that have reduced polysorbate degradation. [Background technology]

[0002] In the past 25 years, antibodies have gained great significance in the pharmaceutical industry. Strickley et al., “A review of Formulations of Commercially Available Antibodies”, J Pharm Sci. 2021; Jul; 110(7): 2590-2608 provides a review of 126 commercially available antibodies approved globally from 1986 to 2021, highlighting their importance for pharmaceutical protein formulations.

[0003] Polysorbates are surfactants that are added to pharmaceutical protein formulations to minimize interfacial stress, which can lead to protein aggregation or precipitation.Therefore, polysorbates are important stabilizers in pharmaceutical protein formulations.The most common polysorbates used in pharmaceutical protein formulations are polysorbate 20 (PS20) and polysorbate 80 (PS80), which consist of a core of dehydrated sorbitol, a mixture of sorbitan and isosorbide, which are polyethoxylated and esterified with fatty acids, mainly lauric acid in PS20 and oleic acid in PS80.

[0004] However, polysorbates such as PS20 and PS80 are susceptible to degradation in the drug substance (DS) or drug product (DP) during storage. Degradation of polysorbates can have a negative impact on product quality in terms of (a) generation of visible and / or subvisible particles due to insoluble polysorbate degradation products, (b) adverse effects on protein quality due to polysorbate degradation such as protein oxidation by lauric acid or peroxides resulting in induced protein aggregation, (c) reduced concentrations of surfactants that may lead to insufficient protection of proteins especially against interfacial stresses, and (d) potential differences in product safety considerations due to, for example, degraded polysorbates.

[0005] Besides isolation from natural sources, therapeutic proteins are mainly produced using mammalian or microbial host cell cultures, in which the host cells express the target protein. In addition to the active protein, the host cells also produce host cell proteins (HCPs), which are partly contained in the host cell culture supernatant, as described, for example, in Jahn, M. et al., PHARMACEUTICAL RESEARCH 2020, 37, 118, or Champion, K., et al., BIOPROCESS INTERNATIONAL, 2005, 52-57. The downstream purification process typically includes various chromatographic and (ultra)filtration steps to capture and polish the therapeutic protein, and the purification process should be able to deplete most of the HCPs, at least to the extent that the impact on patient safety can be excluded. However, due to the natural principles of the separation and purification methods, which are based on equilibrium, 100% depletion of HCPs is not possible. This means that a certain amount of HCPs is present in the bulk drug substance (DS) and therefore also in the DP.

[0006] One underlying cause of polysorbate degradation was found to be degradation catalyzed by lipases present in HCPs. For example, Hall et al., “Polysorbates 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal antibody formulations”. J Pharm Sci. 2016;105:1633-42 reported that the residual catalytic activity of lipase as part of the HCP contained in the final DP formulation may result in polysorbate degradation by hydrolysis of fatty acid ester bonds.

[0007] Therefore, residual lipolytic activity in the DS and DP exerted by lipases present in the HCPs due to suboptimal depletion during the downstream purification process of the target protein may lead to hydrolysis of the polysorbate surfactant, resulting in a reduced quality of the final therapeutic drug product.

[0008] Potential options to resolve the issue of polysorbate degradation due to residual lipolytic activity are: 1) improving the downstream purification process to efficiently remove lipolytic enzymes, 2) generating knockout cell lines for expression of active proteins, where the cells lack expression of lipolytic enzymes, and 3) accepting some degree of polysorbate degradation and limiting the shelf life of the DP accordingly.

[0009] However, these potential options have several drawbacks in the sense that 1) additional efforts, time and costs for development work are required, resulting in more complex, time-consuming and more expensive downstream processes; 2) identification of the lipases involved in polysorbate degradation in complex and time-consuming proteomic studies is required, resulting in additional development work to generate knock-out cell lines and potentially reduced viability and efficiency of the resulting cells; and 3) skepticism from health authorities regarding reduced product quality with potential rejection of the submission may be expected or may require the implementation of a complex supply chain for DPs with limited shelf life.

[0010] This ensures that polysorbate degradation in protein formulations is reduced without the need for costly and time consuming measures or accepting a compromised protein formulation as described above.

[0011] Therefore, there is a need to provide a method to easily and effectively reduce polysorbate degradation in protein formulations and to provide protein formulations with reduced polysorbate degradation.

[0012] The inventors have surprisingly found that the use of citric acid or a salt thereof in a particular amount can reduce polysorbate degradation in protein formulations.The above-mentioned needs are therefore met by the subject matter of the present invention. Summary of the Invention

[0013] In one aspect, the invention provides a method for reducing polysorbate degradation in a protein formulation comprising a protein that does not have lipolytic activity and a polysorbate, the method comprising adding citric acid or a salt thereof to the protein formulation at a concentration of at least about 0.01 mM.

[0014] In a further aspect, the invention provides a use of citric acid or a salt thereof to reduce polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and polysorbate, wherein the citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.01 mM.

[0015] In a further aspect, the present invention provides a protein formulation having reduced polysorbate degradation, the protein formulation comprising a protein having no lipolytic activity, a polysorbate, and citric acid or a salt thereof at a concentration of about 0.01 mM to about 9 mM or about 0.01 mM to about 0.9 mM. [Brief description of the drawings]

[0016] [Figure 1] PS80 content in protein formulations with different citrate concentrations after 2 and 4 weeks of incubation at 40° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] definition In order that the present invention may be readily understood, definitions of some of the terms used in the course of the present invention are provided below.

[0018] The term "protein" refers to a macromolecule composed of a sequence of amino acids. Proteins can be naturally occurring proteins, i.e., proteins produced by naturally occurring and non-recombinant cells, or can be produced by genetically engineered or recombinant cells, and can include molecules having the amino acid sequence of a naturally occurring protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the naturally occurring sequence, or molecules having the amino acid sequence of a protein unrelated to a naturally occurring protein. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acid polymer. Proteins can include, for example, antibodies, structural proteins, enzymes, membrane proteins, membrane-associated proteins, and / or transmembrane proteins, transporters, receptors, signaling proteins, and the like. Proteins also encompass modified proteins, such as, for example, conjugated to one or more non-peptide substances, such as drugs, targeting moieties, tags, e.g., visualization tags, and the like. Proteins of the invention can be therapeutic proteins, for example, used in the diagnosis, treatment, and / or prevention of a disease or disorder. In some embodiments, the polysorbates described herein can improve the stability of proteins in pharmaceutical formulations.

[0019] As used herein, the term "antibody" refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antibody fragment (antigen-binding portion) thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibodies of the present invention include monoclonal antibodies (including full-length monoclonal antibodies) and polyclonal antibodies, whole antibodies, antibody-drug conjugates, chimeric antibodies, humanized antibodies, human antibodies or hybrid antibodies with dual or multiple antigen or epitope specificity, hybrid fragments of any immunoglobulin, or hybrid fragments of any natural, synthetic, or engineered protein that acts like an antibody by binding to a specific antigen to form a complex, and antibody fragments and partial fragments, such as Fab, Fab', F(ab')2, fragments, and the like.

[0020] As used herein, the term "comprising" is intended to be interpreted as embracing both "including" and "consisting of," both meanings of which are specifically contemplated and therefore individually disclosed in embodiments according to the invention. "Comprising," in the sense of "including," as well as "having" or "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0021] As used herein, the articles "a" and "an" preceding an element or component are intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of the element or component. Thus, "a" or "an" shall be read to include one or at least one, and the singular form of an element or component also includes the plural, unless the numerical value is clearly meant to be singular. For example, a protein formulation according to the present invention that includes a protein that does not have lipolytic activity does not mean that the protein is necessarily the only protein included in the protein formulation, but it is understood that additional proteins different from the protein that does not have lipolytic activity, e.g., proteins that have lipolytic activity, may also be included in the protein formulation. Thus, as used herein, the term "protein that does not have lipolytic activity" includes one or more "protein(s) that do not have lipolytic activity", e.g., one, two, three, or four proteins that do not have lipolytic activity, preferably one or two, more preferably one.

[0022] As used herein, the term "about" to modify the amount of a substance, ingredient, component, or parameter used refers to variations in the numerical amount that may occur, for example, through typical measuring and handling procedures, such as liquid handling procedures used to make concentrates or solutions. Additionally, variations may result from inadvertent errors in measuring procedures, differences in manufacture, source, or purity of ingredients used to practice the invention. In one embodiment, the term "about" means within 10% of the reported numerical value. In a more specific embodiment, the term "about" means within 5% of the reported numerical value.

[0023] Although use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0024] Use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular terms listed are representative examples and embodiments of the present disclosure that are not intended to be limited to the specific examples referenced or cited, unless expressly stated otherwise.

[0025] In the context of the present invention, "host cell proteins (HCPs)" shall be understood to not refer to and include "proteins without lipolytic activity". Rather, "host cell proteins (HCPs)" are low-level, process-related, undesirable protein impurities derived from the host organism during the production of a "protein without lipolytic activity", such as a therapeutic protein. In the context of the present invention, a "protein without lipolytic activity" may also be referred to as a "target protein", where the target protein is a protein without lipolytic activity. Thus, the terms "target protein" and "protein without lipolytic activity" are used interchangeably herein, where the target protein is without lipolytic activity.

[0026] It is to be understood that all embodiments, advantages, and discoveries described in the context of one particular aspect of the invention, e.g., the method of the invention, should be equally applicable to all other aspects of the invention, such as the use of the invention and the protein formulation of the invention. For example, the surprising discoveries described in the context of the method of the invention equally apply to the use of the invention and the protein formulation of the invention.

[0027] As already outlined above, the present invention relates to a method for reducing polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and polysorbate, the method comprising adding citric acid or a salt thereof to the protein formulation at a concentration of at least about 0.01 mM.

[0028] The present inventors have surprisingly found that by using citric acid or its salt in a specific amount, polysorbate degradation in protein formulations can be reduced.The method according to the present invention thus provides an excellent method for reducing polysorbate degradation in protein formulations.By using the method according to the present invention, the cumbersome root cause investigation of polysorbate degradation can be avoided by directly reducing polysorbate degradation in protein formulations without the need for time-consuming and expensive development of cell lines or purification processes.Therefore, the method according to the present invention provides a convenient and efficient method for reducing polysorbate degradation in protein formulations.

[0029] The present invention further relates to the use of citric acid or a salt thereof to reduce polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and polysorbate, wherein the citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.01 mM.

[0030] The present invention also relates to a protein formulation having reduced polysorbate degradation, the protein formulation comprising a protein having no lipolytic activity, a polysorbate, and citric acid or a salt thereof at a concentration of about 0.01 mM to about 9 mM or about 0.01 mM to about 0.9 mM.

[0031] In one embodiment, the presence of citric acid in a protein formulation comprising a protein that does not have lipolytic activity and a polysorbate reduces the degradation of the polysorbate by at least 5%, preferably at least 10%, more preferably at least 20% after 2 weeks of storage at 40° C., and / or by at least 5%, preferably at least 10%, more preferably at least 25%, even more preferably at least 50% after 4 weeks of storage at 40° C., when compared to a similar formulation without citric acid.

[0032] Specifically, the inventors have surprisingly found that already low concentrations of citric acid or its salts effectively reduce polysorbate degradation in protein formulations.

[0033] According to the present invention, citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.01 mM. Preferably, citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.05 mM. Preferably, citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.1 mM.

[0034] In one embodiment, citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.01 mM, preferably at least about 0.02 mM, preferably at least about 0.03 mM, preferably at least about 0.04 mM, preferably at least about 0.05 mM, preferably at least about 0.06 mM, preferably at least about 0.07 mM, preferably at least about 0.08 mM, preferably at least about 0.09 mM.

[0035] In one embodiment, citric acid or a salt thereof is added to the protein formulation at a concentration of up to about 40 mM, preferably up to about 35 mM, preferably up to about 30 mM, preferably up to about 25 mM, preferably up to about 20 mM, preferably up to about 15 mM, preferably up to about 12.5 mM, preferably up to about 10 mM, preferably up to about 9 mM, preferably up to about 8 mM, preferably up to about 7 mM, preferably up to about 6 mM, preferably up to about 5 mM, preferably up to about 4 mM, preferably up to about 3 mM, preferably up to about 2 mM, preferably up to about 1 mM, or up to about 0.9 mM.

[0036] Any of the lower limits can be combined with any of the upper limits.

[0037] In one embodiment, citric acid or a salt thereof is added to the protein formulation at a concentration of about 0.01 mM to about 40 mM, preferably about 0.05 mM to about 40 mM, for example, about 0.1 mM to about 40 mM. Accordingly, suitable concentrations of citric acid or a salt thereof include about 0.06 mM to about 35 mM, about 0.07 mM to about 30 mM, about 0.08 mM to about 25 mM, or about 0.1 mM to about 20 mM.

[0038] Preferably, citric acid or a salt thereof is added to the protein formulation at a concentration of about 0.1 mM to about 20 mM, for example, about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In one embodiment, citric acid is added to the protein formulation at a concentration of about 0.1 mM to about 20 mM. In one embodiment, citric acid or a salt thereof is added to the protein formulation at a concentration of about 0.01 mM to about 9 mM. In one embodiment, citric acid or a salt thereof is added to the protein formulation at a concentration of about 0.01 mM to about 0.9 mM.

[0039] Therefore, preferred concentrations of citric acid or a salt thereof in the protein formulation include about 0.1 mM to about 20 mM, about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, and about 0.1 mM to about 0.9 mM.

[0040] For the purposes of the present invention, "citric acid or its salts" shall be understood to refer to anhydrous forms of citric acid and salts of citric acid, as well as their hydrated forms, such as monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate. The term "citric acid or its salts" shall be understood to refer to only one type of citric acid or its salts, as well as combinations of different types of citric acid (i.e., different hydrated forms) and / or different salts thereof. The term "citric acid or its salts" shall further be understood to refer to citric acid, one or more salts of citric acid, and any mixtures thereof.

[0041] Suitable salts of citric acid include monobasic, dibasic, and tribasic salts of citric acid, and mixtures thereof, optionally in combination with citric acid. Suitable cations for the citrate salts are sodium, potassium, calcium, magnesium, aluminum, N + R1R2R3R4, wherein R1, R2, R3, and R4 are the same or different and independently represent H, C 1~4 Alkyl, (CH2) n OH; n is 2, 3, or 4.

[0042] In one embodiment, the citric acid or salt thereof is selected from the group consisting of citric acid, sodium citrate, potassium citrate, calcium citrate, and combinations thereof. Suitable sodium citrates include monosodium citrate, disodium citrate, trisodium citrate, and combinations thereof. Suitable potassium citrates include monopotassium citrate, dipotassium citrate, tripotassium citrate, and combinations thereof. Suitable calcium citrates include tricalcium dicitrate.

[0043] Preferably, the citric acid or a salt thereof is selected from citric acid anhydrate, citric acid monohydrate, trisodium citrate dihydrate, and combinations thereof, more preferably from citric acid. Thus, in one embodiment, the present invention relates to a method for reducing polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and a polysorbate, the method comprising adding citric acid to the protein formulation at a concentration as defined herein, and in all its embodiments, for example at a concentration of at least about 0.01 mM. In one embodiment, citric acid is added to the protein formulation at a concentration of about 0.01 mM to about 40 mM, preferably about 0.05 mM to about 40 mM, for example about 0.1 mM to about 40 mM. In one embodiment, citric acid is added to the protein formulation at a concentration of about 0.06 mM to about 35 mM, about 0.07 mM to about 30 mM, about 0.08 mM to about 25 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to about 7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, or about 0.01 mM to about 0.9 mM.

[0044] In one preferred embodiment, the protein formulation is a liquid protein formulation. In such a preferred embodiment, the method according to the present invention relates to reducing polysorbate degradation in a liquid protein formulation by adding citric acid or a salt thereof to the protein formulation at a concentration of at least about 0.01 mM. Similarly, in such a preferred embodiment, citric acid or a salt thereof is used to reduce polysorbate degradation in a liquid protein formulation by adding citric acid or a salt thereof to the protein formulation at a concentration of at least about 0.01 mM. Thus, in such a preferred embodiment, the protein formulation according to the present invention is a liquid protein formulation comprising citric acid or a salt thereof at a concentration of about 0.01 mM to about 9 mM, or about 0.01 mM to about 0.9 mM.

[0045] In one embodiment, the polysorbate comprises polysorbate-20, polysorbate-80, or a mixture thereof. In one embodiment, the polysorbate is polysorbate-20, polysorbate-80, or a mixture thereof. In one embodiment, the polysorbate is polysorbate-20. In one embodiment, the polysorbate is polysorbate-80. Thus, in one embodiment, reducing polysorbate degradation comprises reducing degradation of polysorbate-20 and / or polysorbate-80.

[0046] The amount of polysorbate in protein formulations can be measured using fluorescent micelle assay (FMA), such as high performance liquid chromatography fluorescent micelle assay (HPLC-FMA).Generally, any FMA that is known in the art and is coupled with HPLC suitable for detecting polysorbates such as PS20 and PS80 can be used.HPLC-FMA can be carried out using, for example, a high performance liquid chromatography (HPLC) system (Waters Alliance e2695) equipped with an isocratic pump, an autosampler, a braided reactor coil (1mL; Supelco #57410-U), and a fluorescence detector (Waters 2475 FLR detector).

[0047] Proteins, especially therapeutic proteins, are mainly produced using mammalian or microbial cell cultures, where the cells express target proteins. In addition to proteins, the cells also produce host cell proteins (HCPs), the part of which is contained in cell culture supernatant. Downstream purification processes typically cannot deplete 100% of HCPs. This indicates that a small percentage of HCPs is present in protein preparations. It is known that one underlying cause of polysorbate degradation is the degradation catalyzed by lipases present in HCPs.

[0048] In one embodiment, polysorbate degradation can be caused by lipolytic activity. In one embodiment, the lipolytic activity can be exerted by a lipase. In one embodiment, the lipase can be derived from a host cell protein (HCP).

[0049] In one embodiment, the protein formulation comprises a host cell protein (HCP). In one embodiment, the lipolytic activity is exerted by a host cell protein (HCP). In one embodiment, the lipolytic activity is exerted by a lipase derived from a host cell protein (HCP).

[0050] Lipolytic activity in the sense of the present invention is undesirable residual lipolytic activity.

[0051] The amount of HCP in a protein formulation is determined, for example, according to USP 39 NF 34 - General Chapter <1132> It can be determined by enzyme-linked immunosorbent assay (ELISA), known to those skilled in the art, according to "Residual Host Cell Protein Measurement in Biopharmaceuticals."

[0052] In one embodiment, the protein formulation contains HCPs as trace impurities, e.g., 100 ppm or less of host cell protein, based on the weight of the protein formulation. In one embodiment, the protein formulation contains 95 ppm or less of host cell protein (HCP), e.g., 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, or 60 ppm or less, based on the weight of the protein formulation. In one embodiment, the protein formulation contains 1 to 100 ppm of host cell protein (HCP), based on the weight of the protein formulation.

[0053] Preferably, the protein formulation comprises 100 ppm or less of host cell protein (HCP) based on the weight of protein without lipolytic activity. When two or more proteins without lipolytic activity are present in the protein formulation, the amount of HCP shall be understood to be based on the combined weight of proteins without lipolytic activity present in the protein formulation. In one embodiment, the protein formulation comprises 95 ppm or less of host cell protein (HCP), for example 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, or 60 ppm or less, based on the weight of protein without lipolytic activity. In one embodiment, the protein formulation comprises 1 to 100 ppm of host cell protein (HCP) based on the weight of protein without lipolytic activity.

[0054] Preferably, the protein formulation comprises 100 ppm or less of host cell protein (HCP) based on the weight of the target protein. When two or more target proteins are present in the protein formulation, the amount of HCP is based on the combined weight of the target proteins present in the protein formulation. In one embodiment, the protein formulation comprises 95 ppm or less of host cell protein (HCP) based on the total weight of the target proteins, for example, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, or 60 ppm or less of host cell protein (HCP). In one embodiment, the protein formulation comprises 1 to 100 ppm of host cell protein (HCP) based on the total weight of the target proteins.

[0055] In one embodiment, the protein formulation comprises at least 1 μg / ml of protein with no lipolytic activity. In one embodiment, the protein formulation comprises at least 10 μg / ml of protein with no lipolytic activity. In one embodiment, the protein formulation comprises at least 100 μg / ml of protein with no lipolytic activity. In one embodiment, the protein formulation comprises at least 500 μg / ml of protein with no lipolytic activity. In one embodiment, the protein formulation comprises at least 1000 μg / ml of protein with no lipolytic activity.

[0056] In one embodiment, the protein formulation comprises at least 1 μg / ml of target protein. In one embodiment, the protein formulation comprises at least 10 μg / ml of target protein. In one embodiment, the protein formulation comprises at least 100 μg / ml of target protein. In one embodiment, the protein formulation comprises at least 500 μg / ml of target protein. In one embodiment, the protein formulation comprises at least 1000 μg / ml of target protein.

[0057] The protein formulation preferably has HCPs as the sole source of lipolytic activity.

[0058] According to the present invention, the protein formulation comprises a protein that does not have lipolytic activity. Thus, the protein is not a lipase. Thus, the protein formulation comprises a protein that does not have lipolytic activity and a polysorbate. Thus, the protein formulation comprises a protein that is not a lipase and a polysorbate. In one embodiment, the protein is a therapeutic protein. In such an embodiment, the therapeutic protein does not have lipolytic activity, i.e., is not a lipase. Preferably, the therapeutic protein is an antibody. In one embodiment, the antibody is a monoclonal antibody. Preferably, the antibody is an anti-CD20 antibody. In one embodiment, the antibody is rituximab.

[0059] Protein formulations are typically intended to be administered to subjects in need thereof. The administration route of the protein formulation of the present invention is not particularly limited. In one embodiment, the protein formulation is administered to a subject in need thereof. In one embodiment, the protein formulation is for subcutaneous injection. In one embodiment, the protein formulation is a liquid protein formulation for subcutaneous injection. In one embodiment, the liquid protein formulation is administered to a subject in need thereof as a subcutaneous injection.

[0060] Protein formulations that are typically buffered with high concentrations of citrate have the deficiency of causing injection site pain when administered as subcutaneous (sc) injections. The present invention circumvents this by using low concentrations (e.g., 0.1 mM to 20 mM) of citric acid or its salts, such that injection site pain is unlikely to be observed after sc administration, yet excellent reduction in polysorbate degradation is surprisingly achieved. Thus, when a protein formulation is intended for subcutaneous injection, citric acid or its salts are used in the present invention at low concentrations in the protein formulation to reduce polysorbate degradation, and optionally, an additional buffering agent for sc injections can be included in the protein formulation.

[0061] Thus, in one embodiment, the protein formulation further comprises a buffering agent, a salt, a stabilizer, or a combination thereof.

[0062] Suitable buffering agents include histidine, histidine hydrochloride, sodium succinate, and combinations thereof.Suitable stabilizing agents include trehalose, sucrose, mannitol, sorbitol, methionine, histidine, glycine, arginine, and combinations thereof.

[0063] The above embodiments and items are merely examples of multiple possibilities and should not be understood as forming limitations of these features and configurations. Any possible combination and configuration of the described features can be selected according to the scope of the present invention. All embodiments and preferred embodiments described herein in relation to one particular aspect of the present invention shall apply to all other aspects of the present invention, such as the use or method according to the present invention.

[0064] This invention is further illustrated by the following examples, which should not be construed as limiting the present invention. EXAMPLES

[0065] Below, the effect of different citrate concentrations on polysorbate degradation in protein formulations was investigated.

[0066] material Citric acid monohydrate (ACS reagent, ≥99.0%), sucrose (≥99.5%), and L-histidine (L-His, ≥98.5%) were obtained from Sigma-Aldrich (now Merck KGaA). L-histidine monohydrochloride monohydrate (L-His HCl, 99%) was obtained from Alfa Aesar. Polysorbate 80 (PS80, NF, Multi-Compendial) was obtained from JT Baker.

[0067] Downstream purification sample of Rituximab Protein A at 18 g / L: eluate was obtained as follows. Cell culture material was obtained from a CHO SSI clone expressing rituximab. The cell culture was clarified using a charged depth filter. The product was captured on next generation PrA resin. Protein A eluate material was brought to pH 7.5, filtered through a 0.2 μm membrane, and stored at -20°C. The material was used without further purification.

[0068] General Procedure for Protein Drug Sample Preparation A formulation buffer was prepared containing 25 mM L-His / L-His HCl at pH 6.0, 600 mM sucrose, and 0.05% (w / v) PS80. The solution was filtered through a 0.22 μm PVDF Durapore® sterile filter.

[0069] A second solution of 40 mM citric acid at pH 6.0 was prepared and filtered through a 0.22 μm PVDF Durapore® sterile filter. In addition to the 40 mM citric acid stock solution, dilutions of the 40 mM citric acid solution were prepared with water to target concentrations of 20 mM, 2 mM, and 0.2 mM.

[0070] Five protein formulation samples were each prepared by spiking 320 μL of downstream purification sample (Rituximab Protein A eluate, 18 g / L) into 1680 μL of the above-prepared formulation buffer in five different particle-free Nalgene bottles. Then, 2000 μL of each of the above-prepared citrate solutions (40 mM, 20 mM, 2 mM, and 0.2 mM) was added to four of the five bottles. The final protein formulations with five different citrate concentrations (20 mM, 10 mM, 1 mM, 0.1 mM, and 0 mM) were aliquoted into 2 mL HPLC vials.

[0071] The final composition of the five protein formulation samples was 10.5 mM L-His / L-His HCl, 250 mM sucrose, 0.02% (w / v) PS80, 1.4 mg / mL Rituximab Protein A eluate at a pH of 6 and final citrate concentrations of 20 mM, 10 mM, 1 mM, 0.1 mM, and 0 mM, respectively.

[0072] Analysis of polysorbate degradation in protein formulation samples 1.2 mL of each protein formulation sample was aliquoted into particle-free Nalgene tubes and placed in a 40° C. stability chamber. Samples were analyzed after 2 and 4 weeks of incubation using HPLC-FMA against a standard curve of intact PS80 in water.

[0073] HPLC-FMA measurements were performed using a high performance liquid chromatography (HPLC) system (Waters Alliance e2695) equipped with an isocratic pump, a fluorescence detector (Waters 2475 FLR detector), an autosampler, and a braided reaction coil 1 mL (Supelco #57410-U). Samples were injected into a mobile phase containing 5 μM N-phenyl-1-naptylamine (NPN), 15 ppm (w / v) Brij® 35 dissolved in a solution of 150 mM NaCl, 50 mM TRIS, 5% (v / v) acetonitrile at pH 8. Fluorescence was measured as λ ex = 350 nm and λ em = 420 nm. Peak integration and analysis were performed using an Empower 3 Chromatographic Data System (CDS).

[0074] The results of protein formulations with five different citrate concentrations (20 mM, 10 mM, 1 mM, 0.1 mM, and 0 mM) after storage at 40° C. for 2 and 4 weeks are shown in FIG.

[0075] As can be seen in Figure 1, in protein formulation samples without citrate (0 mM), PS80 concentrations were significantly lower after 2 and 4 weeks of incubation at 40°C compared to protein formulation samples with 0.1 mM, 1 mM, 10 mM, and 20 mM citrate, demonstrating that small amounts of citrate are unexpectedly effective in reducing polysorbate degradation.

Claims

1. 1. A method for reducing polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and polysorbate, the method comprising adding citric acid or a salt thereof to the protein formulation at a concentration of about 0.01 mM to about 9 mM.

2. 2. The method of claim 1, wherein the polysorbate comprises polysorbate-20, polysorbate-80, or a mixture thereof.

3. The method of claim 1 or 2, wherein the protein that does not have lipolytic activity is a therapeutic protein.

4. The method according to any one of claims 1 to 3, wherein the protein formulation is for subcutaneous injection.

5. 1. Use of citric acid or a salt thereof to reduce polysorbate degradation in a protein formulation comprising a protein having no lipolytic activity and polysorbate, wherein the citric acid or a salt thereof is added to the protein formulation at a concentration of at least about 0.01 mM.

6. The use according to claim 5, wherein citric acid or a salt thereof is added to the protein formulation at a concentration of about 0.01 mM to about 40 mM.

7. The use according to claim 5 or 6, wherein the polysorbate comprises polysorbate-20, polysorbate-80, or a mixture thereof.

8. The use according to any one of claims 5 to 7, wherein said protein having no lipolytic activity is a therapeutic protein.

9. The use according to any one of claims 5 to 8, wherein the protein formulation is for subcutaneous injection.

10. 1. A protein formulation having reduced polysorbate degradation, comprising: a protein having no lipolytic activity; a polysorbate; and citric acid or a salt thereof at a concentration of about 0.01 mM to about 0.9 mM.

11. The protein formulation of claim 10, wherein the polysorbate comprises polysorbate-20, polysorbate-80, or a mixture thereof.

12. The protein formulation of claim 10 or 11, wherein the protein that does not have lipolytic activity is a therapeutic protein.

13. The protein formulation of any one of claims 10 to 12, wherein the protein formulation is for subcutaneous injection.

14. 14. The method, use or protein preparation of any one of claims 1 to 13, wherein said protein preparation has host cell proteins (HCPs) as the only source of lipolytic activity.

15. 15. The method, use or protein formulation of claim 14, wherein the protein formulation comprises HCPs in an amount of less than or equal to 100 ppm, based on the weight of the protein formulation.

16. 16. The method, use or protein formulation of any one of claims 1 to 15, wherein said protein formulation comprises said protein having no lipolytic activity of at least 1 μg / ml.

Images