Formulation and drug product with reduced particle formation

Abstract

To provide formulations and drug products formed by methods of reducing particle formation.SOLUTION: Biopharmaceutical compositions and drug products disclosed herein exhibit reduced amounts of subvisible particle formation. Compositions and drug products disclosed herein comprise a protein and a surfactant or a stabilizer including high percentage amounts (e.g., at least 97%) of a long-chain fatty acid ester. Also disclosed herein are methods of preparing and storing such compositions and drug products.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 560,365, filed September 19, 2017, the entirety of which is incorporated herein by reference.

[0002] The present disclosure relates to biopharmaceutical formulations and drug products that exhibit reduced amounts of subvisible particle formation upon storage, and methods for their preparation and storage. In particular, the disclosure relates to formulations and drug products that include a protein and a surfactant or stabilizer that includes a high percentage amount of long chain monounsaturated fatty acid esters, and methods for their preparation and storage. [Background technology]

[0003] Polysorbates are traditionally used in pharmaceutical products containing proteins as active ingredients (also called "DP") to protect the protein from surface-induced (air / liquid or solid / liquid) instability during manufacturing, storage, handling, and administration. It has been found that lipases co-purified with the protein of interest (POI) can hydrolyze the fatty acid esters of polysorbates to free fatty acids when included in a formulation with polysorbates. Non-enzymatic hydrolysis of fatty acid esters of polysorbates can also occur at a slower rate in the formulation. Free fatty acids resulting from hydrolysis can aggregate and form particulates over time in pharmaceutical products containing such formulations. Particulates (both visible and subvisible) can affect product stability, shorten the shelf life of pharmaceutical products due to failure to meet official particulate matter specifications (e.g., US FDA specifications), and can have clinical consequences, e.g., immunogenic reactions upon administration. Summary of the Invention [Problem to be solved by the invention]

[0004] Hydrophobic interaction chromatography (HIC) or affinity chromatography of POI can reduce or remove lipase co-purified with POI, thus reducing the hydrolysis of fatty acid esters. However, adding a HIC or affinity chromatography step to the preparation of protein formulations requires additional equipment, materials, preparation, protocols, and protocol validation, for example, to the manufacturing process, resulting in additional time and cost. Furthermore, HIC or affinity chromatography steps to remove enzymes do not help prevent non-enzymatic hydrolysis of fatty acid esters in the formulation. A method to reduce or prevent the formation of subvisible and visible particulates in protein compositions, for example, without using additional HIC or affinity chromatography steps, is therefore desired. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 is a diagram of the chemical structure of polyoxyethylene (20) sorbitan monooleate, the major fatty acid ester of polysorbate 80. [Diagram 2] 1 is a graph showing the number of subvisible fine particles (≧10 μm) measured by a membrane microscope particle concentration method in protein pharmaceuticals containing various polysorbate 80. [Diagram 3] 1 is a graph showing the number of subvisible fine particles (≧10 μm) measured by microflow imaging (MFI) in protein pharmaceuticals containing various polysorbate 80s. [Figure 4] 1 is a graph showing the measured concentrations of free fatty acids in protein pharmaceuticals containing various types of polysorbate 80. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the embodiments of the present disclosure. Any feature (e.g., composition, formulation, method, etc.) of an embodiment or example described herein may be combined with any other embodiment or example and are encompassed by the present disclosure.

[0007] As used herein, the terms "comprise," "comprising," or any other variations thereof, are intended to cover a non-exclusive inclusion, and a process, method, article, or apparatus that includes a list of elements may include not only those elements, but also other elements not expressly listed or inherent in such process, method, article, or apparatus. The term "exemplary" is used in the sense of "example," not "ideal." With respect to the terms "for example" and "such as," and their grammatical equivalents, unless otherwise specified, it is understood that they are followed by the phrase "and not limited to."

[0008] As used herein, the term "about" is intended to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about" unless otherwise noted, whether or not the term is explicitly used. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0009] All numerical values ​​disclosed herein (including all disclosed values, limits, and ranges) may have a variation of + / - 10% from the disclosed numerical value (unless a different variation is specified). Further, in the claims, any value, limit, and / or range means that value, limit, and / or range + / - 10%.

[0010] The present disclosure is not limited to the specific compositions, formulations, manufacturers of materials, pharmaceuticals, methods, or experimental conditions disclosed herein, as many variations are possible within the expertise of one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0011] Unless otherwise defined, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are described herein. All publications mentioned are incorporated herein by reference.

[0012] As used herein, the term "protein" refers to any amino acid polymer having more than about 20 amino acids covalently linked through amide bonds. A protein comprises one or more amino acid polymer chains, commonly known in the art as "polypeptides." Thus, a polypeptide may be a protein, and a protein may comprise multiple polypeptides to form a single functional biomolecule in a single conformation. Disulfide bridges (e.g., forming cystine between cysteine ​​residues) may be included in some proteins. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, protamines, etc.

[0013] In addition to disulfide bond formation, proteins may undergo other post-translational modifications, including lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., the addition of glycosyl groups at arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., the addition of a phosphate group to serine, threonine, tyrosine, and / or histidine).

[0014] As used herein, the term "protein" includes biopharmaceutical proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Proteins may be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).

[0015] As used herein, the term "antibody" includes immunoglobulins that are composed of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Typically, antibodies of the present disclosure have a molecular weight of more than 100 kDa, e.g., between 130 kDa and 200 kDa, e.g., about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which may be interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

[0016] For example, a class of immunoglobulins called immunoglobulin G (IgG) is commonly found in human serum and contains four peptide chains, two light chains and two heavy chains. Each light chain is linked to one heavy chain via a disulfide bond of cystine, and the two heavy chains are linked to each other via disulfide bonds of two cystine. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass differs in its constant region and, as a result, may have different effector functions.

[0017] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtained, synthetic, or recombinant polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be obtained from complete antibody molecules using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques, including, for example, the manipulation and expression of DNA encoding the variable and optionally constant domains of an antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or may be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, to introduce codons, to create cysteine ​​residues, to modify, add, or delete amino acids, and the like.

[0018] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include, for example, in the CDRs and particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0019] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least a functional portion of the CH2 and CH3 regions of an immunoglobulin. A "functional portion" refers to a CH2 and CH3 region that can bind to an Fc receptor (e.g., FcγR, or FcRn, i.e., neonatal Fc receptor) and / or participate in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that render them incapable of binding to any Fc receptor or activating complement, then the CH2 and CH3 regions are not functional.

[0020] Fc-containing proteins can include modifications of immunoglobulin domains, including those that affect one or more effector functions of the binding protein, such as modifications that affect FcγR binding, FcRn binding, and thus half-life and / or CDC activity, including, but not limited to, the following modifications and combinations thereof, according to the EU numbering of immunoglobulin constant regions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 352, 353, 354, 355, 356, 358, 360 09, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0021] For example, and not by way of limitation, the binding protein may be an Fc-containing protein, may exhibit an increased serum half-life (compared to the same Fc-containing protein without the listed modification(s)), and may have modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at 250 and / or 428, or modifications at 307 or 308 (e.g., 308F, V308F), and 434. In another example, the modifications can include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), or 307 and / or 308 modifications (e.g., 308F or 308P).

[0022] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusiani, etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, eg, is a retinal cell (eg, a PER.C6™ cell) that expresses a viral gene.

[0023] The term "fatty acid ester" refers to any organic compound that includes a fatty acid chain linked to a head group via an ester bond. An ester bond occurs when a hydroxyl group (e.g., an alcohol or a carboxylic acid) is replaced with an alkoxy group. As used herein, the hydroxyl group can be a moiety of a carboxylic acid, more specifically a fatty acid, and / or an alcohol, e.g., glycerol, sorbitol, sorbitan, isosorbide, etc. The alcohol group is generally referred to herein as the head group.

[0024] Examples of fatty acid esters generally include phospholipids, lipids (e.g., whose head group is glycerol, including monoglycerides, diglycerides, and triglycerides), and surfactants and emulsifiers, including, for example, polysorbates, such as the nonionic surfactants polysorbate 20, polysorbate 60, and polysorbate 80. Surfactants and emulsifiers are useful as cosolvents and stabilizers. They function to maintain the dispersion and structural stability of inclusions, such as proteins, by associating with both hydrophilic and lipophilic surfaces. Surfactants are primarily added to protein formulations to enhance the stability of proteins against mechanical stress, such as partial unfolding and self-association induced by air / liquid and solid / liquid interfaces. Without surfactants, proteins can sometimes become structurally unstable in solution and form multimeric aggregates, which can eventually become invisible particles.

[0025] The term "fatty acid" or "fatty acid chain" refers to a carboxylic acid having an aliphatic tail. The aliphatic tail is simply a hydrocarbon chain that contains carbon and hydrogen, and optionally oxygen, sulfur, nitrogen, and / or chlorine substitutions. The aliphatic tail can be saturated (such as in saturated fatty acids), i.e., all carbon-carbon bonds are single bonds (i.e., alkanes). The aliphatic tail can be unsaturated (such as in unsaturated fatty acids), where one or more carbon-carbon bonds are double bonds (alkenes) or triple bonds (alkynes).

[0026] Fatty acids are generally designated as short chain fatty acids having less than 6 carbons in the aliphatic tail, medium chain fatty acids having 6-12 carbons, long chain fatty acids having 13-21 carbons, and very long chain fatty acids having an aliphatic tail of 22 or more carbons. As mentioned above, fatty acids are also classified by the degree of saturation, which correlates to stiffness and melting point. Common fatty acids include caprylic acid (8 carbons:0 double bond, 8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), myristoleic acid (14:1), palmitic acid (16:0), palmitoleic acid (16:1), sapienic acid (16:1), stearic acid (18:0), oleic acid (18:1), elaidic acid (18:1), vaccenic acid (18:1), linoleic acid (18:2), linelaedic acid (18:3), oleic acid (18:4), oleic acid (18:5), oleic acid (18:6), oleic acid (18:7), oleic acid (18:8), oleic acid (18:9), oleic acid (18:1), oleic acid (18:1), oleic acid (18:1), oleic acid (18:1), oleic acid (18:2), oleic acid (18:1 ... Acids that are essential for the synthesis of fatty acids include: alpha-linolenic acid (18:2), alpha-linolenic acid (18:3), arachidic acid (20:0), arachidonic acid (20:4), eicosapentaenoic acid (20:5), behenic acid (22:0), erucic acid (22:1), docosahexaenoic acid (22:6), lignoceric acid (24:0), and cerotic acid (26:0).

[0027] As mentioned above, polysorbates are fatty acid esters useful as nonionic surfactants and protein stabilizers. Polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 are widely used as stabilizers and emulsifiers in the pharmaceutical, cosmetic, and food industries. Polysorbate 20 primarily comprises the monolaurate ester of polyoxyethylene (20) sorbitan. Polysorbate 40 primarily comprises the monopalmitate ester of polyoxyethylene (20) sorbitan. Polysorbate 60 primarily comprises the monostearate ester of polyoxyethylene (20) sorbitan. Polysorbate 80 primarily comprises the monooleate ester of polyoxyethylene (20) sorbitan (shown in Figure 1).

[0028] The quality of commercial grade polysorbates varies depending on the supplier. Polysorbates are therefore often mixtures of various chemicals, consisting mostly of polyoxyethylene (20) sorbitan monoesters (as described above), with occasional isosorbide ester contaminants. They may also contain, for example, polyethylene glycols (PEGs), intermediate structures, and fatty acid reactants. The head group (in this case polyoxyethylene (20) sorbitan) contains sorbitan (a mixture of dehydrated sorbitols, including 1,4-anhydrosorbitol, 1,5-anhydrosorbitol, and 1,4,3,6-dianhydrosorbitol) with three of its alcohol groups substituted to form ether bonds with three polyoxyethylene groups. The fourth alcohol group is substituted with a fatty acid to form a fatty acid ester.

[0029] In some commercial batches of polysorbate, the polysorbate contains monoesters of isosorbide. Isosorbide is a heterocyclic derivative of glucose, which is also prepared by dehydration of sorbitol. It is a diol, i.e., it has two alcohol groups that can participate in the formation of one or two ester bonds. Thus, for example, some lots of polysorbate 80 may contain significant amounts of oleic acid monoesters and diesters of isosorbide.

[0030] In addition to variation in the head group, preparations of polysorbates contain variable amounts of other fatty acid esters. For example, analysis of one particular source of polysorbate 80 showed <5% myristic acid, <16% palmitic acid, >58% oleic acid, <6% stearic acid, and <18% linoleic acid. Analysis of another source of polysorbate 80 showed approximately 70% oleic acid with the remainder being other fatty acid esters and impurities. Analysis of yet another source of polysorbate 80 showed approximately 86-87% oleic acid. Analysis of a more recently developed source of polysorbate 80 showed ≥99% oleic acid.

[0031] Non-ionic surfactants such as polysorbate 20 or polysorbate 80 help stabilize large molecules such as antibodies and other proteins and help prevent the formation of oligomeric complexes and other aggregates. The aggregates can become nanoparticles or sub-visible particles in the 10-100 micron (10-100 μm) range or 2-100 micron (2-100 μm) range, which can interfere with pharmaceutical stability and shelf life and induce immunogenicity. Thus, the stability of protein formulations depends in some cases on the stability of non-ionic surfactant additives. However, as discussed further herein, polysorbate 20 and polysorbate 80 can in some cases contribute to the formation of aggregates, nanoparticles, and sub-visible particles.

[0032] The phrases "subvisible particles" (SVPs) and "subvisible particulates" according to the present disclosure refer to particles that are not visible to the naked eye, particularly in liquids. In other words, a solution or other liquid containing SVPs, but not visible particles, does not appear cloudy. SVPs generally include particles with diameters of 100 microns (100 μm) or less, but in some cases, particles less than 150 microns (150 μm) (Narhi et al., "A critical review of analytical methods for subvisible and visible particles," Curr Pharm Biotechnol 10(4):373-381 (2009)). SVPs may be the result of contamination, protein aggregation, or aggregation of other components of the DP. SVPs may include, among others, silicone oil droplets (oil droplets), free fatty acids (amorphous particles and / or oil droplets), aggregated proteins (amorphous particles), and / or protein / fatty acid complexes (amorphous particles).

[0033] SVPs can be detected by any one or more of a variety of methods. USP standards specify light obscuration and light microscopy protocols. Other methods include microflow image (MFI) analysis, Coulter counting, and submicron particle tracking. Some measurement and characterization methods for SVPs (e.g., light obscuration, flow microscopy, electrical detection zone, and flow cytometry) are discussed, for example, in Narhi et al., “Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy,” J. Pharma. Sci. 104:1899-1908 (2015).

[0034] Light obscuration has been criticized for underestimating protein aggregates and other amorphous structures. Flow imaging, e.g., microflow imaging (MFI) (Brightwell Technologies, Ottawa, Ontario), is a more sensitive method for detecting irregularly shaped, fragile, transparent proteinaceous SVPs and distinguishing these types of particles from silicone microdroplets, air bubbles, and other foreign bodies (Sharma et al., “Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations,” AAPS J. 12(3):455-464(2010)). In general, SVP measurement and characterization by light obscuration analysis is less sensitive than MFI, so the number of particles detected by MFI tends to be higher than the number of particles detected by light obscuration analysis. Briefly, MFI is a flow microscopy method in which successive bright-field images are acquired and analyzed in real time. Image analysis algorithms are applied to the images to identify air bubbles, silicone oil droplets, and proteinaceous aggregates. Volumes from about 250 microliters up to tens of milliliters can be analyzed. Depending on the system used, particles in the range of 2-300 microns (2-300 μm) or 1-70 microns (1-70 μm) can be detected (ibid.).

[0035] The FDA and other government regulatory agencies have set limits on the amount of SVP permitted in parenteral formulations, with the major and obvious concerns being the uncertainty surrounding potential immunogenicity and downstream adverse effects in patients receiving the drug (Singh et al., “An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics,” J. Pharma. Sci. 99(8):3302-21 (2010)). For small volumes of parenteral drug products (e.g., 100 mL or less), the Pharmacopeia limits SVPs 10 microns (10 μm) or greater to less than 6,000 SVPs per container and 25 microns (25 μm) or greater to less than 600 SVPs per container as measured by light obscuration analysis, and limits SVPs 10 microns (10 μm) or greater to less than 3,000 SVPs per container and 25 microns (25 μm) or greater to less than 300 SVPs per container as measured by membrane microscopic particle concentration testing (United States Pharmacopeia and National Formulary (USP 40-NF 28), <787> Subvisible Particulate Matter in Therapeutic Protein Injections. For eye drops, the SVP limits are 50 particles per mL 10 microns (10 μm), 5 particles per mL 25 microns (25 μm), and 2 particles per mL 50 microns (50 μm). <789> Particulate Matter in Ophthalmic Solutions). Regulatory agencies are increasingly expecting manufacturers to establish specifications for SVPs 2 microns (μm) and larger (see Singh et al., “An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics,” J. Pharm. Sci. 99(8):3302-21 (2010)).

[0036] The term "esterase" refers to an enzyme that catalyzes the hydrolysis of ester bonds to produce acids and alcohols. Esterases are a diverse category of enzymes that include acetyl esterases (e.g., acetylcholinesterase), phosphatases, nucleases, thiolesterases, lipases, and other carboxylester hydrolases (EC 3.1). The name implies carboxylester hydrolases (also known as carboxylesterases, carboxylic ester hydrolases, and EC 3.1.1.1), and therefore hydrolyze carboxylic esters to alcohols and carboxylic acids using water. Lipases are carboxylester hydrolases that catalyze the hydrolysis of lipids, including triglycerides, fats, and oils, to fatty acid and alcohol head groups. For example, triglycerides are hydrolyzed by lipases, such as pancreatic lipase, to produce monoacylglycerol and two fatty acid chains.

[0037] Phospholipases are lipases that hydrolyze phospholipids into fatty acids and other products. Phospholipases are classified into four broad categories: phospholipase A (including phospholipase A1 and phospholipase A2), phospholipase B, and the phosphodiesterases phosphodiesterase C and phosphodiesterase D. In addition to standard phospholipases, phospholipase B-like enzymes present in the lysosomal lumen are thought to be involved in lipid catalysis. For example, phospholipase B-like 2 (PLBL2) is hypothesized to have esterase activity based on sequence homology and subcellular localization (Jensen et al., “Biochemical characterization and liposomal localization of the mannose-6-phosphate protein p76,” Biochem. J. 402:449-458 (2007)).

[0038] An enzymatic activity associated with the destabilization of polysorbates (including polysorbate 20 and polysorbate 80) has been discovered. The activity was found to be associated with esterases, such as polypeptides comprising the amino acid sequences of Table 1. A BLAST search of these peptide sequences revealed identity with a putative phospholipase B-like 2 (PLBL2). PLBL2 is highly conserved in hamster, rat, mouse, human, and cow. Applicants envision that PLBL2, which co-purifies in certain processes with some classes of proteins of interest (POIs) produced in mammalian cell lines, has the esterase activity responsible for the hydrolysis of polysorbates 20 and 80. Applicants envision that other esterase species, of which PLBL2 is an example, may contribute to polysorbate instability depending on the genetic / epigenetic background of the particular protein of interest and / or the host cell.

[0039] Ester hydrolysis of polysorbate 80 has recently been reported (see Labrenz, SR, “Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after observation of visible particulate in mAb formulations,” J. Pharma. Sci. 103(8):2268-77 (2014)). The paper reported the formation of visible particles in formulations containing IgG. The authors hypothesized that colloidal IgG particles result from enzymatic hydrolysis of the oleate ester of polysorbate 80. Although no esterase was directly identified, the authors speculated that a lipase or tweenase copurified with IgG, which was responsible for the degradation of polysorbate 80 (ibid., 7). As stated in the paper, the formation of particles in the presence of polysorbate 80 is a concern, as such particles may affect the stability and efficacy of IgG drug products.

[0040] [Table 1]

[0041] As used herein, the phrase "fatty acid ester hydrolysis percentage" refers to the molar ratio of hydrolyzed fatty acid ester.Since the hydrolysis of fatty acid ester releases free fatty acid, the hydrolysis percentage of fatty acid ester can be determined by measuring the free fatty acid in a sample.Therefore, the hydrolysis percentage of fatty acid ester can be determined by calculating the moles of free fatty acid divided by the total moles of free fatty acid plus the moles of fatty acid ester.In the case of the hydrolysis percentage of polysorbate 80 or polysorbate 20, the number can be determined by calculating the moles of free fatty acid and dividing by the total moles of remaining polysorbate plus the moles of free fatty acid.

[0042] The term "esterase inhibitor" refers to any chemical that reduces, inhibits, or blocks the activity of esterase. Applicants envision that the inclusion of an esterase inhibitor in a protein formulation containing a fatty acid ester surfactant may help maintain protein stability and also help reduce SVP formation. Common esterase inhibitors known in the art include orlistat (tetrahydrolipistatin, an inhibitor of carboxylesterase 2 and lipoprotein lipase), diethylumbelliferyl phosphate (cholesterol esterase [lipase A] inhibitor), URB602 ([1-1'-biphenyl]-3-tl-carbamic acid cyclohexyl ester, a monoacylglycerol lipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor of hormone-sensitive lipase). The inclusion of an esterase inhibitor during purification of a protein of interest or in the final formulation may prevent or slow the hydrolysis of non-ionic surfactants such as polysorbate 80, which is expected to prevent or reduce the formation of subvisible particles. However, the inclusion of an esterase inhibitor may also adversely affect the function of the active ingredient or other ingredients in the final formulation.

[0043] The term "buffer" refers to a buffer or buffering agent that stabilizes the pH of a solution. Buffers generally contain a weak acid and its conjugate base, or a weak base and its conjugate acid. Buffering a protein solution at or near its optimal pH allows for proper protein folding and function. An optimal buffer can be identified, for example, by measuring the thermodynamic stability (DSC) of a protein (e.g., antibody) solution at various pHs, as well as the high molecular weight variants (SEC) and charge variants (CEX) after accelerated storage / incubation. Measuring the circular dichroism of a protein (e.g., antibody) solution at various pHs can also help identify a buffer. Circular dichroism (CD) is one method used to identify protein conformational changes (unfolding) (S. Beychok, “Circular dichroism of biological macromolecules,” Science 154(3754):1288-99(1966); Kemmer and Keller, “Nonlinear least-squares data fitting in Excel spreadsheets,” Nat Protoc. 5(2):267-81(2010)). Some proteins have the ability to act as a buffer (i.e., so-called “self-buffering”), so they may not require the addition of exogenous buffers to maintain a stable pH (Gokarn et al., “Self-buffering antibody formulations,” J Pharm Sci. 97(8):3051-66(2008)). Examples of commonly used buffers are listed in Table 2. For a more complete discussion of buffers of biological fluids, see Irwin H. Segel, Biochemical Calculations (2nd ed. 1976), or Remington, The Science and Practice of Pharmacy 244 (Paul Beringer et al. eds., 21st ed. 2006).

[0044] [Table 2]

[0045] The term "thermal stabilizer" refers to an excipient or other additive included in a biopharmaceutical formulation to protect a protein from thermal degradation, denaturation, and loss of biological activity. In general, a thermal stabilizer helps maintain a protein (e.g., an antibody) in its native conformation and prevents aggregation under conditions of thermal stress. Thermal stress can result from freeze-thaw cycles, high temperature exposure, or long-term storage. Thermal stabilizers include sugars and other carbohydrates, polyols such as sugar alcohols and polyethylene glycol, and amino acids such as glycine. Examples of sugars or sugar alcohols useful as thermal stabilizers include sucrose, trehalose, and mannitol.

[0046] The term "hydrophobic interaction medium" refers to a combination of a support structure and a hydrophobic moiety, the hydrophobic moiety being fixed to the support structure. The medium can be in the form of a chromatography medium, e.g., beads or other particles held in a packed bed column format, in the form of a membrane, or in any format capable of accommodating a liquid containing the protein of interest and contaminants. Thus, support structures include agarose beads (e.g., sepharose), silica beads, cellulosic membranes, cellulosic beads, hydrophilic polymer beads, etc. The hydrophobic moiety binds to the hydrophobic surface of hydrophobic molecules and proteins. The degree of hydrophobicity of the medium can be controlled by selecting the hydrophobic moiety. Hydrophobic interaction media are used in a process known as hydrophobic interaction chromatography (HIC) to separate the protein of interest from product and process related contaminants. When the protein of interest is produced in and / or purified from a host cell, the product and process related contaminants are called host cell proteins (HCPs). HCPs derived from Chinese Hamster Ovary (CHO) cells, a common host cell for biopharmaceutical manufacturing, may be referred to as CHOP (Chinese Hamster Ovary Protein). In some cases, a mixture containing a protein of interest (POI) and HCPs is applied to the HIC medium in a buffer designed to promote binding of the hydrophobic groups of the POI to the hydrophobic moieties of the HIC medium. The POI is fixed to the HIC medium by binding to the hydrophobic moieties, and some HCPs are unbound and come out in a wash buffer. The POI is then eluted with a buffer that promotes dissociation of the POI from the hydrophobic moieties of the HIC medium, thereby separating the POI from unwanted HCPs.

[0047] In some cases, the hydrophobic portion of the HIC binds some of the contaminants, such as HCPs, and the POI is recovered from the flow-through of the HIC. In some cases, affinity chromatography designed to bind specific proteins with lipophilic properties is used instead of or in combination with HIC. Some esterases, such as lipases in general, or phospholipases in particular, bind to triglycerides or phospholipids, so molecules that mimic these lipids may be used to capture the esterases. For example, "myristoylated ADP-ribosylation factor 1" (also known as "myrARF1") can be used to capture lipases, leaving the POI unbound in the flow-through.

[0048] As used herein, the term "container" is meant to include a primary packaging component, such as a syringe (such as in the case of a prefilled syringe), a vial (e.g., a 2.5 mL glass vial for storage of a biopharmaceutical formulation), or any vessel or means for containing a solid, liquid, or gaseous substance. Here, the term "container" is used to refer to a vessel that contains a biopharmaceutical formulation, as that term is used by, among others, the FDA and USP in their guidance on subvisible particle limits (United States Pharmacopeia and National Formulary (USP 40-NF 28), <787> Subvisible Particulate Matter in Therapeutic Protein Injections).

[0049] As used in this disclosure, the terms "composition," "formulation," and "formulated drug substance" (FDS) refer to a combination of two or more drug components for inclusion in a pharmaceutical product. A composition, formulation, or FDS can be, for example, a liquid composition that includes an active drug component, e.g., an antibody, and an excipient, e.g., a stabilizer or surfactant. A composition, formulation, or FDS can include multiple excipients. A composition, formulation, or FDS can also include other components, e.g., proteins that are co-purified with the antibody.

[0050] As used in this disclosure, the term "pharmaceutical product" (DP) refers to a dosage form that includes an amount of FDS for packaging, shipping, or administration. For example, the pharmaceutical product may be a pre-filled syringe that holds an amount of FDS for administration to a patient.

[0051] As mentioned above, it is hypothesized that HCPs such as PLBL2 that co-purify with some POIs exhibit esterase-like activity against fatty acid esters of polysorbates used in formulations and drug products containing those POIs. It is believed that this esterase-like behavior leads to the formation of free fatty acids that can then aggregate to give rise to SVPs. While HIC and / or affinity chromatography can be used to purify POIs and remove HCPs from drug products or formulations, thus reducing esterase-like behavior against fatty acid esters, the addition of a HIC or affinity chromatography step requires additional equipment (e.g., hydrophobic interaction media), materials, preparations, protocols, and protocol validation to the drug product manufacturing process, representing additional time, resources, experiments, and costs. Therefore, it would be desirable to have another way to reduce SVP formation in formulations and drug products containing POIs, polysorbates, and co-purified HCPs.

[0052] FDS and drug products comprising a POI and polysorbate 80 having a high percentage (e.g., >98%) oleate content have been found to form less measurable SVPs over time than FDS and drug products comprising polysorbate 80 having a relatively low percentage (e.g., 70% or 86-87%) oleate content, even when the POI has not been subjected to HIC or affinity chromatography to remove HCPs with esterase-like behavior toward fatty acid esters.

[0053] Embodiments of the present disclosure relate to FDSs and pharmaceutical products comprising a POI (e.g., an antibody) and polysorbate 80 with an oleate content of >98%, where the POI has not been subjected to a HIC or affinity chromatography step to remove HCPs with esterase-like behavior. In some embodiments of the present disclosure, the FDSs and pharmaceutical products exhibit formation of less than 3,000 particles having a diameter of 10 μm or more when stored in a container, for example, at a temperature of 5° C. for at least 6 months. In some embodiments, the FDSs and pharmaceutical products exhibit formation of less than 2,000, 1,500, 1,000, 800, 600, 500, 400, 300, 290, 275, 270, or 250 particles having a diameter of 10 μm or more when stored in a container, for example, at a temperature of 5° C. for at least 6 months. In some aspects, embodiments of the present disclosure relate to methods of preparation of such FDSs and pharmaceutical products.

[0054] In an embodiment of the present disclosure, the FDS or pharmaceutical product comprises a POI. In some embodiments, the POI is an antibody, for example, a human monoclonal antibody. In some embodiments, the POI is an immunoglobulin, for example, an IgG. In some embodiments, the protein is an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the FDS or pharmaceutical product comprises two or more POIs (for example, the FDS or pharmaceutical product comprises a co-formulation of two or more POIs).

[0055] In embodiments of the present disclosure, the POI may be purified by a purification step known in the art. For example, if the POI is an immunoglobulin, it may be purified using a Protein A or Protein G affinity purification step. In some embodiments, one or more HCPs or other impurities may be co-purified with the POI during this purification step. For example, in some embodiments, the FDS or pharmaceutical product includes an esterase co-purified with the POI. In some embodiments, the esterase is a phospholipase B-like protein, such as PLBL2.

[0056] In an embodiment of the present disclosure, the POI concentration in the FDS or pharmaceutical product may be in the range of about 40 mg / mL to about 250 mg / mL, for example, about 50 mg / mL to about 160 mg / mL, about 80 mg / mL to 100 mg / mL, about 100 mg / mL to 160 mg / mL, or about 125 mg / mL to 155 mg / mL.

[0057] In an embodiment, the FDS or pharmaceutical product comprises an amount of a surfactant or stabilizer. In some embodiments, the surfactant or stabilizer is polysorbate 80 comprising a mixture of fatty acid esters, and the oleate content is at least 97%, 98%, or 99%. In some embodiments, the surfactant or stabilizer is polysorbate 80 comprising a mixture of fatty acid esters, and the oleate content is >98%. In further embodiments, the surfactant or stabilizer is polysorbate 80 comprising a mixture of fatty acid esters, and the oleate content is ≧99%. In an embodiment, the concentration of the surfactant or stabilizer in the FDS or pharmaceutical product is 0.005% to 1.00% (w / v), for example, 0.5% (w / v).

[0058] In some embodiments, the volume of the FDS or pharmaceutical product is about 0.25 mL to 3 mL, for example, 0.25 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.25 mL, 2.5 mL, or 3 mL. In some embodiments, the pharmaceutical product comprises a volume of the FDS packaged in a container.

[0059] In some embodiments, the FDS or pharmaceutical comprises additional excipients, such as a buffer, a heat stabilizer, or an esterase inhibitor. In some embodiments, the FDS or pharmaceutical product is stored for at least 6 months at a temperature between about 2-8° C. In other embodiments, the FDS or pharmaceutical product is stored at a temperature between about 5° C., 15° C., 22° C., 24° C., or 30-50° C., e.g., between about 35° C., 40° C., 45° C., or 50° C.

[0060] In some embodiments, the FDS or pharmaceutical product is stored for up to, e.g., 2-4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months, or 36 months. For example, in some embodiments, the FDS or pharmaceutical product is stored at a temperature of about 5° C. for up to 24 months. In other embodiments, the FDS or pharmaceutical product is stored at a temperature of about 30-50° C. for up to 5 months. EXAMPLES

[0061] Example 1 The storage stability of an IgG4 antibody drug product prone to free fatty acid-based subvisible particulates resulting from degradation of polysorbate by co-purified host cell protein (HCP) lipase was evaluated in different DP samples. Each DP sample was 2.136 mL in volume and contained the same concentration of IgG4 antibody (150 mg / mL) and 0.2% (w / v) of one of several lots of PS80. Each of the PS80 lots had one of three different content percentages of oleate ester content (70%, 87%, and ≥99%). The table below summarizes the oleate ester content percentage of PS80 in each FDS sample.

[0062] [Table 3]

[0063] DP samples were stored in glass prefilled syringes at 2–8° C. for up to 24 months. Particulates were measured in each DP sample every 6 months for a total of 24 months by both microscopic particle concentration and microflow imaging (MFI).

[0064] FIG. 2 shows in graphical form the number of SVPs per container having a diameter of ≧10 μm as measured by the microscopic particle concentration method. FIG. 3 shows in graphical form the number of SVPs per container having a diameter of ≧10 μm as measured by the MFI. As shown in FIGS. 2 and 3, DP B and DP C (the two DP samples containing PS80 with an oleate content of ≧99%) showed the lowest number of SVPs over the entire 24-month period as measured by both the microscopic method (FIG. 2) and the MFI (FIG. 3). DP A, containing PS80 with an oleate content of 87%, showed the next lowest number of subvisible particles over the 24-month period (specifically, showing 800-1200 particles at 24 months). DP D, E, and F all showed well over 3000 particles per container (measured by both methods) at least at the 18-month time point.

[0065] The low particle count in DPs A, B, and C (compared to the higher number of particles in DPs D, E, and F) was hypothesized to be the result of using PS80 with a higher percentage of oleic acid (or long chain fatty acid) ester content. Oleic acid is a long chain fatty acid with one unsaturated bond (see FIG. 1). It therefore has a melting temperature below ambient temperature of about 13° C. The precursor to the formation of subvisible and visible free fatty acid (FFA) particulates is the aggregation of individual FFA chains into aggregates, which then precipitate in the form of particles. Oleic acid can arise, for example, by enzymatic hydrolysis of fatty acid esters of polysorbate 80 during storage of the formulation at 5° C. This oleic acid may also form SVPs, but due to its low melting temperature, such particles are likely to be present as an oily liquid in the protein formulation at room temperature (about 22° C.) where the analysis is performed, and therefore are not present as subvisible particulates at room temperature. In contrast, a high content of non-oleic esters in the formulation leads to the formation of the corresponding FFAs upon hydrolysis and, due to their high melting temperatures, the formation of subvisible and visible amorphous particulates is present at ambient temperature during analysis.

[0066] Furthermore, oleate esters are better solubilizers / stabilizers than esters of shorter chain fatty acids due to their high hydrophobicity, which allows them to solubilize free fatty acids and protein particulates, thereby maintaining product stability. Thus, polysorbate 80 with a high oleate content (>98%) can provide improved stability of protein formulations and pharmaceutical products compared to polysorbate 80 with a low oleate content.

[0067] Example 2 The concentration (micrograms / mL) of various free fatty acids in each sample DP (DP A-F) was evaluated after the samples were stored at 5° C. for 18 months. Samples DP A-F were prepared as described in Example 1. Free fatty acid concentrations were measured by LC-MS at 18 months. Figure 4 shows the results in graphical form. As shown, DP B and C (the two DP samples containing PS80 with oleate ester content > 99%) showed the highest oleic acid concentration and the lowest other FFA concentration. This indicates the uniformity of FFA (i.e., oleic acid) in DP B and C.

[0068] The technical ideas that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] A method for reducing the formation of sub-visible and visible particles in a pharmaceutical product, comprising: comprising at least 100 mg / mL of an IgG antibody in said pharmaceutical product; and including in said pharmaceutical product a mixture of polyoxyethylene sorbitan fatty acid esters; The method, wherein the oleic acid ester content in the mixture is greater than 98% of the total fatty acid esters in the mixture.

[0069] [Appendix 2] The method further comprises storing the pharmaceutical at a temperature of 30°C to 50°C for 1 to 5 months; 2. The method of claim 1, wherein after said storage, fewer than 3000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product as detected by one of flow imaging microscopy or membrane microscopy.

[0070] [Appendix 3] The pharmaceutical product is further stored at a temperature of 2°C to 8°C for 18 to 36 months; 2. The method of claim 1, wherein after said storage, fewer than 3000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product as detected by one of flow imaging microscopy or membrane microscopy.

[0071] [Appendix 4] The method of Appendices 1, further comprising adding an agent to the pharmaceutical agent that reduces viscosity. [Appendix 5] The method described in Appendix 1, wherein the IgG antibody is an IgG4 antibody.

[0072] [Appendix 6] The method of Appendices 1, wherein the IgG antibody can be co-purified with a lipase, and the pharmaceutical comprises the lipase. [Appendix 7] The method described in Appendix 1, wherein the IgG antibody is purified using an affinity purification step prior to inclusion in the pharmaceutical product.

[0073] [Appendix 8] The method of Appendices 1, wherein the step of including at least 100 mg / mL of the IgG antibody in the pharmaceutical comprises including at least 150 mg / mL of the IgG antibody in the pharmaceutical.

[0074] [Appendix 9] The method of Appendices 1, wherein the IgG antibody has not been purified using hydrophobic interaction chromatography (HIC) prior to inclusion in the pharmaceutical product. [Appendix 10] The method of Appendices 1, wherein the IgG antibody is an IgG4 antibody and the pharmaceutical agent comprises phospholipase B-like 2 protein.

[0075] [Appendix 11] The method of Appendices 1, further comprising purifying the IgG antibody using a Protein A purification step prior to including the IgG antibody in the pharmaceutical product. [Appendix 12] The method of appendix 1, wherein the oleic acid ester content in the mixture is at least 99% of the total fatty acid esters in the mixture.

[0076] [Appendix 13] The method of Appendices 1, wherein the pharmaceutical agent further comprises an esterase. [Appendix 14] A pharmaceutical product prepared according to the method described in Appendix 1. [Appendix 15] The method of appendix 1, wherein the oleic acid ester content in the mixture is determined by one of gas-liquid chromatography, liquid chromatography, colorimetry, or fluorescence analysis.

[0077] [Appendix 16] The method of appendix 1, wherein the pharmaceutical agent is configured for parenteral administration. [Appendix 17] A method for reducing particulate formation in a pharmaceutical comprising an IgG antibody and an esterase, comprising: comprising: including in the pharmaceutical product a mixture of polyoxyethylene sorbitan fatty acid esters, wherein an oleic acid ester content in the mixture is greater than 98% of the total fatty acid esters in the mixture; The method does not include purifying the IgG antibody using hydrophobic interaction chromatography.

[0078] [Appendix 18] The method described in Appendix 17, wherein the IgG antibody is an IgG4 antibody and the esterase is a phospholipase B-like 2 protein. [Appendix 19] At least 100 mg / mL of IgG antibody; and a mixture of fatty acid esters, The formulation, wherein the oleic acid ester content in the mixture is greater than 98% of the total fatty acid esters in the mixture.

[0079] [Appendix 20] The formulation of Appendices 19, comprising at least 150 mg / mL of the IgG antibody. [Appendix 21] The formulation of Appendices 19, wherein the mixture of fatty acid esters is polysorbate 80.

[0080] [Appendix 22] The formulation of Appendices 19, wherein the IgG antibody is an IgG4 antibody, and the formulation comprises phospholipase B-like 2 protein. [Appendix 23] A pharmaceutical product comprising the formulation described in Appendix 19.

[0081] [Appendix 24] The pharmaceutical product of Appendix 23, wherein fewer than 3,000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product by one of flow imaging microscopy or membrane microscopy after the pharmaceutical product has been stored at a temperature of 30°C to 50°C for 1 to 5 months.

[0082] [Appendix 25] The pharmaceutical product of Appendix 23, wherein fewer than 3,000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product by one of flow imaging microscopy or membrane microscopy after the pharmaceutical product has been stored at a temperature of 2°C to 8°C for 18 to 36 months.

Claims

1. At least 100 mg / mL of an IgG antibody; a mixture of fatty acid esters of polyoxyethylene sorbitan, the polyoxyethylene sorbitan having an oleate content of greater than 98%; A formulation comprising: A formulation wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is present in an amount ranging from about 0.005% to 1.00% based on the total weight of the formulation.

2. 10. The formulation of claim 1 comprising at least 150 mg / mL of IgG antibody.

3. 2. The formulation of claim 1, wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is polysorbate 80.

4. 2. The formulation of claim 1, wherein the IgG antibody is an IgG4 antibody.

5. 10. The formulation of claim 1 further comprising an esterase.

6. 6. The formulation of claim 5, wherein the esterase is a phospholipase B-like 2 protein.

7. 2. The formulation of claim 1, wherein the polyoxyethylene sorbitan has an oleate content of at least 99%.

8. 2. The formulation of claim 1, wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is present in an amount ranging from about 0.5% to 1.00% based on the total weight of the formulation.

9. At least 100 mg / mL of an IgG antibody; a mixture of fatty acid esters of polyoxyethylene sorbitan, the polyoxyethylene sorbitan having an oleate content of greater than 98%; A pharmaceutical comprising: A pharmaceutical product, wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is present in an amount ranging from about 0.005% to 1.00%, based on the total weight of the pharmaceutical product.

10. 10. The pharmaceutical product of claim 9, wherein less than 3000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product by one of flow imaging microscopy or membrane microscopy after the pharmaceutical product is stored at a temperature of 30°C to 50°C for 1 to 5 months.

11. 10. The pharmaceutical product of claim 9, wherein less than 3000 particles having a diameter of 10 microns or greater are detectable in the pharmaceutical product by one of flow imaging microscopy or membrane microscopy after the pharmaceutical product has been stored at a temperature of 2°C to 8°C for 18 to 36 months.

12. 10. The pharmaceutical product of claim 9 comprising at least 150 mg / mL of IgG antibody.

13. The pharmaceutical product of claim 9 , wherein the IgG antibody is an IgG4 antibody.

14. 10. The pharmaceutical product of claim 9, wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is polysorbate 80.

15. 10. The pharmaceutical product of claim 9, wherein the volume of the pharmaceutical product is from about 0.25 mL to about 3 mL.

16. 10. The pharmaceutical product of claim 9, wherein the polyoxyethylene sorbitan has an oleate content of at least 99%.

17. The pharmaceutical product of claim 9 further comprising a phospholipase B-like 2 protein.

18. The pharmaceutical product of claim 9 further comprising an esterase.

19. 10. The pharmaceutical product of claim 9, wherein the mixture of fatty acid esters of polyoxyethylene sorbitan is present in an amount ranging from about 0.5% to 1.00% based on the total weight of the pharmaceutical product.

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