Methods of separating host cell lipases from Anti-LAG3 antibody production

Abstract

To provide methods of separating host cell lipases from an anti-LAG3 antibody or antigen binding fragment in chromatographic processes.SOLUTION: Provided is a method of separating a host cell lipase from a composition comprising an anti-LAG3 antibody or antigen-binding fragment and a host cell lipase through a hydrophobic interaction chromatographic (HIC) process, the method comprising: (a) passing a load fluid comprising the composition through HIC resin under a loading operating condition; and (b) collecting the anti-LAG3 antibody or antigen-binding fragment in a flowthrough. A separation factor (α) is the ratio of the partition coefficient (Kp) for the lipase to Kp for the anti-LAG3 antibody or antigen-binding fragment, log α is larger than 0.5 under the loading operating condition, and the anti-LAG3 antibody or antigen binding fragment comprises (a) light chain CDRs of specific sequences, and (b) heavy chain CDRs of specific sequences.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 967,347, filed January 29, 2020. No. 6,299,499, filed on Dec. 1, 2003, which is hereby incorporated by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing This application has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on January 19, 2021, contains the sequence listing. It is named 4955WOPCT-SEQTXT-19JAN2021.txt and is of size It is 14.2 kilobytes.

[0003] Herein, the chromatography process is used to separate the anti-LAG3 antibody from the host cell protein. Also provided herein are methods for isolating a human chondroitin protein (HCP) (e.g., lipase). Using a chromatography process, anti-LAG3 antibodies (e.g., monoclonal antibodies) By separating the HCP (e.g., lipase) from the anti-LAG3 antibody preparation (e.g., Improved stability of polysorbate-80 (PS-80) in drug substance or pharmaceutical formulations A method for improving this is also provided. [Background technology]

[0004] LAG-3 (lymphocyte activation gene 3) is a gene that activates T cells, B cells, NK cells, and LAG-3 is a cell surface molecule expressed on CD4 and plasmacytoid dendritic cells. It is structurally similar to LAG-3 and binds to MHC class II molecules as an inhibitory receptor. In addition to negatively regulating T cell activation and proliferation, it also inhibits other inhibitory receptors on tumor-infiltrating lymphocytes. LAG3 expression was shown to be co-expressed with the T cell phenotype. vinegar. Goldberg MV1,Drake CG.Curr.Top.Microbi See ol.Immunol.2011;344:269-78.

[0005] In the bioprocessing and manufacturing of antibodies (e.g., monoclonal antibodies), host cell proteins HCPs (e.g., lipases) are impurities that are often difficult to remove from antibodies. Such impurities have various effects on the safety and efficacy of biopharmaceuticals. Regulatory authorities around the world are increasingly demanding that biopharmaceuticals be tested for impurities at low levels. meet specific acceptance criteria, including tests for the detection and quantification of impurities, Several anti-LAG3 antibodies are in clinical development, and HC It is desirable to develop an efficient and effective process for removing P (e.g., lipase). I wish. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Goldberg MV1,Drake CG.Curr.Top.Microbiol.Immunol.2011;344:269-78 Summary of the Invention

[0007] The present disclosure provides methods for isolating anti-LAG3 antibodies or antigen-binding fragments by a chromatographic process. methods for separating HCPs (e.g., lipases) and hydrophobic interaction (HIC) chromatography using a chromatographic process or a cation exchange (CEX) chromatographic process. by separating the HCP (e.g., lipase) from the anti-LAG3 antibody or antigen-binding fragment. Therefore, PS-8 in anti-LAG3 antibody preparations (e.g., drug substance preparations or pharmaceutical preparations) The present disclosure provides methods for improving the stability of HCPs (e.g., lipases) and anti-LAG3 The antibody or antigen-binding fragment and the separation factor (α) and / or HC between the two proteins Partition coefficient (K p ) reaches a certain range of values ​​under certain operating conditions. This invention is based, at least in part, on the discovery that compounds can be isolated.

[0008] In one embodiment, the lipase is PLBL2. In yet another embodiment, the lipase is In one embodiment, the lipase is LP-PLA2. , HCP is clusterin.

[0009] In another embodiment, the anti-LAG3 antibody or antigen-binding fragment is combined with less than 2 ppm of host cell lipase. The present disclosure also provides pharmaceutical compositions comprising the anti-L. AG3 antibody or antigen-binding fragment and polysorbate 80 (PS80) or polysorbate and PS20 (PS80) at 2-8°C for 3 months. Or a pharmaceutical composition in which the concentration of PS20 is maintained at 90% or more of the concentration at the time of formulation. Provide something. [Brief explanation of the drawings]

[0010] [Figure 1] Log KP values ​​for PLBL2 or LPLA2 are shown for a range of HIC conditions representative of modulation of binding by salt concentration. [Figure 2]Figure 1 shows a comparison of the log KP values ​​on HIC resin for PLBL2, LPLA2, and two different mAbs, mAb2 (Ab6) and mAb3. mAb3 has very similar binding to HIC compared to PLBL2 and LPLA2, but mAb2 binds much weaker than mAb3, PLBL2, and LPLA2, resulting in a higher separation potential for PLBL2 and LPLA2 from mAb2 than for mAb3. [Figure 3] PS-80 concentrations are shown for Ab6 AEX pooled drug substance (AEX DS), and Ab6 HIC bound and eluted pooled drug substance (HIC B&E DS), or Ab6 HIC flow-through drug substance (HIC FT DS) at 2, 4, 6, and 14 week intervals at 5±3° C. [Figure 4] Figure 1 shows the PS-80 concentration of the Ab6A drug product of Example 6 at 3 months at 5°C ± 3°C (inverted), accelerated conditions at 25°C (25°C ± 2°C, 60% relative humidity, inverted), and stressed conditions at 40°C (40°C ± 2°C, 75% relative humidity, inverted). DETAILED DESCRIPTION OF THE INVENTION

[0011] definition Certain technical and scientific terms are specifically defined below. All other technical and scientific terms used herein unless expressly defined has the meaning commonly understood by a person of ordinary skill in the art to which this disclosure pertains. In all cases, the present specification, including definitions, will control.

[0012] The terms "operating conditions" are used interchangeably herein. ion), "operation condition", "processing condition ( "processing condition" or "process c "Operation conditions" refer to the conditions for operating a chromatography process. The operating conditions include equilibration conditions, loading conditions, washing conditions and / or elution conditions. The operating conditions include, but are not limited to, the type of chromatography resin, the resin structure, pH of the working solution, composition of the working solution, concentration of each component of the working solution, working solution Conductivity of the solution, ionic strength of the working solution, cation strength of the working solution, anion strength of the working solution or a combination of two or more of the above factors.

[0013] The term "operating solution" refers to the solution used in operating a chromatography process. Operating solutions include equilibration solutions, loading or feeding solutions, wash solutions, and / or Or it may be an elution solution or the like.

[0014] As used herein, the term "partition coefficient" or "K p " is a chromatography resin The concentration of bound protein (Q) and the amount of bound protein remaining in solution at equilibrium under specific operating conditions are The partition coefficient of a particular protein is given by It can be calculated as: K p =Q / C.

[0015] As used herein, the term "separation factor" or "α" refers to the partition coefficient of a first protein. (K p、タンパク質1 ) and the partition coefficient of the second protein (K p、タンパク質2 ) The resolution factor is the separation of the chromatographic tree between two proteins under specific operating conditions. This is used to quantify the selectivity of the chromatographic resin under its operating conditions. The degree of separation between two proteins can be predicted by the The coefficient can be calculated as follows: α=K p、タンパク質1 / K p、タンパク 質2 ; or log α = log K p、タンパク質1 -log K p、タンパク質2 .

[0016] As used herein, "eluent" refers to the liquid that passes through the chromatography. In some embodiments, the eluate is the flow-through of the loading solution. In this case, the eluate is divided into the eluent that passes through the chromatography and the eluate that leaves the chromatography. and any additional ingredients that are provided.

[0017] As used herein, "polysorbate-80 stability" or "PS-80 stability" means , general savings over a period of time (e.g., 1 week, 1 month, 6 months, 1 year, 2 years, etc.) Storage conditions (e.g., 5°C ± 3°C, 25°C ± 3°C, relative humidity (RH) 60% ± 5%, 40°C ±2°C, relative humidity (RH) 75% ±5%), physical, chemical and / or biological This refers to the state of PS-80 remaining stable. PS-80 stability is not limited to However, mass spectrometry (MS), liquid chromatography-mass spectrometry (LCMS), and liquid chromatography LC-MRM-MS or charged aerosol detection Various methods, including solid phase extraction (SPE) using an HPLC system with a Cadaver (CAD), were used. measured by the amount of intact PS-80 molecules and / or the amount of degradation products using It can be done.

[0018] The term "about" does not include amounts of substances or compositions (e.g., mM or M), percentages of formulation components (e.g., v / v or w / v), pH of the solution / formulation, or parameters characterizing the process steps When modifying the value of, for example, the preparation, characterization and / or use of a substance or composition Typical measurement, handling and sampling procedures involved in the use of A misuse of a product to make or use a composition or to perform a procedure by misuse of a product. Refers to variations in numerical quantities that may occur due to differences in manufacturing, source, or purity of ingredients used. In certain embodiments, "about" means ±0.1%, 0.5%, 1%, 2%, 3%, or 4% of the value. This may mean a variation of %, 4%, 5% or 10%.

[0019] Used in the context of measuring PS80 stability or PS20 stability after a period of time In this case, "the concentration of the compound is maintained at 80, 85, 90, 95 or 99% or more of the concentration at the time of formulation." The phrase "has" refers to an assay variation of ±10% for the measurement of PS80 or PS20 concentrations. Take gender into consideration.

[0020] As used herein, "Ab6 variants" refer to variants of Ab6 at positions located outside of the light chain CDRs. three, two, or one conservative amino acid substitutions, and six located outside the heavy chain CDRs; Antibody A except that it has five, four, three, two or one conservative amino acid substitutions. b6 means a monoclonal antibody containing heavy and light chain sequences substantially identical to those of (See below and WO 2016028672, which is incorporated by reference in its entirety.) As described above), for example, the variant position may be and may have a deletion of the C-terminal lysine residue of the heavy chain. In other words, Ab6 and Ab6 variants contain identical CDR sequences, but each has a full-length Conserved amino acids at three or six or fewer other amino acid positions in the light chain sequence and the full-length heavy chain sequence The Ab6 variants differ from each other due to the presence of amino acid substitutions. Binding affinity to human LAG3 and ability to block binding of human LAG3 to human MHC class II It is essentially the same as Ab6 in terms of potency.

[0021] As used herein, the term "antibody" refers to an antibody that exhibits a desired biological activity or binding activity. Therefore, the term "antibody" is used in the broadest sense and without limitation. Examples include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and Clonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, The term "parent antibody" specifically encompasses camelized single domain antibodies and camelized single domain antibodies. Modifying an antibody for use, e.g., prior to humanizing an antibody for use as a human therapeutic , antibodies obtained by exposing the immune system to an antigen.

[0022] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two polypeptide chains. Each pair contains one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50 kDa). The amino-terminal portion of each chain contains approximately 100-1000 ribonucleotides that are primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain contains a variable region of 10 or more amino acids. Typically, human light chains are kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, and a The antibodies are classified as IgM, IgD, or IgF, respectively. Within the light and heavy chains, variable and constant regions are defined as: The heavy chains are joined by a "J" region of about 12 or more amino acids, and the heavy chains also have a "J" region of about 1 It contains a "D" region of 0 or more amino acids. Generally, Fundamental Immun ology Ch.7(Paul,W.,ed.,2nd ed.Raven Pres. See s, NY (1989).

[0023] The variable regions of each light / heavy chain pair form the antibody binding site. A direct antibody has two binding sites. Except for bifunctional or bispecific antibodies, The two binding sites are generally the same.

[0024] Typically, the variable domains of both the heavy and light chains share a relatively conserved framework. Three hypervariable regions, also called complementarity-determining regions (CDRs), located within the complementarity-determining region (FR) The CDRs are usually aligned by framework regions and provide the targeting for a particular epitope. Generally, from N-terminus to C-terminus, the light chain variable domain and the heavy chain variable domain are Both variable domains have FR1, CDR1, FR2, CDR2, FR3, CDR3 and The amino acid assignments for each domain are generally given in the Sequences of Proteins of Immunological Interest,Kaba t,et al.;National Institutes of Health,B ethesda,Md.;5 th ed.;NIH Publ.No.91-3242( 1991);Kabat(1978)Adv.Prot.Chem.32:1-75;K abat, et al., (1977) J.Biol.Chem.252:6609-6 616;Chothia,et al.,(1987)J Mol.Biol.196: 901-917 or Chothia, et al., (1989) Nature 34 2:878-883.

[0025] As used herein, unless otherwise specified, "antibody fragment" or "antigen-binding fragment" refers to a " refers to an antigen-binding fragment of an antibody, i.e., a fragment that specifically binds to the antigen bound by a full-length antibody. "Antibody fragments" refers to antibody fragments that retain the ability to bind to a target molecule, for example, fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; antibody fragments Nanobodies and multispecific antibodies formed from

[0026] A "chimeric antibody" is an antibody in which a portion of the heavy and / or light chain is unmixed, so long as it exhibits the desired biological activity. , derived from a particular species (e.g., human) or specific antibody class or subclass. The sequence of one or more chains is identical or homologous to the corresponding sequence of the antibody to which it belongs. The remainder are derived from another species (e.g., mouse) or from another antibody class or subclass. Antibodies that are identical or homologous to the corresponding sequences of antibodies belonging to the same class, as well as sequences of such antibodies refers to a fragment.

[0027] A "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. is produced in mice, in mouse cells, or in hybridomas derived from mouse cells Similarly, a "mouse antibody" or a "rat antibody" may contain mouse carbohydrate chains. " refers to antibodies that contain only mouse or rat immunoglobulin sequences, respectively.

[0028] A "humanized antibody" is an antibody that contains sequences derived from a non-human (e.g., murine) antibody and a human antibody. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains. wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin; All or substantially all of the FR regions are from human immunoglobulin sequences. It also comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc) of a humanized antibody. The prefix "hum," "hu," or "h" is used when necessary to distinguish the antibody from its parent rodent counterpart. " is added to the name of the antibody clone. Humanized forms of rodent antibodies are generally Contains the same CDR sequences of the antibody, but to increase affinity and stability of the humanized antibody Or, for other reasons, certain amino acid substitutions may be included.

[0029] "comprising" or variations, e.g., "comprise ), "comprises" or "consist of" "of")" is used throughout the specification and claims in a comprehensive sense, i.e., Although the present invention identifies the presence of certain features, it does not substantially affect the operation or utility of any of the embodiments of the present invention. Although the term "feature" is used in a way that does not preclude the presence or addition of further features that may be advantageously enhanced, , unless the context, by express language or necessary implication, requires otherwise.

[0030] "Conservatively modified variants" or "conservative substitutions" are those that do not alter the biological activity or or other desired properties, e.g., antigen affinity and / or specificity. Similar characteristics (e.g., charge, side chain size, hydrophobicity) can be frequently added to / hydrophilicity, main-chain conformation and rigidity) Those skilled in the art will generally understand a single amino acid substitution in a non-essential region of a polypeptide. It is recognized that the acid substitutions do not substantially alter biological activity (e.g., Wats on et al.(1987)Molecular Biology of the Gene,The Benjamin / Cummings Pub.Co.,p.224 (4th Ed.) Furthermore, substitution of structurally or functionally similar amino acids is , are unlikely to disrupt biological activity. Exemplary conservative substitutions are shown in Table 1 below. [Table 1]

[0031] As used throughout this specification and claims, "consisting essentially of" refers to ts essentially of)" and variations such as "consisting essentially of ( "consist essentially of" or "consist essentially of" "sisting essentially of" refers to a specified dosing regimen, method, or any recited element that does not materially alter the basic or novel characteristics of the composition. or the inclusion of a group of elements and any other elements of similar or different nature to the listed elements. Inclusion is shown. Non-limiting examples include anti-LAG3 antibodies consisting essentially of the listed amino acid sequences: The antibody or antigen-binding fragment may also contain one or more nucleotides that do not substantially affect the properties of the binding compound. It may contain one or more amino acids containing substitutions of the above amino acid residues.

[0032] As used herein, "framework region" or "FR" refers to the region of an immunoglobulin excluding the CDR regions. It means an immunoglobulin variable region.

[0033] As used herein, "Kabat" refers to Elvin A. Kabat (1991) Sequences of Proteins of Immunological I nterest,5th Ed.Public Health Service,Nat ional Institutes of Health,Bethesda,Md.) refers to the immunoglobulin alignment and numbering system developed by do.

[0034] Human LAG3 has the following amino acid sequence: MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEG APA QLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPR RYT VLSVGPGGLR SGRLPLQPRV QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRA LSC RLRLRLGQAS MTASPPGSLR ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESP HHH LAESFLFLPQ VSPMDSGPWG CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSR VGL PCRLPAGVGT RSFLTAKWTP PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHL QEQ QLNATVTLAI ITVTPKSFGS PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPW LEA QEAQLLSQPW QCQLYQGERL LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILG VLS LLLLVTGAFG FHLWRRQWRP RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEP EPEP EPEQL (SEQ ID NO: 1); see also Uniprot accession number P18627 Residues 1-22 are the natural leader sequence.

[0035] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a refers to a population of antibodies that is substantially homogeneous, i.e., the antibody molecules that comprise the population are present in small amounts. The amino acid sequences are identical except for possible naturally occurring mutations that may occur. Conventional (polyclonal) antibody preparations typically contain large amounts of the variable domains, particularly those These include many different antibodies with different amino acid sequences in the CDRs of the The modifier "monoclonal" refers to the antibody being substantially homogeneous. It characterizes antibodies obtained from a population and requires the production of antibodies by some specific method. For example, the monoclonal antibodies used in accordance with the present invention may be First published by Kohler et al. (1975) Nature 256:495 They may be made by the hybridoma method described herein, or by recombinant DNA methods (e.g., Monoclonal antibodies can be produced by the methods described in U.S. Pat. No. 4,816,567. " Also, for example, Clackson et al. (1991) Nature 352 :624-628 and Marks et al. (1991) J. Mol. Biol. from a phage antibody library using the techniques described in 222:581-597 Presta (2005) J. Allergy Clin. Immuno See also l.116:731.

[0036] As used in this specification, including the appended claims, the singular forms of words, e.g., "a" and "b" are used interchangeably. "," "an," and "the" are used interchangeably unless the context clearly dictates otherwise. Unless the context otherwise requires, singular terms include the plural, Plural terms shall include the singular.

[0037] As used herein, the terms "at least one" or "one or more" items represents a single item selected from a list, and two or more items selected from a list, respectively. Includes a mixture of

[0038] Any word following the term "eg" or "for example" The example(s) are not meant to be exhaustive or limiting.

[0039] Unless expressly stated otherwise, all ranges cited herein are Ranges are inclusive, i.e., include the upper and lower values ​​of the range and everything in between. By way of example, the temperature ranges, percentages, equivalent ranges, etc. set forth herein include values ​​in the range All ranges include the upper and lower limits of the range and any value in the continuum between them. is also intended to include all subranges included, but not necessarily explicitly stated. For example, the pH range of 4.0 to 5.0 is not listed as pH 4.0, 4.1. , 4.13, 4.2, 4.1-4.6, 4.3-4.4, and 5.0. Furthermore, as used herein, the term "or" refers to combinations where appropriate. That is, the term "or" refers to each of the alternatives listed separately, and combinations thereof.

[0040] Aspects or embodiments of the present disclosure may be classified as Markush groups, or other groups of alternatives. When described in terms of a group, the disclosure applies to the entire group listed as a whole, not just the entire group. Each member of the group individually, and all possible subgroups of the main group Not only includes, but also the main group where one or more of the group members are absent The present disclosure also encompasses any of the group members in the claims. Assume one or more explicit exclusions.

[0041] Exemplary methods and materials are described herein, however, the methods and materials described herein Methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0042] Anti-LAG3 antibody In one embodiment, the anti-LAG3 antibody is Ab6 or an Ab6 variant.

[0043] Ab6 has the following antibody components: A light chain immunoglobulin having the following amino acid sequence: DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWY LQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO:2); A heavy chain immunoglobulin having the following amino acid sequence: QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQA RGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAY MELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO:3); A light chain immunoglobulin variable domain having the following amino acid sequence: DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWY LQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK (SEQ ID NO: 4); A heavy chain immunoglobulin variable domain having the following amino acid sequence: QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQA RGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAY MELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS (SEQ ID NO: 5); and the following CDRs: CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 6); CDR-L2: GASNLES (SEQ ID NO: 7); CDR-L3: QQSTEDPRT (SEQ ID NO: 8); CDR-H1:DYNVD (SEQ ID NO:9); CDR-H2: DINPNDGGTIYAQKFQE (SEQ ID NO: 10); and CDR-H3: NYRWFGAMDH (SEQ ID NO: 11) In some preferred embodiments of the methods of the present invention, the anti-LAG3 antibody or its antigen-binding The fragments comprise: (a) light chain CDRs, SEQ ID NOs: 6, 7, and 8; and (b) heavy chain CDRs, SEQ ID NO: 9. , 10 and 11.

[0044] In another preferred embodiment of the methods of the invention, the anti-LAG3 antibody or antigen-binding fragment thereof (a) a heavy chain variable region comprising SEQ ID NO:5; and (b) a light chain variable region comprising SEQ ID NO:4. In another preferred embodiment of the method of the present invention, the anti-LAG3 antibody comprises: (a) a polypeptide having the sequence SEQ ID NO: 3; and (b) a heavy chain comprising SEQ ID NO: 2. Another preferred embodiment of the method of the present invention In one embodiment, the anti-LAG3 antibody has two heavy chains and two light chains, and (a) the heavy chain has a sequence represented by SEQ ID NO: 3, and (b) the light chain consists of SEQ ID NO:2.

[0045] In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a human constant region. A human heavy chain constant region, such as a γ1, γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. In another embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises a light chain constant region, e.g., a human LAG3 antibody. The human light chain constant region may comprise a human light chain constant region, such as a lambda or kappa human light chain region or a variant thereof. By way of example and not limitation, the human heavy chain constant region can be γ4 and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody has a Ser228Pro mutation. γ4 (Schuurman, J et.al., Mol.Immunol.38: 1-8,2001).

[0046] In some embodiments, various constant domains are derived from the CDRs provided herein. Humanized V L Region and humanized V H For example, the antibody of the present invention (or If the specific intended use of the polypeptide (or fragment) requires an altered effector function, Heavy chain constant domains other than IgG1 may be used, or hybrid IgG1 / I may be used. gG4 may be utilized.

[0047] Chromatography Process Chromatographic separation of host cell lipase from anti-LAG3 antibody or antigen-binding fragment The roughy process may be a CEX chromatography process. The chromatography process is an HIC chromatography process. The lithography processes are CEX, AEX, mixed mode IEX, mixed mode AEX, and mixed Combined mode CEX, affinity chromatography process, Protein A or Protein Tein G Affinity Chromatography Process, Immobilized Metal Affinity Chromatography IMAC (Integrated Micro-Analysis) process and HAC chromatography process In one embodiment, the CEX or HIC chroma Protein A chromatography followed by AEX chromatography was performed before the chromatographic process. In one embodiment, the CEX or HIC chromatography process Protein A chromatography performed in bind and elute mode followed by flow chromatography It is preceded by AEX chromatography performed in low-through mode.

[0048] IEX chromatography separates molecules based on their net charge. The counterions of the target charged molecule and the oppositely charged ligand groups on the IEX chromatography resin The strength of binding of molecules to the IEX resin is affected by pH and It depends on the net charge of the molecule, which is affected by operating conditions such as the on-state strength. These include AEX resins and CEX resins. AEX resins are diethylaminoethyl ( DEAE group, trimethylaminoethyl (TMAE) group, quaternary aminoethyl (QAE) group CEX resins may contain substituents such as carboxymethyl groups and quaternary amine (O) groups. (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and and sulfonate (S). Cellulose-based IEX resins such as CM-23, CM-32 and CM-52 are manufactured by Whatman Available from Sephade Ltd. Maidstone, Kent, UK. x-based IEX resins and crosslinked IEX resins are also known. For example, DEAE-, QAE-, CM- and SP-Sephadex, and DEAE-, Q-, CM-, and S-Se GE Healthcare is a registered trademark of GE Healthcare. In addition, both DEAE and CM are available from Pharmacia, Inc., Piscataway, NJ. Ethylene glycol-methacrylate copolymers derived from, for example, TOYOPE ARL™ DEAE-650S or M, and TOYOPEARL™ CM- 650S or M from Toso Haas Co., Philadelphia, PA POROS™ HS, POROS™ HQ, and POROS™ XS is a trademark of Thermo Fisher Scientific, Waltham, MA It is available from A.

[0049] HIC chromatography separates molecules based on their hydrophobicity. The hydrophobic regions of the ATP bind to the HIC resin through hydrophobic interactions. The strength of the interaction is determined by the H depends on operating conditions such as ionic strength and salt concentration. Generally, HIC resins are hydrophobic. A base matrix (e.g., a hydroxy group) to which ligands (e.g., alkyl or aryl groups) are attached. Non-limiting examples of HIC resins include: Contains Phenyl SEPHAROSE™ 6 FAST FLOW™ (P harmacia LKB Biotechnology,AB,Sweden);Ph enyl SEPHAROSE (trademark) High Performance (Pharm acia LKB Biotechnology,AB,Sweden);Octyl SEPHAROSE(TM) High Performance (Pharmacia LKB Biotechnology,AB,Sweden);Fractogel(Commercial) FRACTOGEL™ EMD Propyl or FRACTOGEL™ EMD Phenyl Merck, Germany); MACRO-PREP™ Methyl or M ACRO-PREP(TM)t-Butyl Supports(Bio-Rad,CA );WP HI-Propyl(C3)(trademark)(JTBaker,NJ);TOY OPEARL™ ether, phenyl or butyl (TosoHaas, PA); and and Tosoh-Butyl-650M (Tosoh Corp., Tokyo, Japan) an) are examples.

[0050] HAC chromatography is carried out using the formula [Ca 10 Insoluble hydrochloride of (PO4)6(OH)2 HAC is used as both a matrix and a ligand. The functional groups on the resin are positively charged calcium ions (C sites) and negatively charged phosphate sites. The C site interacts with a carboxylate residue on the protein surface. The P site can interact with basic protein residues, whereas the P site can interact with The strength of the bond between the protein and the HAC resin varies depending on the pH, ionic strength, and solution composition. The composition depends on the operating conditions, including the concentration of each component of the composition, the pH gradient, the gradient of component concentrations, etc. CHT™ Ceramic Hydroxyapatite and CFT™ Various HAC resins are commercially available, including Ceramic Fluoroapatite .

[0051] Affinity chromatography is a highly specific method for determining the affinity between the molecule of interest and the functional groups on the resin. Interactions between antigens and antibodies, enzymes and substrates, receptors and ligands, or proteins Separating molecules based on interactions between them and nucleic acids. Affinity chromatography resins include Protein A resins for antibody purification or or Protein G resin, avidin for purifying biotin / avidin and their derivatives Ginbiotin resin, glutathione resin for purifying GST-tagged recombinant proteins , heparin resin for separating plasma coagulation proteins, and These include IMAC resins for purifying proteins. The operating conditions of the fluorographic procedure depend on the mechanism of interaction and the factors that affect the interaction. Commercially available affinity chromatography resins include, but are not limited to, Ma bSelect Sure, UNOsphere SUPrA(TM), Affi-Ge 1® and Affi-Prep®.

[0052] A mixed mode is any two or more of the functions or features described above or understood by those skilled in the art. Combinations of structures, e.g., IEX and HIC combinations (e.g., AEX / HIC or CEX / HIC), a combination of AEX and CEX (AEX / CEX), or HIC, AEX and An example of a mixed-mode crossover is a combination of HIC and CEX (HIC / AEX / CEX). Chromatography resins include, but are not limited to, OminPac PCX-500, Primesep, Oblisc R, Oblisc N, Acclaim Trin ity P1, Acclaim Trinity P2, Capto Adhere, C apto Adhere Impres, Capto MMC, Capto MMC I mpres, Capto Core 700, PPA Hypercel, HEA Hy percel, MEP Hypercel, Eshmuno HCX, Toyopear l MX-Trp-650M, Nuvia C Prime, CHT Type I and and CHT Type II.

[0053] Partition coefficient (K p ) and separation factor (α) Partition coefficient (K p ) and separation factor (α) depend on the operating conditions of the chromatographic process. Two specific thermodynamic parameters that must be achieved through the process under operating conditions This can be used to quantify the separation that can be achieved.

[0054] partition coefficient K P is a known liquid concentration of a protein (or other molecule of interest) in a known volume. in a liquid that is mixed with the chromatography resin and in equilibrium with the proteins bound to the resin. is determined by calculating the ratio of the protein remaining in the P =q / c=[result [Combined] / [Free].

[0055] The distribution is generally expressed as log K P It has been reported that log K P is herein The log K can be accurately quantified down to about 0-2 using the UV method described. P Scree The general rules for training are as follows: log K P ≥1.5, strong bond to resin; log K P <1, conditions under which elution is predicted for the bound elution regime; 0.5 <log K P <1, weak interaction condition indicating some binding; log K P <0.5, little or no binding;

[0056] log K between different species p The difference in values ​​is used to calculate the separation factor α as follows: The separation of species can be predicted by: α = K P , タンパク質1 / K P , タンパク 質2 ;log α=log K P , タンパク質1 -log K P , タンパク質2 , where , log α is farther from 0, indicating better separation. An absolute value of log α greater than 0.2 indicates good separation between the two species. In embodiments, an absolute value of log α greater than 0.3 indicates good separation between the two species. In other embodiments, an absolute value of log α greater than 0.5 indicates good correlation between the two species. In other embodiments, the absolute value of log α greater than 1.0 indicates good separation of the two Shows good separation between species.

[0057] HCP The various methods provided herein are applicable to a wide variety of HCPs. Host cells during bioprocessing of intracellularly expressed anti-LAG3 antibodies or antigen-binding fragments ( The HCP may be any endogenous protein derived from the HCP (e.g., CHO cells). Examples include structural proteins, functional proteins, secreted proteins, enzymes, e.g., lipases. In some embodiments, the HCP is In certain embodiments, the HCP is a functional protein. In one embodiment, the HCP is a secreted protein. In yet another embodiment, the HCP is an enzyme. In one embodiment, the HCP is a lipase. In another embodiment, the HCP is a protease. In yet another embodiment, the HCP is a kinase. , HCP is clusterin.

[0058] In certain embodiments, the lipase is PLBL2, LPL, LPLA2, LP-PL In one embodiment, the lipase is selected from the group consisting of PLBL2 and LAL. In another embodiment, the lipase is LPL. In yet another embodiment, the lipase is LPLA2. In one embodiment, the lipase is LP-PLA2. In another embodiment, the lipase is LAL. In yet another embodiment, the lipase comprises 6, 7, 8, 9, 10 or more different lipases. In this study, lipases were synthesized from PLBL2, LPL, LPLA2, LP-PLA2, and LAL. In one embodiment, the lipase comprises two, three, four or five different lipases selected from the group consisting of: In another embodiment, the lipase comprises PLBL2 and LPL. In yet another embodiment, the lipase comprises PLBL2 and LPLA2. In another embodiment, the lipase comprises PLBL2 and LA In one embodiment, the lipase comprises LPL and LPLA2. In yet another embodiment, the lipase comprises LPL and LP-PLA2. In another embodiment, the lipase includes LPL and LAL. In one embodiment, the lipase comprises LPLA2 and LAL. In another embodiment, the lipase includes LP-PLA2 and LAL. In an embodiment, the lipase includes PLBL2, LPL, and LPLA2. In one embodiment, the lipase comprises PLBL2, LPL, and LP-PLA2. In another embodiment, the lipase comprises PLBL2, LPL, and LAL. In yet another embodiment, the lipase comprises: In another embodiment, the lipases include PLBL2, LPLA2, and LAL. In one embodiment, the lipase comprises PLBL2, LP-PLA2, and LAL. In another embodiment, the lipase comprises LPL, LPLA2, and LP-PLA2. In yet another embodiment, the lipase comprises LPL, LPLA2, and LAL. In another embodiment, the lipase comprises LPLA2, LPLA3, LPLA4, LPLA5, LPLA6, LPLA7, LPLA8, LPLA9, LPLA10, LPLA11, LPLA22, LPLA13, LPLA14, LPLA15, LPLA16, LPLA17, LPLA18, LPLA19 ... In one embodiment, the lipase comprises PLBL2, LPL, LAL. In another embodiment, the lipase comprises PLBL2, L In yet another embodiment, the lipase comprises PLBL, LPLA2, and LAL. In another embodiment, the lipase includes: PLBL2, LPLA2, LP-PLA2, and LAL. In the present study, lipases include PLBL2, LPL, LPLA2, LP-PLA2, and LAL. nothing.

[0059] The host cell can be any cell used to express exogenous proteins. Common host cells used in the production of biopharmaceuticals include, but are not limited to, CH O cells, baby hamster kidney (BHK21) cells, mouse myeloma NS0 cells, mouse bone Myeloma Sp2 / 0 cells, human embryonic kidney 293 (HEK293) cells, fibrosarcoma HT-1080 cells cells, PER.C6 cells, HKB-11 cells, CAP cells, HuH-7 cells, mouse C1 27 cells and their naturally occurring or genetically engineered variants. In certain embodiments, the host cell is a CHO cell. In another embodiment, the host cell is a mouse. In yet another embodiment, the host cell is a murine myeloma Sp2 / 0 cell. In yet another embodiment, the host cells are human embryonic kidney 293 (HEK293) cells. In certain embodiments, the host cells are fibrosarcoma HT-1080 cells. In some embodiments, the host cells are PER.C6 cells. In yet another embodiment, the host cells are HKB-11 cells. In yet another embodiment, the host cells are CAP cells. In yet other embodiments, the host cell is a HuH-7 cell. In some embodiments, the host cell is a mouse C127 cell. In other embodiments, the host cell is a naturally occurring variant of the host cell. It is a genetically modified mutant.

[0060] In certain embodiments, the CHO cell lipase is PLBL2, LPL, LPLA2, In one embodiment, the CHO cell line is selected from the group consisting of LP-PLA2 and LAL. In another embodiment, the CHO cell lipase is LPL. In yet another embodiment, the CHO cell lipase is LPLA2. In another embodiment, the CHO cell lipase is LP-PLA2. In another embodiment, the CHO cell lipase is 2, 3, 4, 5, 6, 7, 8 , 9, 10 or more different CHO cell lipases. In CHO cell lipases, PLBL2, LPL, LPLA2, LP-PLA2 and LAL In one embodiment, the CHO cell lipase comprises PLBL2 and LPL. In this embodiment, the CHO cell lipases include PLBL2 and LPLA2. In this embodiment, the CHO cell lipase comprises PLBL2 and LP-PLA2. In another embodiment, the CHO cell lipase comprises PLBL2 and LAL. In another embodiment, the CHO cell lipase comprises LPL and LPLA2. O-cell lipases include LPL and LP-PLA2. CHO cell lipases include LPL and LAL. In one embodiment, CHO cell lipases include LPLA2 and LP-PLA2. In another embodiment, the CHO cell lipase comprises LPLA2 and LAL. In yet another embodiment, the CHO cell lipase comprises PLB In another embodiment, the CHO cell lipase includes: L2, LPL, and LPLA2. In one embodiment, the lipases include CHO cell lipases, PLBL2, LPL, and LP-PLA2. In another embodiment, the CHO cell lipase comprises PLBL2, LPL, and LAL. , PLBL2, LPLA2, and LP-PLA2. In yet another embodiment, CH O-cell lipases include PLBL2, LPLA2, and LAL. In one embodiment, CHO cell lipases include PLBL2, LP-PLA2, and LAL. In this study, CHO cell lipases include LPL, LPLA2, and LP-PLA2. In an embodiment, the CHO cell lipases include LPL, LPLA2, and LAL. In this embodiment, the CHO cell lipases include LPL, LP-PLA2 and LAL. In another embodiment, the CHO cell lipase is LPLA2, LP-PLA2 and LA In one embodiment, the CHO cell lipase comprises PLBL2, LPL, LPLA2, and In another embodiment, the CHO cell lipase comprises PLBL2, L In yet another embodiment, the CHO cell lipase comprises PL, LPLA2, and LAL. In another embodiment, the present invention comprises C HO cell lipases include PLBL2, LPLA2, LP-PLA2, and LAL. In yet another embodiment, the CHO cell lipase is PLBL2, LPL, LPLA2, Includes LP-PLA2 and LAL.

[0061] Screening of operating conditions for separating host cell lipase from anti-LAG3 antibody method The present disclosure provides a method for the preparation of anti-LAG3 antibodies or antigen-binding proteins by the chromatography process of the present invention. Screening of operating conditions for separating HCPs (e.g., lipases) from their soluble fragments Provide the law.

[0062] salt, type of salt, salt concentration, other components in the solution (e.g., counterions), and the concentration of each component; or Design numerous operating conditions, including pH, with or without load protein concentration, etc. Consider a CP (e.g., lipase) or an anti-LAG3 antibody or antigen-binding fragment. The operating conditions screened are those commonly used for the selected resin. The processing conditions used, such as equilibration conditions, loading conditions, washing conditions, elution conditions or storage conditions, It may be a ripping condition or the like.

[0063] HCP (e.g., lipase) and the K p The value is determined by methods disclosed in the specification or commonly understood by those skilled in the art. The log α between the HCP (e.g., lipase) and the anti-LAG3 antibody or antigen-binding fragment Values ​​are calculated using the methods described herein. Generally, HCPs (e.g., lipases) and greater than 0.5 for good separation between the anti-LAG3 antibody or antigen-binding fragment The absolute value of log α is preferred.

[0064] In one embodiment, the screening is carried out according to the method described by Welsh et al., Biotechno As disclosed in Prog. 30(3):626-635(2014), resin This is done using the slurry plate method, e.g., different combinations of pH, salts, and feeds. The mixture was then transferred to a 96-well filter plate (e.g., P / N MSBVN1250, Mi Add to a chromatographic column (Lipore Sigma, Burlington, MA). The resin volume ranges from 2 to 50 μL, and the liquid supply volume is 200 μL. In some embodiments, 16 to 32 conditions are tested for each resin. 24 to 96 conditions are tested. Separation of the resin and liquid is achieved by vacuum filtration. First, the resin was incubated with equilibration buffer for 10 minutes, and the equilibration step was repeated three times. The resin is then incubated with the feed for 60 minutes. The resin is then stripped. The equilibration step is repeated twice. The 60 minute time period for feed mixing allows for buffer exchange from the solution. This allows for pseudo-equilibration between the resin ligand and the protein. Determine the final liquid protein concentration, c, as measured by UV absorbance at 280-320 nm. The bound concentration of protein, q, was determined by mass balance between c and the known feed concentration, c0. It was determined.

[0065] In another embodiment, the screening is carried out using the method described by Welsh et al., Biotechn ol Prog.30(3):626-635(2014) or Petroff et al.,Biotech Bioeng.113(6):1273-1283(201 5) using a mini-column method. For example, pH, salt and The mixture of different combinations of the feed and the eluate was then passed through a 0.6 mL column with a bed height of 3 cm. Screening on a single mat. Screen up to 8 columns in parallel. The miniature column format is used for the typical column at about 300 cm / h. By reducing the linear flow rate to approximately 45 cm / h, a typical residence time of approximately 4 min was achieved. All other typical parameters for chromatographic screening are maintained. The eluate fractions are collected in a 96-well plate for pooling and preservation. or collected as fractions to produce chromatograms similar to those in laboratory-scale studies It is possible.

[0066] Procedures for separating HCPs (e.g., lipases) from anti-LAG3 antibodies or antigen-binding fragments Once the operating conditions are determined, the load fluid and / or The resin conditions can be adjusted by, for example, using a solution to bring the resin to the required operating conditions. The resin can be equilibrated by washing with HCl.

[0067] Method for separating host cell lipase from anti-LAG3 antibody The present disclosure further provides a method for the preparation of anti-LAG3 antibodies or antigen-binding fragments by a chromatography process. A method for separating the HCP (eg, lipase) from the fragments is provided.

[0068] In one embodiment, anti-LA is obtained by a hydrophobic interaction chromatography (HIC) process. and isolating host cell lipase from a composition comprising the G3 antibody or antigen-binding fragment and the host cell lipase. A method of separating, comprising the steps of: (a) passing a load fluid containing the composition through the HIC resin under loading operating conditions; That, and (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; The separation factor (α) is calculated by the partition coefficient of lipase (K p ) anti-LAG3 antibody or antigen-binding fragment thereof K p and log α is greater than 0.5 under loading conditions. and (a) a light chain CDR of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10, and 11. .

[0069] In another embodiment, anti-L A composition comprising the AG3 antibody or antigen-binding fragment and a host cell lipase. 1. A method for separating (a) passing a load fluid containing the composition through an HIC resin; and (b) Elution solution was used to remove anti-LAG3 antibody from the chromatography resin under the elution operating conditions. and eluting the antibody or antigen-binding fragment, The separation factor (α) is calculated by the partition coefficient of lipase (K p ) an anti-LAG3 antibody or antigen-binding fragment thereof K p The ratio of log α to anti-LA is greater than 0.5 under the elution operating conditions. G3 antibody or antigen-binding fragment comprising: (a) the light chain CDRs of SEQ ID NOs: 6, 7, and 8; and (b) and heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

[0070] In a further embodiment, the anti-LAG3 antibody or A method for separating host cell lipase from a composition comprising an antigen-binding fragment and host cell lipase. There was, (a) passing a load fluid containing the composition through a CEX resin; and (b) Elution solution was used to remove anti-LAG3 antibody from the chromatography resin under the elution operating conditions. and eluting the antibody or antigen-binding fragment, The separation factor (α) is calculated by the partition coefficient of lipase (K p ) an anti-LAG3 antibody or antigen-binding fragment thereof K p The ratio of log α to anti-LA is greater than 0.5 under the elution operating conditions. G3 antibody or antigen-binding fragment comprising: (a) the light chain CDRs of SEQ ID NOs: 6, 7, and 8; and (b) and heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

[0071] In certain embodiments, log α is greater than 1.0 under loading operating conditions. stomach.

[0072] In some embodiments, the log K of the lipase p is 1 under loading operation conditions. In another embodiment, the log K of the lipase is greater than 0. p is the loading operation condition Below 1.5 and above.

[0073] In certain embodiments, under loading operating conditions, log α is greater than 0.5. Lipase log K p is greater than 1.0. Under the operating conditions, the log α is greater than 0.5 and the log K p is 1.5 In other embodiments, under loading operating conditions, log α is greater than 1.0. The log K of lipase is also large. p is greater than 1.0. Under loading operating conditions, log α is greater than 1.0 and the log K of lipase p teeth Greater than 1.5.

[0074] In certain embodiments, the lipase is PLBL2, LPL, LPLA2, LP-PL In one embodiment, the lipase is selected from the group consisting of PLBL2 and LAL. In another embodiment, the lipase is LPL. In yet another embodiment, the lipase is LPLA2. In one embodiment, the lipase is LP-PLA2. In another embodiment, the lipase is LAL. In yet another embodiment, the lipase comprises 6, 7, 8, 9, 10 or more different lipases. In this study, lipases were synthesized from PLBL2, LPL, LPLA2, LP-PLA2, and LAL. In one embodiment, the lipase comprises two, three, four or five different lipases selected from the group consisting of: In another embodiment, the lipase comprises PLBL2 and LPL. In yet another embodiment, the lipase comprises PLBL2 and LPLA2. In another embodiment, the lipase comprises PLBL2 and LA In one embodiment, the lipase comprises LPL and LPLA2. In yet another embodiment, the lipase comprises LPL and LP-PLA2. In another embodiment, the lipase includes LPL and LAL. In one embodiment, the lipase comprises LPLA2 and LAL. In another embodiment, the lipase includes LP-PLA2 and LAL. In an embodiment, the lipase includes PLBL2, LPL, and LPLA2. In one embodiment, the lipase comprises PLBL2, LPL, and LP-PLA2. In another embodiment, the lipase comprises PLBL2, LPL, and LAL. In yet another embodiment, the lipase comprises: In another embodiment, the lipases include PLBL2, LPLA2, and LAL. In one embodiment, the lipase comprises PLBL2, LP-PLA2, and LAL. In another embodiment, the lipase comprises LPL, LPLA2, and LP-PLA2. In yet another embodiment, the lipase comprises LPL, LPLA2, and LAL. In another embodiment, the lipase comprises LPLA2, LPLA3, LPLA4, LPLA5, LPLA6, LPLA7, LPLA8, LPLA9, LPLA10, LPLA11, LPLA22, LPLA13, LPLA14, LPLA15, LPLA16, LPLA17, LPLA18, LPLA19 ... In one embodiment, the lipase comprises PLBL2, LPL, LAL. In another embodiment, the lipase comprises PLBL2, L In yet another embodiment, the lipase comprises PLBL, LPLA2, and LAL. In another embodiment, the lipase includes: PLBL2, LPLA2, LP-PLA2, and LAL. In the present study, lipases include PLBL2, LPL, LPLA2, LP-PLA2, and LAL. nothing.

[0075] In some embodiments of the various methods provided herein, the lipase is a CHO cell lipase. In certain embodiments, the CHO cell lipase is PLBL2, LPL, In one embodiment, the LPLA2, LP-PLA2, and LAL are selected from the group consisting of: In another embodiment, the CHO cell lipase is PLBL2. In yet another embodiment, the CHO cell lipase is LPLA2. In another embodiment, the CHO cell lipase is LP-PLA2. In another embodiment, the CHO cell lipase is LAL. , 6, 7, 8, 9, 10 or more different CHO cell lipases. In another embodiment, the CHO cell lipase is PLBL2, LPL, LPLA2, LP-P two, three, four or five different CHs selected from the group consisting of LA2 and LAL In one embodiment, the CHO cell lipase is PLBL2 and LP In another embodiment, the CHO cell lipase comprises PLBL2 and LPLA2. In yet another embodiment, the CHO cell lipase is PLBL2 and LP-PLA2 In another embodiment, the CHO cell lipase comprises PLBL2 and LAL. In one embodiment, the CHO cell lipases include LPL and LPLA2. In another embodiment, the CHO cell lipases include LPL and LP-PLA2. In another embodiment, the CHO cell lipases include LPL and LAL. HO cell lipases include LPLA2 and LP-PLA2. In another embodiment, CHO cell lipases include LPLA2 and LAL. In yet another embodiment, the enzymes include LP-PLA2 and LAL. In another embodiment, the enzymes include PLBL2, LPL, and LPLA2. Lipases include PLBL2, LPL, and LP-PLA2. In another embodiment, the cellular lipases include PLBL2, LPL, and LAL. Alveolar lipases include PLBL2, LPLA2, and LP-PLA2. In some embodiments, CHO cell lipases include PLBL2, LPLA2, and LAL. In embodiments, the CHO cell lipases include PLBL2, LP-PLA2, and LAL. In one embodiment, the CHO cell lipase is capable of catalyzing LPL, LPLA2, and LP-PLA2. In another embodiment, the CHO cell lipase includes LPL, LPLA2, and LAL. In yet another embodiment, the CHO cell lipase is selected from the group consisting of LPL, LP-PLA2 and L In another embodiment, the CHO cell lipase comprises LPLA2, LP-PLA In one embodiment, the CHO cell lipase comprises PLBL2, LPL, In another embodiment, the CHO cell lipase comprises P In yet another embodiment, the CHO cells contain LBL2, LPL, LPLA2, and LAL. Alveolar lipases include PLBL2, LPL, LP-PLA2, and LAL. In terms of morphology, CHO cell lipases are PLBL2, LPLA2, LP-PLA2 and LA In yet another embodiment, the CHO cell lipase comprises PLBL2, LPL, Includes LPLA2, LP-PLA2 and LAL.

[0076] In certain embodiments of the various methods provided herein, the operating conditions include adding salt. It further includes adjusting the ionic strength and / or conductivity by In embodiments, the effect of adding salt is to achieve a desired log α. In this state, the effect of adding salt is to increase the desired log K p The goal is to achieve this. In another embodiment, the effect of adding salt is to reduce the desired log α of the lipase. K pThus, in one embodiment, the operating conditions are such that salt is added. In another embodiment, the operating conditions further include achieving the desired log α by The desired log K of lipase can be achieved by adding salt. p It further includes achieving In yet another embodiment, the operating conditions are such that the desired log α is achieved by adding salt. , the desired log K of the lipase p The present invention further includes achieving the following.

[0077] In some embodiments, the salt in the working solution is sodium chloride, sodium acetate, phosphorus From the group consisting of sodium sulfate, ammonium sulfate, sodium sulfate and Tris-HCl In one embodiment, the salt is sodium chloride. In another embodiment, the salt is vinegar. In yet another embodiment, the salt is sodium phosphate. In an embodiment, the salt is ammonium sulfate. In one embodiment, the salt is sodium sulfate. In another embodiment, the salt is Tris-HCl.

[0078] In one embodiment, the concentration of sodium chloride in the working solution is about 100 mM to about 225 mM. The chromatography resin was CEX, and the operating conditions were pH 4.5 to 8.0. In another embodiment, the concentration of sodium chloride in the working solution is from about 150 mM to about 180 mM. The chromatography resin was CEX, and the operating conditions were pH 5.0 to 8. In one embodiment, the concentration of sodium chloride in the working solution is from about 100 mM to about 2.0 mM. The pH was 25 mM, the chromatography resin was CEX, and the operating conditions were pH 5.0 to 6.0. In another embodiment, the concentration of sodium chloride in the working solution is about 150 mM ~ about 180 mM, the chromatography resin is CEX, and the operating conditions are pH about 5 .0 to approximately 6.0.

[0079] In a further aspect, anti-LAG3 is isolated by a hydrophobic interaction chromatography process. A composition comprising an antibody or antigen-binding fragment and PLBL2 or PLBL2. or LPLA2, comprising the steps of: (a) passing a load fluid containing the composition through a hydrophobic interaction chromatography resin; That, and (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; The load fluid has a conductivity of about 25 to 80 mS / cm and is a carrier of an anti-LAG3 antibody or an antigen-binding protein. The combined fragment comprises (a) the light chain CDRs of SEQ ID NOs: 6, 7, and 8, and (b) the light chain CDRs of SEQ ID NOs: 9, 10, and 11. and 11 heavy chain CDRs.

[0080] In a further aspect, anti-LAG3 is isolated by a hydrophobic interaction chromatography process. A composition comprising an antibody or antigen-binding fragment and PLBL2 or PLBL2. or LPLA2, comprising the steps of: (a) passing a load fluid containing the composition through an HIC resin; and (b) Eluting the anti-LAG3 antibody or antigen-binding fragment from the HIC resin using an elution solution. wherein the elution solution has a conductivity of about 25 to 80 mS / cm; 10. An anti-LAG3 antibody or antigen-binding fragment comprising: (a) a light chain CDR of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10, and 11. .

[0081] In another specific embodiment, the concentration of sodium sulfate in the working solution is about 500 mM to The concentration was about 620 mM, the chromatography resin was HIC, and the operating conditions were pH about 7. In yet another specific embodiment, the concentration of sodium sulfate in the working solution is about 5 The concentration is 10 mM to about 560 mM, the chromatography resin is HIC, and the operating conditions are p H is about 7.

[0082] In one embodiment of the HIC chromatography process, the load fluid or elution solution is about 5 In another embodiment, the load fluid or elution solution has a conductivity of 0 to 70 mS / cm. In another embodiment, the load comprises about 300 mM to about 650 mM of a monovalent salt or a divalent salt. The fluid or elution solution contains about 300 mM to about 650 mM monovalent or divalent salt and has a pH In another embodiment, the salt is about 500 to 620 mM sodium sulfate. In a further embodiment, the salt is 560 mM sodium sulfate. The pH of the load fluid or elution solution is approximately 7.

[0083] The separation methods provided herein may be used in combination with any method described herein or commonly known in the art. In one embodiment, one or more separation steps may be used in combination. The separation step precedes the methods described herein. In another embodiment, one or more of the separation steps In yet another embodiment, the one or more separation steps follow the methods described herein. In another embodiment, one or more separations are performed between the two methods described herein. The steps may be performed before, after and / or during the methods described herein. Separate steps or methods that can be or are combined There is no limit to the order of law.

[0084] In further embodiments of the various methods provided herein, the load fluid is In one embodiment, the eluate is obtained from a pre-chromatographic process. The filtration process comprises affinity chromatography. The previous chromatographic process was affinity chromatography followed by immunoprecipitation. In yet another embodiment, affinity chromatography is used. In another embodiment, the chromatography is Protein A chromatography. In yet another embodiment, the exchange chromatography is AEX chromatography. The pre-chromatography process is Protein A chromatography, followed by Includes AEX chromatography.

[0085] Methods for improving PS-80 stability in anti-LAG3 antibody formulations The present disclosure further provides a method for detecting anti-LAG3 antibodies or antigen binding sites using a chromatographic process. By separating the HCP (e.g., lipase) from the ligated fragments, anti-LAG3 antibody or anti- PS-80 stability in original binding fragment formulations (e.g., drug substance formulations or pharmaceutical formulations) Provide ways to improve.

[0086] In yet another embodiment, the anti-LAG3 antibody or antigen-binding fragment formulation contains polysorbate 80. 1. A method for improving PS-80 stability, comprising: (a) Binding of host cell lipase to anti-LAG3 antibody or antigen under loading conditions passing a load fluid containing the fragments through an HIC resin; (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; and (c) The anti-LAG3 antibody or antigen-binding fragment preparation is a PS-80-containing solution. formulating a LAG3 antibody or antigen-binding fragment thereof, The separation factor (α) is calculated by the partition coefficient of lipase (K p ) an anti-LAG3 antibody or antigen-binding fragment thereof K p and log α is greater than 0.5 under loading conditions. and (a) a light chain CDR of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10, and 11. The improvement in PS-80 stability was observed when steps (a), (b) and (c) were combined as compared to step (c) alone. This is in response to c).

[0087] In yet another embodiment, a method of formulating an anti-LAG3 antibody or antigen-binding fragment formulation includes: There was, (a) Binding of host cell lipase to anti-LAG3 antibody or antigen under loading conditions passing a load fluid containing the fragments through an HIC resin; (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; and (c) Addition of PS-80 to the formulation enhances the anti-LAG3 antibody or antigen-binding fragment formulating the composition, The separation factor (α) is calculated by the partition coefficient of lipase (K p ) an anti-LAG3 antibody or antigen-binding fragment thereof K p and log α is greater than 0.5 under loading conditions. and (a) a light chain CDR of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10, and 11. .

[0088] In certain embodiments, log α is greater than 1.0 under loading operating conditions. stomach.

[0089] In some embodiments, the log K of the lipase p is 1 under loading operation conditions. In another embodiment, the log K of the lipase is greater than 0. p is the loading operation condition Below 1.5 and above.

[0090] In certain embodiments, under loading operating conditions, log α is greater than 0.5. Lipase log K p is greater than 1.0. Under the operating conditions, the log α is greater than 0.5 and the log K p is 1.5 In other embodiments, under loading operating conditions, log α is greater than 1.0. The log K of lipase is also large. p is greater than 1.0. Under loading operating conditions, log α is greater than 1.0 and the log K of lipase p teeth Greater than 1.5.

[0091] In another aspect, there is provided a method for improving PS-80 stability in an anti-LAG3 antibody formulation, comprising: , (a) A load fluid containing host cell lipase and anti-LAG3 antibody was passed through the HIC resin. To do so, (b) Elution solution was used to remove anti-LAG3 antibody from the chromatography resin under the elution operating conditions. eluting the body, and (c) Formulating the anti-LAG3 antibody so that the anti-LAG3 antibody formulation is a PS-80-containing solution. This includes: α is the K pof anti-LAG3 antibody p is the ratio of the elution Provided herein is a method for improving PS-80 stability, which is greater than 0.5 under operating conditions. is for steps (a), (b) and (c) compared to step (c) alone.

[0092] In another aspect, there is provided a method of formulating an anti-LAG3 antibody formulation, comprising: (a) A load fluid containing host cell lipase and anti-LAG3 antibody was passed through the HIC resin. To do so, (b) Elution solution was used to remove anti-LAG3 antibody from the chromatography resin under the elution operating conditions. eluting the body, and (c) Formulating an anti-LAG3 antibody by adding PS-80 to the formulation. fruit, α is the K p of anti-LAG3 antibody p is the ratio of the elution Provided herein is a method whereby the .alpha.-to-.alpha.

[0093] In certain embodiments, log α is greater than 1.0 under elution operating conditions.

[0094] In some embodiments, the log K of the lipase p is greater than 1.0 under the elution operating conditions. In another embodiment, the log K p is greater than 1.5 under the elution operating conditions. big.

[0095] In certain embodiments, under elution operating conditions, log α is greater than 0.5 and the reactivity is Log K of p is greater than 1.0. In some embodiments, under elution operating conditions , log α is greater than 0.5, and the log K of lipase pis greater than 1.5. In this embodiment, under the elution operating conditions, the log α is greater than 1.0 and the lo of the lipase is gK p In yet another embodiment, under elution operating conditions, log α is greater than 1.0 and the log K of lipase p is greater than 1.5.

[0096] In certain embodiments, the lipase is PLBL2, LPL, LPLA2, LP-PL In one embodiment, the lipase is selected from the group consisting of PLBL2 and LAL. In another embodiment, the lipase is LPL. In yet another embodiment, the lipase is LPLA2. In one embodiment, the lipase is LP-PLA2. In another embodiment, the lipase is LAL. In yet another embodiment, the lipase comprises 6, 7, 8, 9, 10 or more different lipases. In this study, lipases were synthesized from PLBL2, LPL, LPLA2, LP-PLA2, and LAL. In one embodiment, the lipase comprises two, three, four or five different lipases selected from the group consisting of: In another embodiment, the lipase comprises PLBL2 and LPL. In yet another embodiment, the lipase comprises PLBL2 and LPLA2. In another embodiment, the lipase comprises PLBL2 and LA In one embodiment, the lipase comprises LPL and LPLA2. In yet another embodiment, the lipase comprises LPL and LP-PLA2. In another embodiment, the lipase includes LPL and LAL. In one embodiment, the lipase comprises LPLA2 and LAL. In another embodiment, the lipase includes LP-PLA2 and LAL. In an embodiment, the lipase includes PLBL2, LPL, and LPLA2. In one embodiment, the lipase comprises PLBL2, LPL, and LP-PLA2. In another embodiment, the lipase comprises PLBL2, LPL, and LAL. In yet another embodiment, the lipase comprises: In another embodiment, the lipases include PLBL2, LPLA2, and LAL. In one embodiment, the lipase comprises PLBL2, LP-PLA2, and LAL. In another embodiment, the lipase comprises LPL, LPLA2, and LP-PLA2. In yet another embodiment, the lipase comprises LPL, LPLA2, and LAL. In another embodiment, the lipase comprises LPLA2, LPLA3, LPLA4, LPLA5, LPLA6, LPLA7, LPLA8, LPLA9, LPLA10, LPLA11, LPLA22, LPLA13, LPLA14, LPLA15, LPLA16, LPLA17, LPLA18, LPLA19 ... In one embodiment, the lipase comprises PLBL2, LPL, LAL. In another embodiment, the lipase comprises PLBL2, L In yet another embodiment, the lipase comprises PLBL, LPLA2, and LAL. In another embodiment, the lipase includes: PLBL2, LPLA2, LP-PLA2, and LAL. In the present study, lipases include PLBL2, LPL, LPLA2, LP-PLA2, and LAL. nothing.

[0097] In some embodiments of the various methods provided herein, the lipase is a Chinese In certain embodiments, the lipase is a CHO cell lipase. Lipases are a group consisting of PLBL2, LPL, LPLA2, LP-PLA2 and LAL In one embodiment, the CHO cell lipase is PLBL2. In yet another embodiment, the CHO cell lipase is LPL. In one embodiment, the CHO cell lipase is LP-PLA2. In another embodiment, the CHO cell lipase is LAL. O-cell lipases can be expressed as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different CH In yet another embodiment, the CHO cell lipase is PLBL 2, two selected from the group consisting of LPL, LPLA2, LP-PLA2 and LAL; In one embodiment, the CHO cell lipases include three, four, or five different CHO cell lipases. The lipase includes PLBL2 and LPL. In another embodiment, the CHO cell lipase is In yet another embodiment, the CHO cell lipase comprises In another embodiment, the CHO cell lipase In one embodiment, the CHO cell lipase comprises LPL and LAL. In another embodiment, the CHO cell lipase comprises LPL and LPLA2. In yet another embodiment, the CHO cell lipase comprises LPL and LAL. In another embodiment, the CHO cell lipase comprises LPLA2 and LP-PLA In one embodiment, the CHO cell lipase comprises LPLA2 and LAL. In this embodiment, the CHO cell lipases include LP-PLA2 and LAL. In an embodiment, the CHO cell lipases include PLBL2, LPL and LPLA2. In another embodiment, the CHO cell lipase is PLBL2, LPL, and LP-PLA In one embodiment, the CHO cell lipase comprises PLBL2, LPL, and LAL. In another embodiment, the CHO cell lipase comprises PLBL2, LPLA2 and LP- In yet another embodiment, the CHO cell lipase comprises PLBL2, LPL In another embodiment, the CHO cell lipase includes PLBL2, A2, and LAL. In one embodiment, the CHO cell lipase includes LPL, LP-PLA2, and LAL. In another embodiment, the CHO cell lipase comprises L In yet another embodiment, the CHO cell lipase comprises PL, LPLA2, and LAL. In another embodiment, the CHO cell lipase comprises: The enzymes include LPLA2, LP-PLA2, and LAL. Lipases include PLBL2, LPL, LPLA2, and LP-PLA2. In certain embodiments, CHO cell lipases include PLBL2, LPL, LPLA2, and LAL. In yet another embodiment, the CHO cell lipase is PLBL2, LPL, LP-PLA2 In another embodiment, the CHO cell lipase includes PLBL2, LP In yet another embodiment, the CHO cells Lipases include PLBL2, LPL, LPLA2, LP-PLA2 and LAL.

[0098] In certain embodiments of the various methods provided herein, the operating conditions include adding salt. The method further includes adjusting the ionic strength and / or conductivity of the operating solution by In one embodiment, the operating conditions include adjusting the ionic strength of the operating solution by adding salt. In another embodiment, the operating conditions further include: In yet another embodiment, the operating conditions further include adjusting the conductivity. It further includes adjusting the ionic strength and conductivity of the operating solution by In this embodiment, the effect of adding salt is to achieve the desired log α. In this form, the effect of adding salt is to increase the desired log K of the lipase. p The goal is to achieve In yet another embodiment, the effect of adding salt is to balance the desired log α and the desired lo of lipase. gK p The goal is to achieve this.

[0099] In some embodiments, the salt in the working solution is sodium chloride, sodium acetate, phosphorus From the group consisting of sodium sulfate, ammonium sulfate, sodium sulfate and Tris-HCl In one embodiment, the salt is sodium chloride. In another embodiment, the salt is vinegar. In yet another embodiment, the salt is sodium phosphate. In an embodiment, the salt is ammonium sulfate. In one embodiment, the salt is sodium sulfate. In another embodiment, the salt is Tris-HCl.

[0100] In another specific embodiment, the concentration of sodium sulfate in the working solution is about 500 mM to The concentration was about 620 mM, the chromatography resin was HIC, and the operating conditions were pH about 7. be.

[0101] In yet another specific embodiment, the concentration of sodium sulfate in the working solution is about 510 The chromatography resin is HIC, and the operating conditions are pH It is about 7.

[0102] In further embodiments of the various methods provided herein, the load fluid is In one embodiment, the eluate is obtained from a pre-chromatographic process. The filtration process comprises affinity chromatography. The previous chromatographic process was affinity chromatography followed by non- In yet another embodiment, affinity chromatography is used. In another embodiment, the chromatography is Protein A chromatography. Affinity chromatography is AEX chromatography. In embodiments, the prior chromatography process is Protein A chromatography. In one embodiment, the load fluid comprises a binding and Protein A chromatography performed in flow-through mode followed by Protein A chromatography in flow-through mode This is the eluate from AEX chromatography performed at 100°C.

[0103] Pharmaceutical Composition The present disclosure also provides a method for producing an anti-LAG3 antibody or antigen-binding fragment in a culture medium containing less than 2 ppm of host cell lipids. 1. A pharmaceutical composition comprising an anti-LAG3 antibody or antigen-binding fragment thereof, the anti-LAG3 antibody or antigen-binding fragment comprising: (a) SEQ ID NO: (b) the light chain CDRs of SEQ ID NOs: 6, 7, and 8, and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10, and 11. The present invention provides a pharmaceutical composition comprising:

[0104] In certain embodiments, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment and 1p In another embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody and an anti-LAG3 antibody. and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and less than 0.8 or 0.9 ppm of host cell lipase. The product comprises an anti-LAG3 antibody or antigen-binding fragment and less than 0.1 ppm of host cell lipase. In another embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment and 0.2 In yet another embodiment, the pharmaceutical composition comprises an anti-L AG3 antibody or antigen-binding fragment and less than 0.3 ppm of host cell lipase; and In another embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment and 0.4 ppm In yet another embodiment, the pharmaceutical composition comprises an anti-LA lipase. G3 antibody or antigen-binding fragment and less than 0.5 ppm of host cell lipase. In one embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment and less than 0.6 ppm of a soluble form of the compound. In another embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen. In yet another embodiment, the lipase comprises a binding fragment and less than 0.7 ppm of host cell lipase. The pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment and less than 0.8 ppm of host cell lipoproteins. In another embodiment, the pharmaceutical composition comprises an anti-LAG3 antibody or antigen-binding fragment thereof. and less than 0.9 ppm host cell lipase.

[0105] In certain embodiments of the pharmaceutical composition, the lipase is PLBL2, LPL, LPLA2 In one embodiment, the lipase is selected from the group consisting of LP-PLA2, LP-PLA2, and LAL. In another embodiment, the lipase is LPL. In one embodiment, the lipase is LPLA2. In one embodiment, the lipase is LP-PLA2. In another embodiment, the lipase is LAL. In yet another embodiment, the lipase is 2, and / or containing 3, 4, 5, 6, 7, 8, 9, 10 or more different lipases. In another embodiment, the lipase is PLBL2, LPL, LPLA2, LP-PLA2, and and LAL. In one embodiment, the lipase comprises PLBL2 and LPL. In yet another embodiment, the lipase comprises PLBL2 and LPLA2. In another embodiment, the lipase comprises PLBL2 and LP-PLA2. In one embodiment, the lipase comprises LPL and LPLA2. In another embodiment, the lipase includes LPL and LP-PLA2. In one embodiment, the lipase comprises LPL and LAL. In one embodiment, the lipase comprises LPLA2 and LP-PLA2. In another embodiment, the lipase comprises LP-PLA2 and LAL. In yet another embodiment, the lipase comprises PLBL2, LPL, and LPLA2. In yet another embodiment, the lipase comprises PLBL2, LPL, and LP-PLA2. In an embodiment, the lipases include PLBL2, LPL, and LAL. Lipases include PLBL2, LPLA2, and LP-PLA2. In another embodiment, the lipases include PLBL2, LPLA2, and LAL. In one embodiment, the lipases include PLBL2, LP-PLA2, and LAL. Lipases include LPL, LPLA2, and LP-PLA2. In yet another embodiment, the lipase is: In another embodiment, the lipase is LPL, LP-PLA2, or LAL. In one embodiment, the lipase is PLBL2. In another embodiment, the lipase includes P In yet another embodiment, the lipases include LBL2, LPL, LPLA2, and LAL. In another embodiment, the IL-11 polypeptide comprises PLBL2, LPL, LP-PLA2, and LAL. Lipases include PLBL2, LPLA2, LP-PLA2, and LAL. In another embodiment, the lipase is PLBL2, LPL, LPLA2, LP-PLA2, and and LAL.

[0106] The present disclosure also provides a method for the preparation of an anti-LAG3 antibody or antigen-binding fragment thereof in a composition comprising a soluble ... ) or polysorbate 20 (PS20), and the pharmaceutical composition containing At 3, 6, 9, or 12 months, the concentrations of PS80 or PS20 were The concentration of anti-LAG3 antibody or antigen is maintained at 90%, 95%, or 99% or more. The binding fragment comprises (a) the light chain CDRs of SEQ ID NOs: 6, 7, and 8, and (b) the light chain CDRs of SEQ ID NOs: 9, 10, and 11. and 11 heavy chain CDRs. In one embodiment, the pharmaceutical composition comprises: When formulated, the anti-LAG3 antibody or antigen-binding fragment and the polysorbate are present in a concentration of approximately 0.2 mg / ml. Contains polysorbate 80 (PS80) or polysorbate 20 (PS20) and can be stored at 2-8°C. At 1, 3, 6, 9, or 12 months, PS80 or PS20 concentrations were at least In one embodiment, PS80 is used in the formulation. In one embodiment, PS80 is produced at 2-8°C for 1, 3, 6, 9 or 12 months. In one embodiment, PS80 is maintained at 95% or more of its concentration at the time of formulation. The concentration is maintained at more than 99% of the concentration at the time of conversion.

[0107] The present disclosure also provides, when formulated, a concentration of about 20.0 mg / mL of an anti-LAG3 antibody or anti- original binding fragment, approximately 5.0 mg / mL pembrolizumab, approximately 54 mg / mL sucrose; Approximately 0.2 mg / mL polysorbate 80, pH approximately 5.8, buffered with approximately 10 mM histidine A pharmaceutical composition comprising: a buffer; about 56 mM L-arginine; and about 8 mM L-methionine. or, when formulated, about 25.0 mg / mL of anti-LAG3 antibody or antigen-binding fragment Approximately 50 mg / mL sucrose; approximately 0.2 mg / mL polysorbate 80; pH approximately 5.8, about 10 mM histidine buffer; about 70 mM L-arginine-HCl; and and optionally about 10 mM L-methionine, and At 6, 9, or 12 months, the concentration of PS80 was at least 100% of the formulated concentration. and a pharmaceutical composition in which the purity is maintained at 90%, 95%, 99%, 85% or 80%. .

[0108] In various embodiments of the pharmaceutical compositions described herein, the level of host cell lipase is , liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-multiplex Measured by multiple reaction monitoring (LC-MRM-MS).

[0109] In some embodiments, the pharmaceutical composition is prepared by HIC chromatography comprising the steps of: It can be obtained by the process: (a) passing a load fluid containing the composition through an HIC resin; and (b) an elution solution having a pH of about 5 to about 7.5 and a conductivity of about 25 to 80 mS / cm; or eluting the anti-LAG3 antibody or antigen-binding fragment thereof using (c) a rody having a pH of about 5 to about 7.5 and a conductivity of about 25 to 80 mS / cm; Using the elution operating conditions, the anti-LAG3 antibody or its antigen-binding fragment was released into the flow-through. The collecting process.

[0110] In another embodiment, the pharmaceutical composition is prepared by a HIC chromatography process comprising the steps of: can be obtained by: (a) passing a load fluid containing the composition through an HIC resin; and (b) an elution solution having a pH of about 5 to about 7.5 and a conductivity of about 50 to 70 mS / cm; or eluting the anti-LAG3 antibody or antigen-binding fragment thereof using (c) a rody having a pH of about 5 to about 7.5 and a conductivity of about 50 to 70 mS / cm; Using the elution operating conditions, the anti-LAG3 antibody or its antigen-binding fragment was released into the flow-through. The collecting process.

[0111] In another embodiment, HIC chromatography is preceded by a column operated in bind and elute mode. Protein A chromatography, which is performed in flow-through mode, and AEX chromatography, which is performed in flow-through mode. Chromatography comes first.

[0112] [Example] The examples in this section (Section VI) are provided by way of illustration and not limitation.

[0113] [Example 1] Different species of K P How to determine partition coefficient K P is a known liquid concentration of a protein (or other molecule of interest) in a known volume. The resulting mixture is mixed with the chromatography resin, separating the proteins bound to the resin and the proteins remaining in the liquid. It is determined by calculating the ratio of: K P =q / c=[bound] / [free].

[0114] In subsequent Examples 2-3, the chromatographic volume was 20 μL and the liquid volume was 2 The volume was 0.00 μL and the protein concentration was 0.5 mg / mL. / mL effective resin loading provides a phase ratio of 10:1.

[0115] Screening was performed using 96-well filter plates (P / N MSBVN1250, M The resin and liquid were vigorously mixed in a centrifuge (Sigma, Burlington, MA). The resin was thoroughly mixed and then separated from the liquid by vacuum filtration. It was as follows: (a) Three equilibrations (buffer containing no feed), each with a 10 min incubation. tion; (b) one feed mix, 60 min incubation; and (c) Two stripping conditions, 10 min incubation for each step The equilibration step allows for buffer exchange from the initial resin slurry buffer. The 60 min time for the pseudo-equilibrium between the resin ligand and protein under a given set of conditions is The filtrate from the feed process is measured by UV absorbance at 280-320 nm. The final liquid concentration of the protein, c, was determined by the above procedure. The bound concentration of the protein, q, was determined around c and It was determined by mass balance with a known feed concentration c0 (0.5 mg / mL).

[0116] The distribution is generally expressed as log K P It has been reported that log K P is herein The log K can be accurately quantified down to about 0-2 using the UV method described. P Scree The general rules for training are as follows: log K P ≥1.5, strong bond to resin; log K P <1, conditions under which elution is predicted for the bound elution regime; 0.5 <log K P <1, weak interaction condition indicating some binding; log K P <0.5, little or no binding;

[0117] The log Kp of different species can also be calculated by calculating the separation factor α as follows: is used to predict the separation of species: α = K P、タンパク質1 / K P、タンパク質 2;log α=log K P、タンパク質1 -log K P、タンパク質2 , where: The further log α is from 0, the better the separation. In the following examples, α=K P、 リパーゼ / K P、mAb ;log α=log K P、リパーゼ -log K P、mAb A log α greater than 0.5 indicates a good separation between lipase and monoclonal antibody. A log α of less than -0.5 also indicates good separation between lipase and monoclonal antibody. This indicates good separation from the local antibody.

[0118] [Example 2] K of PLBL2 and mAb under typical treatment conditions P Comparing values K P and α using the method to determine the operating conditions for the anti-LAG3 antibody Ab6. In this study, the known lipase impurity PLBL2 was isolated by various chromatographic processes. The ability to separate was evaluated.

[0119] Table 2 shows the log K values ​​of Ab6 and PLBL2 under several treatment conditions for Ab6. p-values ​​and log α-values ​​are summarized. [Table 2]

[0120] In the Protein A process, PLBL2 does not have affinity, so most of the PLBL2 is expected to flow through the Protein A resin during the loading or wash steps The only PLBL2 present in the pool was due to insufficient washing or Ab It is likely related to 6.

[0121] In the CEX process, Ab6 has lower binding at lower salts, resulting in a higher binding across the salt range. In the AEX process, Ab6 has a significantly more robust log α than PLBL2. It binds strongly to the resin, resulting in a negative log α under loading and washing conditions. When operated in low-through mode, there was no separation potential, indicating that Ab6 The enrichment of PLBL2 in the flow-through due to relatively strong binding may also be demonstrated.

[0122] An additional HIC process was also tested for Ab6. , a relatively high log K across load and elution conditions P Comparison of elution salt concentrations The results suggest that the α values ​​were relatively large in the relatively low salt range. indicates that both Ab6 recovery and PLBL2 isolation are more favorable.

[0123] [Example 3] K of PLBL2 and LPLA2 under various conditions for HIC resins P Value Mapping Various buffers that may be used for downstream processing of Ab6 (mAb2) and mAb3 The partition coefficients of PLBL2 and LPLA2 were calculated for HIC resins with the following conditions. (Table 3). [Table 3]

[0124] Adjust the sodium sulfate concentration to a buffer condition of 20 mM sodium phosphate (pH 7.0) By this, PLBL2 and L were added to HIC resin, Tosoh Butyl-650M. Partitioning studies of PLA2 were performed (Table 3, Figure 1). Both lipases exhibit strong binding in high salt conditions. Typical HIC behavior (log K P >1.5, 250mM for PLBL2 (more than 400 mM sodium sulfate for LPLA2, and 400 mM sodium sulfate for LPLA2), low salt In this case, the distribution is reduced (log K P <1, <150 mM sulfate for PLBL2 sodium, and 200 mM sodium sulfate for LPLA2).

[0125] The HIC resin, Tosoh Butyl-650M (Figure 2), was used under the conditions listed in Table 3. The partitioning of the antibody and lipase was also compared. This condition resulted in little separation between 3 and PLBL2, and 300 mM sulfuric acid Only sodium provides some separation, with a log α of about 0.3. Sodium sulfate from 0 to 400 mM gives somewhat better separation with a log α of about 0.5. In contrast, Ab6 is much less hydrophobic than mAb3, PLBL2, or LPLA2. Therefore, the log K of 1.5 is maintained until sodium sulfate exceeds 600 mM. P Exceeding For Ab6 and PLBL2, the binding to the HIC resin is 1.5-2 A log α value of 0.0 can be achieved with 300-500 mM sodium sulfate, which is It has a very wide salt range with promising separation capabilities for operation in aqueous solutions. Similarly, log α values ​​greater than 1 are observed in this same salt range.

[0126] [Example 4] Hydrophobic interaction chromatography purification of an anti-LAG3 antibody preparation by flow-through method The harvested cell culture medium containing Ab6 was subjected to Protein A affinity chromatography as described in Example 2. ion exchange chromatography and anion exchange chromatography, and hydrophobic interactions The product was subjected to hydrophobic interaction chromatography (Tosoh To The Yopearl Butyl-650M process has a target loading of 150g / L resin. The virus filtrate containing the anti-LAG3 antibody Ab6 was run in flow-through mode at room temperature. The permeate was adjusted to 560 mM Na2SO4 with 1.4 M Na2SO4, and g of virus filtered product was adjusted to 0.77 kg of 1.4M Na2SO4. After adding Na2SO4, the feed was diluted with 1 M Tris base to a target pH of 7.0. Titrate and obtain the HIC load. Table 4 shows the HIC chromatography: column equilibration, HI C chromatographic process operating steps and parameters are detailed. Column effluent The absorbance of the solution was monitored online at a wavelength of 280 nm to measure the unadjusted HIC product. The unadjusted HIC product was diluted to a target pH of 5.8 with 1 M acetic acid solution. After pH adjustment, 1 kg of the HIC product was diluted with 2 kg of 10 mM histidine, 7 Diluted with 0 mM arginine pH 5.8 and Millipore SHC 0.5 / 0 The solution was filtered through a 0.2 μm filter and then ultrafiltered (Ultrafiltration The unfiltered and difiltrated (UFDF) load was obtained. [Table 4]

[0127] Liquid chromatography-multiple reaction monitoring (LC-MR) was performed as described below. Lipase identification by M-MS was compared with the corresponding chromatographic strip samples. Both in-process intermediates of the above batches were tested (Table 5). PLBL2 was the load sample. was found in the HIC flow-through sample but was absent from the HIC flow-through sample.

[0128] Multiple reaction monitoring mass spectrometry (MRA) was performed using a Waters TQS triple quadrupole MS. Reversed-phase ultra-high performance liquid chromatography (RP-UPLC-MRM MS) was used. An 8-minute LC-MRM assay was developed to quantify CHO lipases PLBL2 and LPLA2. MS methods are used to characterize bioprocess intermediates and / or biopharmaceutical drug substances (ng / mg or p It is a lipase-specific quantitative assay that provides absolute quantification of two lipases in each The assay quantification range of lipase from 1 to 500 ng / mg was determined for CHO recombinant PLBL2 and LPLA2 (MyBioSource) was used as a protein standard for Ab6 drug substances. and PLBL2(H2N-LTFPTGR( 13 C6, 15 N4-OH) SEQ ID NO: 12 and LPLA2(H2N-IPVIGPLK( 13 C6, 15 N2)-OH SEQ ID NO: 1 3) (New England Peptide) C13- and N15-heavy labeled peptides This is achieved by adding a nucleotide as an internal standard (IS). The standards were denatured, the S—S bonds reduced and alkylated, and trypsinized prior to LC-MS analysis. The digested samples were analyzed by a Waters Acquity UPLC BEH Load onto a C18 column (50 × 2.1 mm, 1.7 μm) at a flow rate of 0.2 mL / min. The separation was performed using a gradient of 10 to 35% mobile phase B (0.1% formic acid in acetonitrile) Mobile phase A was an aqueous solution of 0.1% formic acid. (Isotopic / IS) vs. analyte concentration), and 1 / x for linear regression 2 PLB by weighting coefficient For the quantification of L2 and LPLA2, MR of surrogate peptides generated by trypsin digestion was used. M transition, m / z 39 of PLBL2 peptide LTFPTGR (SEQ ID NO: 12) 6.5 (precursor ion) -> m / z 430.3 (fragment ion), and LPLA 2. Peptide IPVIGPLK (SEQ ID NO: 13) m / z 419.1 (precursor ion) -> m / z 362.3 (fragment ion) was used. [Table 5]

[0129] [Example 5] Hydrophobic interaction chromatography purification of an anti-LAG3 antibody preparation by bind-and-elute method The harvested cell culture medium containing Ab6 was purified by Protein A affinity chromatography as described in Example 2. -chromatography and anion exchange chromatography, as well as hydrophobic phases The resulting mixture was subjected to interaction chromatography at room temperature with a target loading of 30 g / L resin. and elution mode in a hydrophobic interaction chromatography step (Tosoh™). Toyopearl Butyl-650M resin) was used. The virus filtrate was adjusted with 1.4M Na2SO4 to obtain 1 kg of virus filtrate. Adjust the amount of NaSO4 to 2 kg. The feed is titrated with 1 M Tris base to a target pH of 7.0 to obtain the HIC load. The HIC load was filtered through a Millipore SHC 0.5 / 0.2 μm filter. The column was then loaded with HCl. Table 6 shows the results of HIC chromatography: column equilibration, and details the operating steps and parameters of the HIC chromatography process. The absorbance of the column effluent was monitored online at a wavelength of 280 nm, and the unadjusted HI The unadjusted HIC product was purified by 5.5% acetic acid using 1 M acetic acid solution. The pH was adjusted to a target pH of 8. After pH adjustment, 1 kg of the HIC product was added to 2 kg of 10 mM HisCl Diluted with 70 mM arginine, pH 5.8, Millipore SH The solution was filtered through a 0.5 / 0.2 μm filter and then ultrafiltered (U FDF) load was obtained. [Table 6]

[0130] Regarding the identification of lipases by liquid chromatography-mass spectrometry (LC-MS), In-process intermediates from the above batches were tested along with chromatography strip samples. PLBL2 and clusterin were detected in the load and strip samples (Table 7). was found in the elution pool sample but was absent from the HIC elution pool sample.

[0131] HC including HCP identification and relative quantification of bioprocess intermediates and drug substances (DS) HCP proteomics by LC-MS / MS to provide P profiling Tandem MS data are acquired in data-dependent acquisition or DDA mode) Denaturation, DTT reduction, IAA alkylation, and trypsin digestion were performed using HIC column load. Samples including the elution pool, HIC column elution pool, and HIC column strip sample were provided. Then, a Waters H-class UPLC-Thermo QE Orbitrap system was used. The digested samples were analyzed by LC-MS / MS (DDA) performed using a chromatographic system. ACQUITY UPLC PEPTIDE CSH C18 Column (130 Å, 1.7 μm, 1 × 150 mm) was used for separation, and an aqueous solution of 0.1% FA and 0.1% A solution of FA in ACN and FA in HCl were used as mobile phases A and B. For protein identification, Th The CHO database was searched using ermo PD 2.2 (MS mass accuracy ≤ 1 0 ppm, and for MS / MS ≤ 0.02 Da; ≤ 1% FDR; per protein ≥ 2 unique peptide IDs). Relative quantification of HCPs was performed using the corresponding peptides extracted by PD 2.2. This is achieved by the Σ XIC MS1 peak area of ​​the unique peptide(s). do. [Table 7]

[0132] [Example 6] PS-80 stability increased as host cell lipase was removed Ab6A injection is a sterile, preservative-free solution that requires dilution for intravenous infusion. Ab6A is a combination of the anti-LAG3 antibody Ab6 and the anti-PD-1 antibody MK-3475 (pembrolizumab ) and each single-use vial contains 40 mg in a 2.0 mL fill. The pharmaceutical composition contains 20.0 mg / mL of Ab6 and 10 mg of MK-3475. Ab6, 5.0 mg / mL MK-3475, 54 mg / mL sucrose; 0.2 m g / mL polysorbate 80, pH 5.8, 10 mM histidine buffer; 56 mM L-arginine; and 8 mM L-methionine. Ab6 drug from Example 4 The substance was used to formulate the Ab6A drug product.

[0133] For Ab6A drug products, stability was assessed using polysorbate 80 (PS-80) for up to 3 months. At 5°C, there was almost no change in the PS-80 content (%) at 3 months (Fig. 4). At 25°C, no degradation of PS-80 was observed at 3 months (0.19 mg / ml). A slight decrease was observed (0.18 mg / ml), and at 40°C, a slight increase was observed over the same interval. A significant decrease was observed in (0.16 mg / ml).

[0134] Post column switch and charged aerosol A mixed-mode column (Waters Oasis Max) coupled with chromatographic detection (CAD) was used. High-performance liquid chromatography (HPLC) with a column, 2.1 × 20 mm, 30 μm ) was used to determine polysorbate 80. Corona CAD was used to determine the amount of polysorbate 80 dissolved from the column. Responds to essentially all non-volatile compounds and some semi-volatile compounds in the sample A mass-sensitive detector was used. The gradient setting was 1 mL / min. Mobile phase A: 0.5% (v / v) Aqueous acetic acid solution, and Mobile phase B: 0.5% (v / v) acetic acid in isopropyl alcohol solution The calculation of polysorbate 80 concentration was performed using a quadratic fitted calibration line ( quadratic fit calibration line) and the sample Report as polysorbate 80 concentration in solution (mg / mL).

[0135] PS-80 stability was assessed using two Ab6 purified from two-column and three-column purification schemes. A comparison was made between two drug substance (DS) samples. The two-column purification scheme included Protein A and The resulting AEX pool (AEX) was diluted with 25 mg / mL of Ab6; 50 mg / mL of Ab6; 0.2 mg / mL sucrose; 0.2 mg / mL polysorbate 80; pH 5.8, 10 m 70 mM histidine buffer; and 70 mM L-arginine-HCl, formulated in "AEX The three-column purification scheme includes Protein A, AEX, and HIC binding. and elution, or flow-through (HIC B&E DS or HIC FT DS). The resulting HIC pool was diluted with 25 mg / mL Ab6; 50 mg / mL sucrose; 0.2 mg / mL polysorbate 80; pH 5.8, 10 mM L-histidine Buffer: Formulated with 70 mM L-arginine and 10 mM L-methionine. The samples were placed in a stability chamber at 5°C ± 3°C; 25°C ± 3°C; and 60% ± 5% relative humidity (RH). Samples were removed and tested for PS-80 concentrations at 2-, 4-, 6-, and 14-week intervals.

[0136] As shown in Figure 3, the PS-80 concentration in AEX DS increased from 0.20 (week 0) at 5°C. The decomposition of PS-80 increased with increasing storage temperature. For example, at 25°C, the PS-80 concentration in AEX DS increased with increasing temperature. (week 0) to 0.12 mg / mL (week 6). In both the HIC FT DS and the HIC FT DS, the PS-80 concentration changed significantly over time at both temperatures. The assay variability of the PS-80 stability method is ±10%. In this case, a drift of ≤ ±10% from the initial reported value can be considered to be similar. Therefore, the presence of PLBL2 in the AEX pool is related to the 5-25% of the AEX DS. It is hypothesized that this may be one potential cause of the decrease in PS-80 concentration at ℃. Addition of rum effectively removes lipase and stabilizes PS-80 in HIC DS. It can improve sexuality. [Table 8]

[0137] Purified as per Example 4, 25 mg / mL Ab6; 50 mg / mL sucrose; 0 0.2 mg / mL polysorbate 80; pH 5.8, 10 mM L-histidine buffer Ab6 drug substance formulated in 70 mM L-arginine and 10 mM L-methionine Additional PS80 stability tests were performed on vials in a stability chamber at 5°C ± 3°C. Samples were taken and the PS-80 concentration was measured at 1, 3, 6, 9, and 12 month intervals. The PS-80 concentration did not change significantly over time, confirming the PS-80 stability test. Assay variability was within ±10%. [Table 9]

Claims

1. The hydrophobic interaction chromatography (HIC) process was used to isolate anti-LAG3 antibodies or Method for separating host cell lipase from a composition comprising an antigen-binding fragment and host cell lipase And, (a) passing a load fluid containing the composition through an HIC resin under loading operating conditions; and (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; The separation factor (α) is the partition coefficient (K ｐ The anti-LAG3 antibody or antigen of K of the binding fragment ｐ and log α is greater than 0.5 under the loading operation conditions. and the anti-LAG3 antibody or antigen-binding fragment is selected from the group consisting of: (a) SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

2. The hydrophobic interaction chromatography (HIC) process was used to isolate anti-LAG3 antibodies or Method for separating host cell lipase from a composition comprising an antigen-binding fragment and host cell lipase And, (a) passing a load fluid containing the composition through an HIC resin under loading operating conditions; and (b) removing the anti-LAG antibody from the chromatography resin using an elution solution under elution operating conditions.

3. Eluting the antibody or antigen-binding fragment; The separation factor (α) is the partition coefficient (K ｐ The anti-LAG3 antibody or antigen of K of the binding fragment ｐ and log α is greater than 0.5 under the elution conditions. the anti-LAG3 antibody or antigen-binding fragment comprises (a) a light chain C of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

3. The anti-LAG3 antibody or antigen-binding fragment is then transferred to the host by a cation exchange (CEX) process.

1. A method for separating a host cell lipase from a composition comprising a host cell lipase, the method comprising: (a) passing a load fluid containing the composition through a CEX resin under loading operating conditions; and (b) removing the anti-LAG antibody from the chromatography resin using an elution solution under elution operating conditions.

3. Eluting the antibody or antigen-binding fragment; The separation factor (α) is the partition coefficient (K ｐ The anti-LAG3 antibody or antigen of K of the binding fragment ｐ and log α is greater than 0.5 under the elution conditions. the anti-LAG3 antibody or antigen-binding fragment comprises (a) a light chain C of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

4. Polysorbate-80 (PS-80) in anti-LAG3 antibody or antigen-binding fragment formulations 1. A method for improving stability, comprising: (a) Host cell lipase and anti-LAG3 antibody or antigen binding under loading operating conditions passing a load fluid containing the fragments through an HIC chromatography resin; (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; and (c) the anti-LAG3 antibody or antigen-binding fragment preparation is a PS-80-containing solution; formulating the anti-LAG3 antibody or antigen-binding fragment thereof; The separation factor (α) is the partition coefficient (K ｐ The anti-LAG3 antibody or antigen of K of the binding fragment ｐ and log α is greater than 0.5 under the loading operation conditions. and the anti-LAG3 antibody or antigen-binding fragment is selected from the group consisting of: (a) SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

5. The method according to any one of claims 1 to 4, wherein log α is greater than 1.0 under elution operating conditions. How to post.

6. The log K of the lipase ｐ is greater than 1.0 How to do it.

7. The log K of the lipase ｐ is greater than 1.5 How to do it.

8. The method of any one of claims 1 to 6, wherein the lipase is a CHO cell lipase. 。

9. The lipase may be phospholipase B-like 2 (PLBL2), lipoprotein lipase (L PL), lysosomal phospholipase A2 (LPLA2), phospholipase A2 VII ( lysosomal acid lipase A (LAL), The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 8, wherein the lipase is PLBL2.

11. The method according to any one of claims 1 to 8, wherein the lipase is LPLA2.

12. The loading conditions or elution solution may contain sodium chloride, sodium acetate, sodium phosphate, thorium sulfate, ammonium sulfate, sodium sulfate, and Tris-HCl.

5. The method according to claim 1, wherein the pH of the solution is about 5 to 7.

5. The method described.

13. 13. The method of claim 12, wherein the salt is potassium chloride or sodium chloride.

14. The concentration of sodium chloride in the working solution is about 100 mM to about 225 mM, and 13. The method of claim 13, wherein the roughy resin is CEX and the operating conditions have a pH of about 5.0 to about 6.

0. The method described below.

15. The concentration of sodium chloride in the working solution is about 150 mM to about 180 mM, and the operating conditions have a pH of about 5.0 to about 6.

0.

14. The method according to claim 13.

16. Anti-LAG3 antibody or antigen binding was achieved by a hydrophobic interaction chromatography process. and isolating PLBL2 or LPLA2 from a composition containing the fragment and PLBL2 or LPLA2. A method of separating (a) passing a load fluid containing the composition through a hydrophobic interaction chromatography resin; and (b) collecting the anti-LAG3 antibody or antigen-binding fragment in the flow-through; The load fluid has a conductivity of about 25 to 80 mS / cm, and the anti-LAG3 antibody or the antigen-binding fragment comprises (a) the light chain CDRs of SEQ ID NOs: 6, 7, and 8; and (b) the light chain CDRs of SEQ ID NO: 9, and heavy chain CDRs 10 and 11.

17. Anti-LAG3 antibody or antigen binding was achieved by a hydrophobic interaction chromatography process. and isolating PLBL2 or LPLA2 from a composition containing the fragment and PLBL2 or LPLA2. A method of separating (a) passing a load fluid containing the composition through a HIC resin; and (b) removing the anti-LAG3 antibody or antigen-binding fragment from the HIC resin using an elution solution. wherein the elution solution has a conductivity of about 25 to 80 mS / cm; the anti-LAG3 antibody or antigen-binding fragment comprises: (a) a light chain CD4 receptor of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

18. 17. The load fluid or elution solution has a conductivity of about 50-70 mS / cm. Or the method according to 17.

19. The load fluid or elution solution contains about 300 mM to about 650 mM of a monovalent or divalent salt. The method according to claim 16 or 17.

20. the salt is about 500-620 mM sodium sulfate and the pH is about 5-7.5; 20. The method of claim 19.

21. the salt is 560 mM sodium sulfate, and the pH of the load fluid or elution solution is 21. The method of claim 20, wherein the pH is about 7.

22. The load fluid is a Protein A chromatography column run in bind and elute mode. , followed by the eluate from AEX chromatography performed in flow-through mode.

22. The method according to any one of claims 1 to 21.

23. Compositions comprising an anti-LAG3 antibody or antigen-binding fragment and less than 2 ppm of host cell lipase 1. The method of claim 1, wherein the anti-LAG3 antibody or antigen-binding fragment comprises: (a) a polypeptide selected from the group consisting of SEQ ID NOs: 6, 7, and 8; and (b) the heavy chain CDRs of SEQ ID NOs: 9, 10 and 11.

24. 24. The composition of claim 23, comprising less than 1 ppm host cell lipase.

25. The lipase is derived from PLBL2, LPL, LPLA2, LP-PLA2 and LAL. The composition of any one of claims 23 to 24, selected from the group consisting of:

26. The composition according to any one of claims 23 to 24, wherein the lipase is PLBL2.

27. The host cell lipase levels are determined by liquid chromatography-mass spectrometry (LC-MS). or measured by liquid chromatography-multiple reaction (LC-MRM-MS), The composition according to any one of claims 23 to 26.

28. (a) Incubating the anti-LAG3 antibody or antigen-binding fragment with host cell lymphocytes under loading conditions. passing a load fluid containing a composition comprising a lysate and a lysate over the HIC resin; and (b) an elution solution having a pH of about 5 to about 7.5 and a conductivity of about 25 to 80 mS / cm; or eluting the anti-LAG3 antibody or antigen-binding fragment thereof using (c) a rody having a pH of about 5 to about 7.5 and a conductivity of about 25 to 80 mS / cm; and using flow-through operating conditions to induce the anti-LAG3 antibody or antigen-binding fragment thereof to pass through the flow-through. The process of collecting 28. The method according to claim 23, wherein the method is obtainable by a HIC process comprising: The composition described.

29. (a) combining the anti-LAG3 antibody or antigen-binding fragment with host cells under loading conditions; passing a load fluid containing a composition comprising a cellular lipase through the HIC resin; and (b) an elution solution having a pH of about 5 to about 7.5 and a conductivity of about 50 to 70 mS / cm; or eluting the anti-LAG3 antibody or antigen-binding fragment thereof using (c) a pH of about 5 to about 7.5 and a conductivity of about 50 to 70 mS / cm; and using flow-through operating conditions to induce the anti-LAG3 antibody or antigen-binding fragment thereof to pass through the flow-through. The process of collecting 28. The method according to claim 23, wherein the method is obtainable by a HIC process comprising: The composition described.

30. HIC chromatography was preceded by Protein A chromatography operated in bind and elute mode. and AEX chromatography operated in flow-through mode.

30. The composition of any preceding claim 28 or 29.

31. Anti-LAG3 antibody or antigen-binding fragment and polysorbate 80 (PS80) or polysorbate 80 (PS80) and PS20, and the pharmaceutical composition is maintained at 2-8°C for 1 month, 3 months, 6 months, or At 1 month, 9 months, or 12 months, the concentrations of PS80 or PS20 were The anti-LAG3 antibody or antigen-binding fragment is maintained at 90% or more of its initial concentration, and a) light chain CDRs of SEQ ID NOs: 6, 7 and 8; and (b) heavy chains of SEQ ID NOs: 9, 10 and 11. and a pharmaceutical composition comprising the CDR.

32. 32. The method of claim 31, wherein the formulation contains about 0.2 mg / mL of polysorbate 80. A pharmaceutical composition comprising:

33. about 20.0 mg / mL of the anti-LAG3 antibody or antigen-binding fragment when formulated, about 5.0 mg / mL pembrolizumab, about 54 mg / mL sucrose; about 0.2 mg / mL of about 10 mM histidine buffer, polysorbate 80, pH about 5.8; about 56 mL 32. The medicament of claim 31, comprising: M L-arginine; and about 8 mM L-methionine. composition.

34. about 25.0 mg / mL of the anti-LAG3 antibody or antigen-binding fragment; about 50 mg / mL Sucrose; about 0.2 mg / mL polysorbate 80; pH about 5.8, about 10 mM Histidine buffer: containing about 70 mM L-arginine-HCl and about 10 mM L-methyl 32. The pharmaceutical composition of claim 31, which may also contain onin.

35. the anti-LAG3 antibody or antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO: 5, The method of any one of claims 1 to 34, wherein the chain comprises a light chain variable region comprising SEQ ID NO:

4. Method, composition or pharmaceutical composition.

36. the anti-LAG3 antibody comprises a heavy chain and a light chain, the heavy chain comprising SEQ ID NO: 3, and the light chain The method, composition or pharmaceutical composition of any one of claims 1 to 34, wherein the Finished product.

37. The method of any one of claims 1 to 34, wherein the anti-LAG3 antibody is an Ab6 variant. Method, composition or pharmaceutical composition.

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