Formulations with reduced particle formation and methods for reducing particle formation

Abstract

To provide an antibody preparation in which particle formation is reduced, and to provide a method for reducing particle formation in an antibody preparation. [Solution] The biopharmaceutical compositions and pharmaceuticals disclosed herein exhibit a reduction in the amount of invisible particle formation. The compositions and pharmaceuticals disclosed herein comprise a protein and a surfactant or stabilizer containing a high percentage (e.g., at least 97%) of long-chain fatty acid esters. Methods for preparing and storing such compositions and pharmaceuticals are also disclosed herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 560,365, filed on September 19, 2017. The entire disclosure of the provisional application is incorporated herein by reference.

[0002] The present disclosure relates to biopharmaceutical formulations and pharmaceuticals that exhibit a reduced amount of invisible particles upon storage, as well as methods for preparing and storing them. Specifically, the present disclosure relates to formulations and pharmaceuticals comprising a protein and a surfactant or stabilizer comprising a high percentage amount of long - chain monounsaturated fatty acid esters, as well as methods for preparing and storing them.

Background Art

[0003] Polysorbates have conventionally been used in pharmaceuticals containing a protein as an active ingredient (also referred to as "DP") to protect the protein from surface - induced (gas / liquid or solid / liquid) instabilities during the processes of manufacture, storage, handling, and administration. Lipase co - purified with the protein of interest (POI) has been found to be able to hydrolyze the fatty acid ester of polysorbate to free fatty acid when included in the formulation together with polysorbate. Non - enzymatic hydrolysis of the fatty acid ester of polysorbate can also occur at a slower rate in the formulation. The resulting free fatty acids can aggregate and form microparticles over time in pharmaceuticals containing such formulations. Microparticles (both visible and invisible to the eye) can affect the stability of the product, shorten the shelf - life of the pharmaceutical due to not meeting official particulate matter specifications (e.g., U.S. FDA specifications), and have clinical effects, such as causing an immunogenic reaction upon administration.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Hydrophobic interaction chromatography (HIC) or affinity chromatography of POIs can reduce or remove lipases co-purified with POIs, thereby reducing the hydrolysis of fatty acid esters. However, adding HIC or affinity chromatography steps to the preparation of protein formulations requires additional steps to the manufacturing process, such as equipment, materials, preparation, protocol, and protocol validation, resulting in increased 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. Therefore, a method is desired in protein compositions that reduces or prevents the formation of invisible and visible particulate matter without using additional HIC or affinity chromatography steps. [Brief explanation of the drawing]

[0005] [Figure 1] This is a diagram of the chemical structure of polyoxyethylene(20) sorbitan monooleate, the main fatty acid ester of polysorbate 80. [Figure 2] This graph shows the number of invisible microparticles (≧10μm) measured by membrane microscopy particle concentration method in various protein pharmaceuticals containing polysorbate 80. [Figure 3] This graph shows the number of invisible particles (≧10 μm) measured by microflow imaging (MFI) in various protein pharmaceuticals containing polysorbate 80. [Figure 4] This graph shows the measured concentrations of free fatty acids in various protein-based pharmaceuticals containing polysorbate 80. [Modes for carrying out the invention]

[0006] The accompanying drawings incorporated herein and forming part thereof illustrate various exemplary embodiments and, together with their descriptions, help to illustrate the principles of the embodiments of this disclosure. Any feature of the embodiments or examples described herein (e.g., compositions, formulations, methods, etc.) may be combined with any other embodiments or examples and are incorporated herein.

[0007] As used herein, the terms “comprise,” “comprising,” or any other variation thereof are intended to describe non-exclusive inclusion, and a process, method, item, or apparatus containing a list of elements may not contain only those elements, but may also contain other elements not expressly described in or specific to such process, method, item, or apparatus. The term “exemplary” is used in the sense of “example,” not “ideal.” The terms “for example” and “such as” and their grammatical equivalents are understood to be followed by the phrase “and not limited to” unless otherwise specified.

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

[0009] All numerical values ​​disclosed herein (including all disclosed values, limits, and ranges) may have a variation of + / - 10% from the disclosed numerical values ​​(unless a different variation is specified). Furthermore, in the claims, values, limits, and / or ranges mean such values, limits, and / or ranges + / - 10%.

[0010] This disclosure is not limited to any specific composition, formulation, material manufacturer, drug, method, or experimental conditions disclosed herein, and many variations are possible within the expertise of those skilled in the art. The terms used herein are for illustrative purposes only and are not intended to limit to any specific embodiment.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the field to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but specific methods and materials are described herein. All publications mentioned herein 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 via amide bonds. A protein contains 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 contain multiple polypeptides to form a single functional biomolecule in a single conformation. Disulfide crosslinks (e.g., forming cystine between cysteine ​​residues) may be present in some proteins. For example, disulfide crosslinks are essential for the proper structure and function of insulin, immunoglobulins, protamines, etc.

[0013] In addition to the formation of disulfide bonds, proteins may undergo other post-translational modifications. These modifications include lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchoring), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of glycosyl groups to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups 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, and the like. Proteins can be produced using recombinant cell lineage production systems, such as insect baculovirus lines, yeast lines (e.g., Pichia species), and mammalian lines (e.g., CHO cells and CHO-K1 cells and other CHO derivatives).

[0015] As used herein, the term “antibody” includes immunoglobulins, which are composed of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Typically, the antibodies of this disclosure have a molecular weight greater than 100 kDa, for example, 130 kDa to 200 kDa, for example, about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain includes a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which may contain more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl 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, immunoglobulins of a class called immunoglobulin G (IgG) are commonly found in human serum and consist of four peptide chains: two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cystine disulfide bond, and the two heavy chains are linked to each other via two cystine disulfide bonds. 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 has a different constant region and, as a result, can have different effector functions.

[0017] As used herein, the term “antibody” includes the antigen-binding fragment of a complete antibody molecule. As used herein, terms such as “antigen-binding portion” and “antigen-binding fragment” of an antibody 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 also be obtained from complete antibody molecules using any appropriate standard techniques, such as proteolytic or recombinant genetic engineering techniques, including, for example, manipulation and expression of DNA encoding the variable domain and optionally constant domain of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (e.g., including phage antibody libraries), or can be synthesized. Such DNA can be sequenced and manipulated chemically or using molecular biological techniques to, for example, arrange one or more variable and / or constant domains into appropriate positions, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[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 present invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDR and particularly CDR3 (mutations introduced, for example, by random or site-directed 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 a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0019] The term "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least the functional portions of the CH2 and CH3 regions of immunoglobulins. "Functional portion" refers to the CH2 and CH3 regions that can bind to an Fc receptor (e.g., FcγR or FcRn, i.e., the neonatal Fc receptor) and / or be involved in complement activation. If the CH2 and CH3 regions are subject to deletion, substitution, and / or insertion or other modifications that prevent them from binding to any Fc receptor and from activating complement, then those CH2 and CH3 regions are considered non-functional.

[0020] Fc-containing proteins may include modifications to the immunoglobulin domain, such as modifications that affect one or more effector functions of the binding protein (e.g., modifications affecting FcγR binding, FcRn binding, and consequently half-life and / or CDC activity). Such modifications include, but are not limited to, the following modifications and their combinations, in accordance with the EU numbering of the immunoglobulin constant region: 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, 3 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, though not for the purpose of limitation, the binding protein may be an Fc-containing protein that exhibits an extended serum half-life (compared to the same Fc-containing protein without the listed modifications(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 modification could include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), or modifications of 307 and / or 308 (e.g., 308F or 308P).

[0022] The term “cell” includes any cell suitable for expressing recombinant nucleic acid sequences. Cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., E. coli, Bacillus species, Streptomyces strains, 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, such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic cells and are 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 cells include one or more viral genes and are, for example, retinal cells expressing viral genes (e.g., PER.C6® cells).

[0023] The term "fatty acid ester" means any organic compound containing 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 carboxylic acid) is replaced by an alkoxy group. As used herein, the hydroxyl group can be part of a carboxylic acid, more specifically a fatty acid, and / or an alcohol, such as 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., those having a glycerol head group, including monoglycerides, diglycerides, and triglycerides), and surfactants and emulsifiers including polysorbates such as polysorbate 20, polysorbate 60, and polysorbate 80, which are nonionic surfactants. 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 mainly added to protein formulations to enhance the stability of proteins against mechanical stresses, such as partial unfolding and self-association induced at the air / liquid interface and the solid / liquid interface. Without surfactants, proteins can sometimes become structurally unstable in solution, forming multimeric aggregates that can ultimately become invisible particles.

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

[0026] Fatty acids are generally classified as short-chain fatty acids with fewer than 6 carbon atoms in the aliphatic tail, medium-chain fatty acids with 6 to 12 carbon atoms, long-chain fatty acids with 13 to 21 carbon atoms, and very long-chain fatty acids with 22 or more carbon atoms in the aliphatic tail. As described above, fatty acids are also classified by their degree of saturation, which correlates with stiffness and melting point. Common fatty acids include caprylic acid (8 carbon: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), and linoleic elaedic acid (linelaedic acid). Examples include arachidic 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 described 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 mainly contains monolaurate ester of polyoxyethylene (20) sorbitan. Polysorbate 40 mainly contains monopalmitate ester of polyoxyethylene (20) sorbitan. Polysorbate 60 mainly contains monostearate ester of polyoxyethylene (20) sorbitan. Polysorbate 80 mainly contains monooleate ester of polyoxyethylene (20) sorbitan (shown in Figure 1).

[0028] The quality of commercially available polysorbates varies depending on the supplier. Polysorbates are therefore often mixtures of various chemicals, mostly consisting of polyoxyethylene(20) sorbitan monoester (as described above), and sometimes containing isosorbide ester admixtures. They may also contain, for example, polyethylene glycol (PEG), intermediate structures, and fatty acid reaction substances. 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) in which three of its alcohol groups are 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 commercially available batches of polysorbate, the polysorbate contains isosorbide monoesters. Isosorbide is a heterocyclic derivative of glucose and can also be prepared by dehydrating sorbitol. It is a diol, meaning it has two alcohol groups that can be involved in the formation of one or two ester bonds. Therefore, for example, some lots of polysorbate 80 may contain a considerable amount of isosorbide oleate monoesters and diesters.

[0030] In addition to variations in the head group, polysorbate preparations contain variable amounts of other fatty acid esters. For example, analysis of a specific 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] Nonionic 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. Aggregates can be nanoparticles or invisible particles ranging from 10 to 100 microns (10 to 100 μm) or 2 to 100 microns (2 to 100 μm), which can impair the stability and shelf life of pharmaceuticals and induce immunogenicity. Therefore, the stability of protein formulations sometimes depends on the stability of the nonionic surfactant additive. However, as will be discussed further herein, polysorbate 20 and polysorbate 80 may, in some cases, contribute to the formation of aggregates, nanoparticles, and invisible particles.

[0032] The terms “subvisible particles” (SVP) and “subvisible microparticles” as used in this disclosure refer to particles that are not visible to the naked eye, particularly in liquids. In other words, solutions or other liquids containing SVPs, rather than visible particles, are not visible due to turbidity. SVPs generally include particles with a diameter of 100 microns (100 μm) or less, but in some cases include particles smaller 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 result from the inclusion of foreign matter, protein aggregation, or aggregation of other components of the DP. SVPs may include, among other things, 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] Subvisible particle counts (SVPs) can be detected by one or more of the following methods. USP standards specify light shielding and optical microscopy protocols. Other methods include microflow imaging (MFI) analysis, Coulter counting, and submicron particle tracking. Several measurement and characterization methods for SVPs (e.g., light shielding, flow microscopy, electrodetection zone method, 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-shielding analysis has been criticized for underestimating protein aggregates and other amorphous structures. Flow imaging analysis, such as microflow imaging (MFI) (Brightwell Technologies, Ottawa, Ontario), is a more sensitive method for detecting irregularly shaped, brittle, transparent proteinaceous vasoconduct (SVPs) and distinguishing these types of particles from silicone droplets, bubbles, and other foreign matter (Sharma et al., “Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations,” AAPS J.12(3):455-464(2010)). Generally, SVP measurement and characterization by light-shielding analysis is less sensitive than MFI, and therefore the number of particles detected by MFI tends to be higher than that detected by light-shielding analysis. In short, MFI is a flow microscopy technique that acquires and analyzes continuous bright-field images in real time. Image analysis algorithms are applied to these images to identify bubbles, silicone droplets, and proteinaceous aggregates. It can analyze volumes ranging from approximately 250 microliters to several tens of milliliters. Depending on the system used, it can detect particles in the range of 2-300 microns (2-300 μm) or 1-70 microns (1-70 μm) (ibid.).

[0035] The FDA and other government regulatory agencies have set limits on the permissible amount of subvisible particles (SVPs) in parenteral formulations. The main clear concern is the uncertainty surrounding the 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 amounts of parenteral medication (e.g., 100 mL or less), the pharmacopoeia limits SVP (Small Particle Validity) of 10 microns (10 μm) or larger to less than 6,000 SVP per container and SVP of 25 microns (25 μm) or larger to less than 600 per container when measured by light shielding analysis, and limits SVP of 10 microns (10 μm) or larger to less than 3,000 SVP per container and SVP of 25 microns (25 μm) or larger to less than 300 per container when measured by membrane microscopy 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 for particles 10 microns (10 μm) or larger, 5 particles per mL for particles 25 microns (25 μm) or larger, and 2 particles per mL for particles 50 microns (50 μm) or larger (ibid.). <789> (Particulate Matter in Ophthalmic Solutions). Regulatory bodies are increasingly expecting manufacturers to establish specifications for subvisible particles (SVPs) larger than 2 microns (2 μm) (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 enzymes that catalyze the hydrolysis of ester bonds, producing acids and alcohols. Esterases are a diverse category of enzymes, including acetylesterases (e.g., acetylcholinesterase), phosphatases, nucleases, thiolesterases, lipases, and other carboxyl ester hydrolases (EC 3.1). The name carboxyl ester hydrolase (also known as carboxyl esterase, carboxylic acid ester hydrolase, and EC 3.1.1.1) includes the meaning of hydrolyzing carboxylic acid esters to alcohols and carboxylic acids using water. Lipases are carboxyl ester 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, producing monoacylglycerols and two fatty acid chains.

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

[0038] Enzymatic activity associated with the destabilization of polysorbates (including polysorbate 20 and polysorbate 80) was discovered. This activity was found to be related to esterases, such as polypeptides containing the amino acid sequences shown in Table 1. BLAST searches of these peptide sequences revealed their identity with putative phospholipase B-like 2 (PLBL2). PLBL2 is highly conserved in hamsters, rats, mice, humans, and cattle. The applicants hypothesize that PLBL2, co-purified with certain classes of target proteins (POIs) produced in mammalian cell lines using a specific process, possesses esterase activity involved in the hydrolysis of polysorbate 20 and 80. The applicants hypothesize that other esterase species, such as PLBL2, may contribute to the destabilization of polysorbates, depending on the specific target protein and / or the genetic / epigenetic background of 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)). That paper reported the formation of visible particles in formulations containing IgG. The authors hypothesized that colloidal IgG particles were produced by the enzymatic hydrolysis of the oleic acid ester of polysorbate 80. Although the esterase was not directly identified, the authors speculated that a lipase or tweenase co-purified with IgG, which caused the degradation of polysorbate 80 (ibid., 7). As stated in that paper, particle formation due to the presence of polysorbate 80 is a concern, as such particles may affect the stability and efficacy of IgG pharmaceuticals.

[0040] [Table 1]

[0041] As used herein, the term “fatty acid ester hydrolysis percentage” refers to the molar ratio of the hydrolyzed fatty acid ester. Since free fatty acids are released during the hydrolysis of fatty acid esters, the fatty acid ester hydrolysis percentage can be determined by measuring the free fatty acids in the sample. Thus, the fatty acid ester hydrolysis percentage can be determined by calculating the number of moles of free fatty acids by dividing the number of moles of free fatty acids by the sum of the number of moles of fatty acid ester. In the case of polysorbate 80 or polysorbate 20 hydrolysis percentage, the number can be determined by calculating the number of moles of free fatty acids and dividing it by the total number of moles of remaining polysorbate plus the number of moles of free fatty acids.

[0042] The term "esterase inhibitor" refers to any chemical substance that reduces, inhibits, or blocks the activity of an esterase. The applicants hypothesize that including an esterase inhibitor in a protein formulation containing a fatty acid ester surfactant may help maintain protein stability and reduce SVP formation. Common esterase inhibitors known in the art include orlistat (tetrahydrolipistatin, an inhibitor of carboxylesterase 2 and lipoprotein lipase), diethylumbelliferyl phosphate (a cholesterol esterase [lipase A] inhibitor), URB602 ([1-1'-biphenyl]-3-tl-cyclohexyl carbamate, a monoacylglycerol lipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor of hormone-sensitive lipase). Including an esterase inhibitor during the purification of the target protein or in the final formulation may prevent or delay the hydrolysis of nonionic surfactants such as polysorbate 80, which is expected to prevent or reduce the formation of invisible particles. However, including 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. The optimal buffer can be identified, for example, by measuring the thermodynamic stability (DSC) of a protein (e.g., antibody) solution at various pH levels after accelerated storage / incubation, as well as high molecular weight variants (SEC) and charge variants (CEX). Measuring the circular dichroism of a protein (e.g., antibody) solution at various pH levels can also be helpful in identifying a buffer. Circular dichroism (CD) is one method used to identify structural changes (unfolding) in proteins (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 function as buffers (i.e., so-called “self-buffering”), so it may not be necessary to add 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 biological fluid buffers, 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 "heat stabilizer" refers to excipients or other additives included in biopharmaceutical formulations to protect proteins from thermal decomposition, denaturation, and loss of biological activity. Generally, heat stabilizers help maintain proteins (e.g., antibodies) in their natural conformation and prevent aggregation under thermal stress conditions. Thermal stress can arise from freeze-thaw cycles, high-temperature exposure, or long-term storage. Heat stabilizers include sugars and other carbohydrates, sugar alcohols and polyols such as polyethylene glycol, and amino acids such as glycine. Examples of sugars or sugar alcohols useful as heat 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 a chromatography medium, e.g., in the form of beads or other particles held in a packed bed column, a membrane, or any other form capable of containing a liquid containing the protein of interest and contaminants. Thus, the support structure includes 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 mediums are used in a process known as hydrophobic interaction chromatography (HIC) to separate the protein of interest from the product and process-related contaminants. When the protein of interest is produced in and / or purified from host cells, the product and process-related contaminants are called host cell proteins (HCPs). HCP 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 the protein of interest (POI) and HCP is applied to the HIC medium in a buffer designed to facilitate the binding of the hydrophobic groups of the POI to the hydrophobic portion of the HIC medium. The POI adheres to the HIC medium by binding to the hydrophobic portion, while some HCP detaches to the wash buffer without binding. The POI is then eluted using a buffer that facilitates the dissociation of the POI from the hydrophobic portion of the HIC, thereby separating the POI from unwanted HCP.

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

[0048] As used herein, the term “container” means any container or means for containing a primary packaging component, such as a syringe (e.g., a pre-filled syringe), a vial (e.g., a 2.5 mL glass vial for storing a biopharmaceutical product), or any other container or means for containing a solid, liquid, or gaseous substance. Here, the term “container” is used, in particular, to refer to a container for containing a biopharmaceutical product, as the FDA and USP use the term in their guidance on limiting invisible particles (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 formulation ingredient” (FDS) refer to a combination of two or more drug components for inclusion in a pharmaceutical product. A composition, formulation, or FDS may be a liquid composition comprising, for example, an active drug component, such as an antibody, and an excipient, such as a stabilizer or surfactant. A composition, formulation, or FDS may comprise multiple excipients. A composition, formulation, or FDS may also comprise other components, such as a protein co-purified with the antibody.

[0050] As used in this disclosure, the term “Pharmaceutical Product” (DP) refers to a dosage form containing a FDS of a quantity for packaging, shipping, or administration. For example, a pharmaceutical product may be a pre-filled syringe holding an FDS of a quantity to be administered to a patient.

[0051] As described above, it is hypothesized that HCPs such as PLBL2, when co-purified with certain POIs, exhibit esterase-like activity towards the fatty acid esters of polysorbates used in formulations and pharmaceuticals containing those POIs. This esterase-like behavior is thought to lead to the formation of free fatty acids that can subsequently aggregate to produce SVPs. While HIC and / or affinity chromatography can be used to purify POIs, remove HCPs from pharmaceuticals or formulations, and thereby reduce esterase-like behavior towards fatty acid esters, the addition of HIC or affinity chromatography steps necessitates additional equipment (e.g., hydrophobic interaction media), materials, preparations, protocols, and protocol validation in the pharmaceutical manufacturing process, resulting in additional time, resources, experiments, and costs. Therefore, it is desirable to have alternative methods for reducing SVP formation in formulations and pharmaceuticals containing POIs, polysorbates, and co-purified HCPs.

[0052] FDSs and pharmaceuticals containing POI and polysorbate 80 with a high percentage (e.g., >98%) of oleic acid ester content have been shown to produce less measurable SVP over time than FDSs and pharmaceuticals containing polysorbate 80 with a relatively low percentage (e.g., 70% or 86-87%) of oleic acid ester content. This is true even when the POI is not subjected to HIC or affinity chromatography to remove HCPs that exhibit esterase-like behavior toward fatty acid esters.

[0053] Embodiments of this disclosure relate to FDS and pharmaceuticals comprising a POI (e.g., an antibody) and polysorbate 80 having an oleate ester content of >98%, wherein the POI is not subjected to a HIC or affinity chromatography step for removing HCP having esterase-like behavior. In some embodiments of this disclosure, the FDS and pharmaceuticals show the formation of fewer than 3,000 particles having a diameter of 10 μm or more when stored in a container at a temperature of 5°C for at least 6 months. In some embodiments, the FDS and pharmaceuticals show the formation of fewer 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 at a temperature of 5°C for at least 6 months. In some aspects, embodiments of this disclosure relate to methods for preparing such FDS and pharmaceuticals.

[0054] In embodiments of this disclosure, the FDS or pharmaceutical 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, IgG. In some embodiments, the protein is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the FDS or pharmaceutical comprises two or more POIs (for example, the FDS or pharmaceutical comprises a co-formulation of two or more POIs).

[0055] In embodiments of this disclosure, the POI may be purified by purification steps known in the art. For example, if the POI is an immunoglobulin, it may be purified using an affinity purification step of protein A or protein G. 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 contains an esterase co-purified with the POI. In some embodiments, the esterase is a phospholipase B-like protein, such as PLBL2.

[0056] In embodiments of this disclosure, the POI concentration in the FDS or pharmaceutical 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 some embodiments, the FDS or pharmaceutical contains a certain amount of surfactant or stabilizer. In some embodiments, the surfactant or stabilizer is polysorbate 80 containing a mixture of fatty acid esters, with an oleic acid ester content of at least 97%, 98%, or 99%. In some embodiments, the surfactant or stabilizer is polysorbate 80 containing a mixture of fatty acid esters, with an oleic acid ester content of >98%. In further embodiments, the surfactant or stabilizer is polysorbate 80 containing a mixture of fatty acid esters, with an oleic acid ester content of ≥99%. In some embodiments, the concentration of the surfactant or stabilizer in the FDS or pharmaceutical 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 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 comprises a certain volume of the FDS packaged in a container.

[0059] In some embodiments, the FDS or pharmaceutical product includes further excipients, such as buffers, heat stabilizers, or esterase inhibitors. In some embodiments, the FDS or pharmaceutical is stored at a temperature of approximately 2–8°C for at least 6 months. In other embodiments, the FDS or pharmaceutical is stored at a temperature of, for example, approximately 5°C, 15°C, 22°C, 24°C, or 30–50°C, for example, approximately 35°C, 40°C, 45°C, or 50°C.

[0060] In some embodiments, the FDS or pharmaceutical is stored for up to, for example, 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 is stored at a temperature of approximately 5°C for up to 24 months. In other embodiments, the FDS or pharmaceutical is stored at a temperature of approximately 30-50°C for up to 5 months. [Examples]

[0061] Example 1 The storage stability of IgG4 antibody drugs, which are prone to generating invisible free fatty acid particles due to polysorbate degradation by co-purified host cell protein (HCP) lipase, was evaluated using 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 lot of PS80 had one of three different oleate ester content percentages (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 pre-filled syringes at 2–8°C for up to 24 months. Microparticles were measured for each DP sample every 6 months for a total of 24 months using both microscopic particle concentration methods and microflow imaging (MFI).

[0064] Figure 2 shows a graph of the number of SVPs per container with a diameter of ≥10 μm, measured by the microscopic particle concentration method. Figure 3 shows a graph of the number of SVPs per container with a diameter of ≥10 μm, measured by MFI. As shown in Figures 2 and 3, DP B and DP C (these two DP samples contain PS80 with an oleate ester content of ≥99%) showed the lowest SVP counts over the entire 24-month period, measured by both microscopy (Figure 2) and MFI (Figure 3). DP A, containing PS80 with an oleate ester content of 87%, showed the next lowest number of invisible particulate matter over the 24-month period (specifically, 800–1200 particles over 24 months). DP D, E, and F all showed well over 3000 particles per container (measured by both methods) at least 18 months.

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

[0066] Furthermore, oleic acid esters are superior solubilizers / stabilizers to esters of shorter-chain fatty acids due to their high hydrophobicity. This hydrophobicity allows oleic acid esters to solubilize free fatty acid and protein particles, thereby maintaining product stability. Therefore, polysorbate 80 with a high oleic acid ester content (>98%) can provide improved stability for protein preparations and pharmaceuticals compared to polysorbate 80 with a low oleic acid ester content.

[0067] Example 2 The concentrations (micrograms / mL) of various free fatty acids in each sample DP (DP A-F) were evaluated after storing the samples 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 after 18 months. Figure 4 shows the results in graph form. As shown, DP B and C (these two DP samples contain PS80 with an oleic acid ester content of ≥99%) showed the highest oleic acid concentration and the lowest other FFA concentration. This indicates the homogeneity of FFA (i.e., oleic acid) in DP B and C.

[0068] The technical concepts that can be understood from the above embodiments are described below as an addendum. [Note 1] A method for reducing the formation of particles invisible to the naked eye and visible to the eye in pharmaceuticals, The aforementioned pharmaceutical product shall contain at least 100 mg / mL of IgG antibody, The aforementioned pharmaceutical product includes a mixture of polyoxyethylene sorbitan fatty acid esters, The method wherein the oleic acid ester content in the mixture exceeds 98% of the total fatty acid esters in the mixture.

[0069] [Note 2] This further includes storing the pharmaceutical product at a temperature of 30°C to 50°C for 1 to 5 months. The method according to Appendix 1, wherein, after the aforementioned storage, the pharmaceutical product is detected by flow imaging microscopy or membrane microscopy, and fewer than 3,000 particles having a diameter of 10 microns or more are detectable in the pharmaceutical product.

[0070] [Note 3] Further includes storing the pharmaceutical product at a temperature of 2°C to 8°C for 18 to 36 months. The method according to Appendix 1, wherein, after the aforementioned storage, the pharmaceutical product is detected by flow imaging microscopy or membrane microscopy, and fewer than 3,000 particles having a diameter of 10 microns or more are detectable in the pharmaceutical product.

[0071] [Note 4] The method according to Note 1, further comprising adding a viscosity-reducing agent to the pharmaceutical product. [Note 5] The method according to Note 1, wherein the IgG antibody is an IgG4 antibody.

[0072] [Note 6] The method according to Note 1, wherein the IgG antibody can be co-purified with lipase, and the pharmaceutical product contains the lipase. [Note 7] The method according to Note 1, wherein the IgG antibody is purified using an affinity purification step before inclusion in the pharmaceutical product.

[0073] [Appendix 8] The method according to Appendix 1, wherein the step of including at least 100 mg / mL of the IgG antibody in the pharmaceutical product is further comprising including at least 150 mg / mL of the IgG antibody in the pharmaceutical product.

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

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

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

[0077] [Note 16] The method according to Note 1, wherein the pharmaceutical product is configured for parenteral administration. [Note 17] A method for reducing the formation of microparticles in pharmaceuticals containing IgG antibodies and esterases, The pharmaceutical product comprises a mixture of polyoxyethylene sorbitan fatty acid esters, wherein the oleic acid ester content in the mixture exceeds 98% of the total fatty acid esters in the mixture. The method, which does not include purifying the IgG antibody using hydrophobic interaction chromatography.

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

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

[0080] [Note 22] The preparation according to Note 19, wherein the IgG antibody is an IgG4 antibody and the preparation contains phospholipase B-like protein 2. [Note 23] Pharmaceuticals including the preparations described in Note 19.

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

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

Claims

1. At least 100 mg / mL of IgG antibody, A mixture of fatty acid esters, An antibody preparation containing, The oleic acid ester content in the mixture exceeds 98% of the total fatty acid esters in the mixture. The aforementioned preparation does not contain an exogenous buffer, and is an antibody preparation that contains a mixture of fatty acid esters, wherein the amount of oleic acid esters in the mixture is less than 98% of the total fatty acid esters in the mixture, and has fewer particles invisible to the naked eye compared to such a preparation.

2. The formulation according to claim 1, wherein the IgG antibody is an IgG4 antibody.

3. The formulation according to claim 1, comprising at least 150 mg / mL of IgG antibody.

4. The formulation according to claim 1, wherein the mixture of fatty acid esters comprises polysorbate 20.

5. The formulation according to claim 1, wherein the mixture of fatty acid esters comprises polysorbate 80.

6. The formulation according to claim 1, wherein the oleic acid ester content in the mixture is at least 99% of the total fatty acid esters in the mixture.

7. The formulation according to claim 1, wherein the exogenous buffer comprises histidine, citrate, glycine, acetate, phosphoric acid, succinic acid, tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), or any combination thereof.

8. The formulation according to claim 1, wherein the mixture of fatty acids is present in an amount ranging from 0.005% to 1.00% of the total weight of the formulation.

9. A pharmaceutical product comprising the formulation according to claim 1.

10. The pharmaceutical product according to claim 9, wherein, after storing the pharmaceutical product at a temperature of 30°C to 50°C for 1 to 5 months, or after storing the pharmaceutical product at a temperature of 2°C to 8°C for 18 to 36 months, fewer than 3,000 particles having a diameter of 10 microns or more are detectable in the pharmaceutical product by one of flow imaging microscopy or membrane microscopy.

11. A method for reducing the formation of invisible and visible particles in an antibody preparation, wherein the method is: The preparation shall contain at least 100 mg / mL of IgG antibody, The formulation includes a mixture of polyoxyethylene sorbitan fatty acid esters, Includes, The oleic acid ester content in the mixture exceeds 98% of the total fatty acid esters in the mixture. The formulation does not contain an exogenous buffer, and the formulation is a formulation containing a mixture of fatty acid esters, wherein the number of particles invisible to the naked eye is lower compared to a formulation containing a mixture of fatty acid esters in which the oleic acid ester content in the mixture is less than 98% of the total fatty acid esters in the mixture.

12. The method according to claim 11, wherein the IgG antibody is an IgG4 antibody.

13. The method according to claim 11, wherein the formulation contains at least 100 mg / mL of IgG antibody, which includes containing at least 150 mg / mL of IgG antibody.

14. The method according to claim 11, wherein the mixture of fatty acid esters comprises polysorbate 20.

15. The method according to claim 11, wherein the mixture of fatty acid esters comprises polysorbate 80.

16. The method according to claim 11, wherein the oleic acid ester content in the mixture is at least 99% of the total fatty acid esters in the mixture.

17. The method according to claim 11, wherein the exogenous buffer comprises histidine, citrate, glycine, acetic acid, phosphoric acid, succinic acid, tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), or any combination thereof.

18. The method according to claim 11, wherein the mixture of fatty acids is present in an amount ranging from 0.005% to 1.00% of the total weight of the preparation.

19. The method according to claim 11 is, A method further comprising storing the formulation at a temperature of 30°C to 50°C for 1 to 5 months, wherein, after storage, the formulation is detected by flow imaging microscopy or membrane microscopy, and fewer than 3,000 particles having a diameter of 10 microns or more are detectable in the formulation.

20. The method according to claim 11 is, A method further comprising storing the formulation at a temperature of 2°C to 8°C for 18 to 36 months, wherein, after storage, the formulation is detected by flow imaging microscopy or membrane microscopy, and fewer than 3,000 particles having a diameter of 10 microns or more are detectable in the formulation.

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