BR122022014045B1 - METHOD TO IDENTIFY NON-COVALENT INTERACTION SITES OR DIMERIZATION INTERFACES IN A PROTEIN DRUG - Google Patents

Abstract

Systems and methods are provided for characterizing dimerization interfaces at the subdomain level of a protein. An exemplary method includes digesting a protein dimer sample into subdomains, labeling the digested protein sample, isolating the labeled dimeric and monomeric subdomain fragments, and mapping the peptides from the labeled sample to determine where the dimer fragments are labeled and where the dimer fragments are labeled. dimer fragments are not labeled. Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface.

Description

“Divided from BR112020013009-5, deposited on 02/01/2019” CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims benefit and priority to US Provisional Patent Application No. 62/625,732 filed on February 2, 2018 and 62/738,051 filed on September 28, 2018, both of which are incorporated herein by reference , in its entirety.

FIELD OF THE INVENTION

[002] The invention is directed, in general, to methods and analytical systems to characterize non-covalent interaction sites in proteins and fragments thereof.

FUNDAMENTALS OF THE INVENTION

[003] Monoclonal antibodies (mAbs) have been successfully employed to target a wide range of therapeutic areas over the past two decades (Walsh G., Nature biotechnology 2014; 32: 992-1000; Lawrence S. Nature biotechnology 2007; 25: 380 -two) . Protein aggregation remains a major concern in the production of monoclonal antibodies and other therapeutic proteins. Due to the possible impact on potency and immunogenicity, it is important to understand the aggregation mechanism so that appropriate control strategies can be implemented to ensure product quality.

[004] Mapping the dimerization interface on mAb molecules remains challenging due to the complexity and heterogeneity of mAb dimers. Although hydrogen-deuterium exchange mass spectrometry (HDX MS) has been successful in studying protein-protein interactions, little success has been achieved in revealing the mAb dimerization interface, presumably due to the limitations of the method in detecting interactions. of the protein side chain.

[005] Thus, it is an object of the invention to provide systems and methods for mapping heterogeneous protein dimerization interfaces.

[006] It is another object of the invention to provide protein drugs with reduced levels of dimerization.

[007] It is yet another object of the invention to provide methods of producing protein drugs with reduced dimerization.

SUMMARY OF THE INVENTION

[008] Systems and methods are provided for characterizing protein dimerization interfaces at the peptide/residue level of a protein. An exemplary method includes digesting a protein dimer sample into subdomains, labeling the digested protein sample mixture, isolating the labeled dimeric and monomeric subdomain fragments, and mapping peptides from the labeled sample to determine and compare the extent of labeling on the dimer and in the monomer. Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface.

[009] In one embodiment, non-covalent dimers were isolated and analyzed by peptide mapping to locate sites of non-covalent interaction. In another embodiment, to locate the non-covalent dimerization interface at the peptide/residue level, the enriched mAb dimer sample was digested in a mixture of F(ab) homodimer [or F(ab')], F(ab') monomers ) [or F(ab')] and Fc, and subjected to a labeling experiment, followed by fractionation, tryptic digestion and LC-MS analysis to quantify the extent of labeling and compare peptide/residue levels. In the following, more details are provided on the steps of the described methods.

[0010] Yet another embodiment provides a method of producing an antibody, including the steps of culturing the cells producing the antibody in a cell culture, obtaining a sample of the cell culture, characterizing the dimerization interfaces in the antibody sample according to the method described above and modifying one or more culture conditions of the cell culture to reduce the amount of dimerization or non-covalent interaction/aggregation of the antibody produced during cell culture. In some embodiments, the sample is collected during cell culture at any interval. In other embodiments, the sample is taken after culture production, after protein extraction, or after purification. The one or more cell culture conditions that are altered to reduce the amount of dimerization or aggregation can be selected from the group consisting of temperature, pH, cell density, amino acid concentration, osmolality, growth factor concentration, agitation, pressure partial gas, surfactants or combinations thereof. Cells can be eukaryotic or prokaryotic. The cells can be Chinese hamster ovary (CHO) cells (e.g. K1 CHO, DXB-11 CHO, Veggie-CHO), COS cells (e.g. COS-7), retinal cells, Vero cells, CV1 cells , kidney cells (e.g. HEK293, EBNA 293, MSR 293, MDCK, HaK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo25 cells, HB 8065 cells, HL-60 cells, lymphocyte cells, e.g. autologous T cells, Jurkat (T lymphocytes) or Daudi (B lymphocytes), A431 cells (epidermal), U937 cells, 3T3 cells, L cells, C127 cells, SP2/0 cells, NS-0 cells, MMT cells, stem, tumor cells and a cell line derived from any of the aforementioned cells. In one embodiment, the cells are hybridoma or quadroma cells.

[0011] Yet another embodiment provides a method of producing an antibody, including the steps of growing the cells producing the antibody in cell culture, then purifying and formulating the antibody to obtain a formulated drug substance (SDS), obtaining a purified antibody sample before and/or after antibody formulation, characterize the dimerization interfaces in the antibody sample or SDS sample according to the method described above, and modify one or more conditions of the antibody purification or formulation to reduce the amount of dimerization or non-covalent interaction/aggregation of the antibody produced during cell culture. The one or more antibody purification conditions that are altered to reduce the amount of dimerization or aggregation can be selected from the group consisting of temperature, pH, affinity matrix, chromatography resin, buffers, filters, or combinations thereof. The one or more conditions of the antibody formulation that are altered to reduce the amount of dimerization or aggregation can be selected from the group consisting of pH, antibody concentration, excipient concentration, buffers, surfactants, stabilizers, or any combination thereof. One or more excipients in the SDS can be altered to reduce the amount of dimerization or aggregation and are selected from any of the excipients known in the art.

[0012] Yet another embodiment provides an antibody produced by the methods described herein.

[0013] Also provided are methods for providing modified protein drugs with reduced dimerization or non-covalent interaction with an unmodified protein drug. In one embodiment, the protein drug is a monoclonal antibody or antigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a workflow diagram of an example limited digestion and single tag strategy for mAb dimerization interface mapping.

[0015] Figure 2 is a graph of glycine ethyl ester (GEE) hydrochloride percentage (monomer dimer) for the indicated chain/residue, showing the graph of differential labeling of GHG between the Fab dimer and the Fab monomer of mAb-1 generated from Lys-C limited digestion of enriched HMW sample.

[0016] Figure 3A is a bar graph of the percentage of GHG for the indicated antibody fragment of mAb-1 at the peptide level. Figure 3B is a bar graph of the percentage of GHG for the indicated antibody fragment of mAb-2 at the amino acid residue level.

[0017] Figures 4A to 4C are scatter plots showing the Poisson fit of oxidized species for a HMW-limited sample of LysC-digested mAb1. Figure 4A represents the Fc region, Figure 4B represents the LC region and Figure 4C represents the Fd region. The line represents the Poisson fit for each graph.

[0018] Figure 5A is a bar graph of the extent of modification with FPOP for the indicated fragment at the peptide level of mAb-1. Figure 5B is a bar graph of the extent of modification with FPOP for the fragment indicated at the mAb-1 residue level.

[0019] Figures 6A and 6B are scatter plots showing the Poisson fit of oxidized species for lysozyme protein with different focus positions and laser power conditions.

[0020] Figures 7A to 7F are scatter plots showing the Poisson fit of oxidized species for lysozyme protein with various scavenger concentrations (500 μM, 350 μM or 200 μM His) and various laser power settings (97 mJ or 156 mJ).

[0021] Figures 8A to 8D are scatter plots showing the Poisson fit of oxidized species for lysozyme protein with various scavenger concentrations (500 μM or 200 μM His) and various protein concentrations (1 mg/mL or 0, 1mg/mL).

[0022] Figures 9A to 9B are scatterplots showing the Poisson fit of oxidized species for mAb1 (Figure 9A) or for deglycosylated mAb1 (Figure 9B).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0023] The use of the terms “an”, “an”, “the/a” and similar references in the context of describing the currently claimed invention (especially in the context of the claims) shall be interpreted to encompass both singular and plural, unless otherwise indicated herein or clearly contradicted by context.

[0024] The repetition of ranges of values described herein is only intended to serve as a method of reference in abbreviated form to each separate value within the range, unless otherwise indicated herein, and each separate value is incorporated in the descriptive report as if it were individually mentioned again here.

[0025] The use of the term “about” is intended to describe values above or below the stated value within a range of approximately +/10%; in other embodiments, the values may vary in value above or below the indicated value within a range of approximately +/- 5%; in other embodiments, the values may vary in value above or below the indicated value within a range of approximately +/- 2%; in other embodiments the values may vary in value above or below the indicated value within a range of approximately +/- 1%. The above ranges are intended to be clarified by context, and no further limitations are implied. All methods described herein may be performed in any proper order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or example language (eg, "how to") provided herein, is merely intended to further clarify the invention and does not represent a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any element not claimed to be essential to the practice of the invention.

[0026] The term "protein" refers to a molecule composed of two or more amino acid residues linked together by a peptide bond. Protein includes polypeptides and peptides and can also include modifications such as glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Proteins can be of scientific or commercial interest, including protein-based drugs, and proteins include, among other things, enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins. Proteins are produced by various types of recombinant cells using known cell culture methods, and are generally introduced into the cell by transfection of genetically engineered nucleotide vectors (e.g., as a sequence encoding a chimeric protein or a codon-optimized sequence, an intron-free sequence, etc.), and the vectors can reside as an episome or be integrated into the genome of the cell.

[0027] The term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains a CH1 domain, a hinge region domain, a CH2 domain, and a CH3 domain. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with more conserved regions, called main chain regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Each VH/CH1 and VL/CL chain associates (eg, by disulfide bonds) to form a pair, or F(ab), having an antigen-binding site at its N-terminus. Thus, after limited enzymatic digestion , an F(ab) [or F(ab')] fragment is an antibody fragment that still binds antigens, but is monovalent, with no Fc portion. An antibody is typically bivalent with respect to antigen binding and as such includes two F(ab) moieties, known as F(ab')2, linked together by disulfide bridges at the top hinge. F(ab')2 fragments are generated by limited digestion of whole antibodies to remove most of the Fc region, leaving part of the hinge region intact, and can be further reduced to F(ab') fragments. The term "antibody" includes reference to immunoglobulins, both glycosylated and non-glycosylated, of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes bispecific antibody, which includes a heterotetrameric immunoglobulin that can bind more than one different epitope. Bivalent antibody constructs can also be bispecific with respect to the specificity of each antigen-binding site for different antigens. Bispecific antibodies are generally described in US Patent Application Publication No. 2010/0331527, which is incorporated by reference herein.

[0028] "Fc fusion proteins" comprise part or all of two or more proteins, one of which is an Fc portion of an immunoglobulin molecule, that are not found together in nature. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al., Proc. Natl. academic ScL USA 88: 10535, 1991; Byrn et al. (1990) Nature 344:677; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992. "Receptor Fc fusion proteins" comprise one or more extracellular domains of a receptor coupled to an Fc moiety which, in some embodiments, comprises a hinge region followed by a CH2 and CH3 domain of an immunoglobulin. In some embodiments, the Fc fusion protein comprises two or more distinct receptor chains that bind one or more ligands. For example, an Fc fusion protein is a trap, such as an IL-1 trap or a VEGF trap.

[0029] The term "Cell Culture" refers to the propagation or proliferation of cells in a vessel, such as a flask or bioreactor, and includes, but is not limited to, fed-batch culture, continuous culture, perfusion culture, and the like .

[0030] The term “MS/MS” refers to a mass spectrometry technique that uses two mass spectrometers in tandem. Between the two analyzers (MS1 and MS2) is a collision gas cell. Precursor ions selected by MS1 collide with a high pressure gas (usually He) in the cell and undergo fragmentation.

[0031] The term “LC-MS” refers to liquid chromatography-mass spectrometry, which is an analytical chemical technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of spectrometry mass (MS).

[0032] The term "a conservative amino acid substitution" refers to a substitution of one amino acid for another that has similar properties. It is expected that this type of substitution rarely results in dysfunction in the corresponding protein.

II. Protein Drugs

[0033] One embodiment provides a protein drug that has been modified to reduce dimerization or non-covalent interaction of the protein drug. The modification may be a conservative amino acid substitution. In a conservative amino acid substitution, one amino acid is replaced with another that has similar properties. It is expected that this type of substitution rarely results in dysfunction in the corresponding protein.

A. Proteins of Interest

[0034] In one embodiment, the protein drug may contain one or more proteins of interest suitable for expression in prokaryotic or eukaryotic cells. For example, the protein of interest includes, but is not limited to, an antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, an ScFv or fragment thereof, an Fc fusion protein or fragment thereof, a growth factor or fragment thereof, a cytokine or fragment thereof, or an extracellular domain of a cell surface receptor or fragment thereof. Proteins of interest can be simple polypeptides consisting of a single subunit or complex multi-subunit proteins comprising two or more subunits. The protein of interest can be a biopharmaceutical, food additive or preservative, or any protein product subject to purification and quality standards.

[0035] In some embodiments, the protein product (protein of interest) is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, a protein-binding antibody fragment antigen, a single-chain antibody, a diabody, triabody, or tetrabody, a specific tetravalent G-type immunoglobulin-like molecule called dual variable domain immunoglobulin (DVD-IG), an IgD antibody, an IgE antibody, an IgM antibody, an antibody IgG, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody comprises a chimeric hinge. In yet other embodiments, the antibody comprises a chimeric Fc. In one embodiment, the antibody is an IgG2/IgG4 chimeric antibody. In one embodiment, the antibody is an IgG2/IgG1 chimeric antibody. In one embodiment, the antibody is an IgG2/IgG1/IgG4 chimeric antibody.

[0036] In some embodiments, the antibody is selected from the group consisting of an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody, as described in US Patent Application Publication No. US2015/0203579A1), an anti-programmed cell death-1 ligand (e.g., an anti-PD-L1 antibody as described in US Patent Application Publication No. US2015/0203580A1), an anti-Dll4 antibody, an anti-Angiopoietin-2 antibody ( for example, an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-Angiopoietin type 3 antibody (for example, an anti-AngPtl3 antibody as described in U.S. Patent No. 9,018,356), an antibody of anti-platelet-derived growth factor receptor (for example, an anti-PDGFR antibody as described in US Patent No. 9,265,827), an anti-Erb3 antibody, an anti-prolactin receptor antibody (for example, anti- PRLR as described in US Patent No. 9,302,015), an anti-Complement 5 antibody (e.g., an anti-C5 antibody as described in US Patent Application Publication No. US2015/0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (for example, an anti-EGFR antibody as described in US Patent No. 9,132,192 or an anti-EGFRvIII antibody as described in US Patent Application Publication No. US2015/0259423A1) , an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (for example, an anti-PCSK9 antibody as described in US Patent No. 8,062,640 or US Patent Application Publication No. US2014/0044730A1), an anti- Growth and Differentiation Factor-8 (for example, an anti-GDF8 antibody, also known as an anti-myostatin antibody as described in US Patent Nos. 8,871,209 or 9,260,515), an anti-glucagon receptor (for example , anti-GCGR antibody as described in US Patent Application Publication Nos. US2015/0337045A1 or US2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, an interleukin 4 receptor antibody (e.g., an antibody anti-IL4R as described in US Patent Application Publication No. US2014/0271681A1 or US Patent Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody such as described in US Patent Nos. 7,582,298, 8,043,617 or 9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 antibody (for example, anti-IL33 antibody as described in US Patent Application Publication No. US2014/0271658A1 or US2014/0271642A1), an anti-Virus antibody respiratory syncytial (e.g., anti-RSV antibody as described in US Patent Application Publication No. US2014/0271653A1), an anti-differentiation Group 3 (e.g., an anti-CD3 antibody, as described in U.S. Patent Application Publication No. US Patent Nos. US2014/0088295A1 and US20150266966A1, and in US Application No. 62/222605), an anti-Differentiation Group 20 antibody (e.g., an anti-CD20 antibody as described in US Patent Application Publication No. s US2014/0088295A1 and US20150266966A1, and in US Patent No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti-Differentiation Group-48 antibody (e.g., anti-CD48 antibody, as described in US Patent No. 9,228,014), an anti-Fel d1 antibody (for example, as described in US Patent No. 9,079,948), an anti-Middle East Respiratory Syndrome virus (for example, an anti- MERS as described in US Patent Application Publication No. US2015/0337029A1), an anti-Ebola Virus antibody (for example, as described in US Patent Application Publication No. US2016/0215040), an anti-Zika Virus antibody, an anti-Lymphocyte Activation Gene 3 antibody (for example, an anti-LAG3 antibody or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (for example, an anti-NGF antibody as described in the Publication of the Application US Patent Nos. US2016/0017029 and US Patent Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-Activin A bispecific antibody. CD3 x anti-CD20 (as described in US Patent Application Publication Nos. US2014/0088295A1 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (for example, an anti-CD3 x anti-Muc16 bispecific antibody ) and an anti-CD3x anti-Prostate Specific Membrane Antigen bispecific antibody (e.g. an anti-PSMA anti-CD3x bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, blzlotent, brodalumab, canakinumab, capromab pendetide, certolizumab pegal, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirumab, evin infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nes vacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, tre vogrumab, ustekinumab and vedolizumab.

[0037] In some embodiments, the protein of interest is a recombinant protein that contains an Fc moiety and another domain ((e.g., an Fc fusion protein). In some embodiments, an Fc fusion protein is an Fc fusion protein of receptor, which contains one or more extracellular domains of a receptor coupled to an Fc moiety. In some embodiments, the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG. In some embodiments, the fusion protein Receptor Fc contains two or more distinct receptor chains that bind a single ligand or multiple ligands. For example, an Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g. , rilonacept, which contains the IL-1RAcP ligand-binding region fused to the extracellular region of IL-1R1 fused to hIgGl Fc; see US Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, comprising the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgGl; see US Patent Nos. 7,087,411 and 7,279,159). In other embodiments, an Fc fusion protein is an ScFv-Fc fusion protein, which contains one or more than one or more antigen-binding domains, such as a variable heavy chain fragment and a variable light chain fragment, from one antibody coupled to an Fc moiety.

B. Protein Production Methods

[0038] In one embodiment, the protein drug is produced in a fed batch cell culture. In protein production, a "fed-batch cell culture" or "fed-batch culture" refers to a batch culture in which cells and culture medium are initially supplied to the culture vessel and additional nutrients from the culture are fed slowly, in discrete increments, to the culture during the culture, with or without periodic collection of cells and/or products before the end of the culture. Fed-batch culture includes “semi-continuous fed-batch culture” whereby periodically all culture (which may include cells and media) is removed and replaced with fresh media. Fed-batch culture is distinguished from simple "batch culture" in that all components for cell culture (including animal cells and all culture nutrients) are provided to the culture vessel at the start of the culture process in the feed culture. batch. Fed-batch culture may differ from "perfusion culture" in that the supernatant is not removed from the culture vessel during a standard fed-batch process, whereas in perfusion culture, cells are restricted in culture by , for example, filtration and the culture medium is continuously or intermittently introduced into and removed from the culture vessel. However, removal of samples for testing purposes during fed-batch cell culture is contemplated. The fed batch process continues until it is determined that maximum working volume and/or protein production has been reached, and the protein is then harvested.

[0039] One embodiment provides a protein drug that is produced in a continuous cell culture. The phrase "continuous cell culture" refers to a technique used to continuously culture cells, usually at a specific stage of growth. For example, if a constant supply of cells is needed, or if production of a specific protein of interest is needed, the cell culture may require maintenance at a specific growth phase. Thus, conditions must be continuously monitored and adjusted accordingly to keep the cells in that particular phase.

[0040] The cell cultures used to produce the protein drug include a cell culture medium. The terms "cell culture medium" and "culture medium" refer to a nutrient solution used for growing mammalian cells that normally provides nutrients needed to enhance cell growth, such as an energy source of carbohydrates, amino acids essential (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and non-essential (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine), trace elements, energy sources, lipids, vitamins, etc. The cell culture medium may contain extracts, for example serum or peptones (hydrolysates), which provide raw materials that support cell growth. Media may contain soy extracts or yeast derivatives rather than extracts of animal origin. Chemically defined medium refers to a cell culture medium in which all chemical components are known (ie, have a known chemical structure). The chemically defined medium is completely free of animal-derived components such as serum or animal-derived peptones. In one embodiment, the medium is a chemically defined medium.

[0041] The solution may also contain components that increase growth and/or survival above the minimal rate, including hormones and growth factors. The solution can be formulated to an optimum pH and salt concentration for the survival and proliferation of the specific cell being cultured.

[0042] A "cell line" refers to a cell or cells that are derived from a specific lineage through serial passage or cell subculture. The term "cells" is used interchangeably with "cell population".

[0043] The term "cell" includes any cell that is suitable for expressing a recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes, such as bacterial cells, mammalian cells, human cells, non-human animal cells, avian cells, insect cells, yeast cells, or cell fusions, such as, for example, hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, simian, hamster, rat or mouse cell. In other embodiments, the cell is selected from the following cells: Chinese Hamster Ovary (CHO) (e.g. K1 CHO, DXB-11 CHO, Veggie-CHO), COS (e.g. COS-7), retinal cells, Vero, CV1, kidney (e.g. HEK293, EBNA 293, MSR 293, MDCK, HaK , BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocytes, e.g. Jurkat (lymphocyte T) or Daudi (B lymphocyte), A431 (epidermal), U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT cell, stem cell, tumor cell and a cell line derived from a mentioned cell previously. In some embodiments, the cell comprises one or more viral genes, for example, a retinal cell that expresses a viral gene (for example, a PER.C6® cell). In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0044] Protein purification methods are well known in the art. The drug may undergo multiple purification steps after cell culture and protein extraction to remove impurities, including host cell proteins and aberrant drug forms, associated with the cell culture process. Forms of chromatography are well known, such as “affinity chromatography”, which is a chromatographic method that uses specific and reversible interactions between proteins rather than general protein properties, such as isoelectric point, hydrophobicity or size, to effect chromatographic separation. "Protein A affinity chromatography" or "Protein A chromatography" refers to a specific affinity chromatographic method that utilizes the affinity of the IgG binding domains of Protein A for the Fc portion of an immunoglobulin molecule. This Fc portion comprises human or animal immunoglobulin constant domains CH2 and CH3 or immunoglobulin domains substantially similar thereto. Other forms of chromatography or separation techniques include, but are not limited to: Protein G affinity chromatography, His-Tagged affinity chromatography, Ion Exchange chromatography (Weak Cation, Weak Anion, Strong Cation, Strong Anion), Size Exclusion, Reverse Phase Chromatography, Normal Phase Chromatography, Hydrophobic Interaction Chromatography, Hydrophilic Interaction Chromatography. Each technique requires optimization of proper conditions, including, but not limited to, resin loading and size, washing conditions, pH, loading volume, loading concentration, and the like. Ultrafiltration and diafiltration techniques are also included in the purification process.

[0045] Protein formulation methods are well known in the art. Medicaments of the present invention include liquid pharmaceutical compositions comprising the antigen-binding molecules (e.g., an antibody) of the present invention. Pharmaceutical compositions of the invention are formulated with suitable vehicles, excipients and other agents that provide improved transfer, delivery, tolerance and the like. A multitude of suitable formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. For example, excipients can include a stabilizer, buffer, toner, surfactant, organic solvent, salt, or combinations thereof. In some embodiments, the stabilizer is selected from the group consisting of a polyol, a sugar, an amino acid, a nonionic surfactant, and combinations thereof. In some embodiments, the toner is selected from the group consisting of a sugar, an amino acid, a salt, and combinations thereof. In some embodiments, the medicament is a liquid formulation or a reconstituted lyophilized formulation of the medicament. In some embodiments, the medicament is a coformulation comprising two or more different antibodies.

III. Methods for Characterizing Dimerization Interfaces in Proteins

[0046] Protein aggregation remains a major concern in the production of monoclonal antibodies. Due to the potential impact on purity, potency and immunogenicity, it is important to understand the aggregation mechanism so that appropriate control strategies can be implemented to ensure product quality. Mapping the dimerization interface on a mAb molecule remains challenging due to the complexity and heterogeneity of mAb dimers. Although hydrogen-deuterium exchange mass spectrometry (HDX MS) has been successful in studying protein-protein interactions, little success has been achieved in revealing the mAb dimerization interface, presumably due to the limitations of the method in detecting interactions. of the protein side chain.

[0047] To overcome these obstacles, new systems and methods are provided for the successful mapping of heterogeneous dimerization interfaces (covalent and non-covalent) of a protein by comprehensive MS-based methods. An exemplary strategy for mapping the dimerization interface is provided in Figure 1. The protein may be an antibody or antigen-binding fragment thereof, a recombinant protein, or a fusion protein.

A. Methods to Characterize Non-Covalent Interaction Sites

[0048] One embodiment provides a method for determining dimerization interfaces at the peptide/residue level of a protein. An exemplary method includes digesting a protein dimer sample into subdomains, labeling the digested protein sample mixture, isolating the labeled dimeric and monomeric subdomain fragments, and mapping peptides from the labeled sample to determine and compare the extent of labeling on the dimer and in the monomer. Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface.

[0049] In one embodiment, peptides were isolated and analyzed by peptide mapping to locate sites of non-covalent interaction. In another embodiment to locate the non-covalent dimerization interface at the peptide/residue level, the enriched mAb dimer sample was digested in a mixture of F(ab) homodimer [or F(ab')], F(ab') monomers ) [or F(ab')] and Fc, then subjected to a covalent labeling experiment, followed by SEC fractionation, tryptic digestion and LC-MS analysis to quantify the extent of labeling and compare peptide/residue levels. In yet another embodiment to locate the non-covalent dimerization interface at the peptide/residue level, the enriched mAb dimer sample was digested in a mixture of F(ab) homodimer [or F(ab')], F(ab') monomers ) [or F(ab')] and Fc, then subjected to a single labeling experiment with FPOP (rapid photochemical oxidation of proteins), followed by SEC fractionation, tryptic digestion and LC-MS analysis for quantification and comparison of the labeling on peptide/residue levels. In the following, more details are provided on the steps of the described methods.

1. Protein Dimer Fragmentation

[0050] In one embodiment, a protein drug, e.g., an antibody, is obtained from a cell culture and enriched for dimers so that the dimers provide an enriched dimer sample. The enriched dimer sample can be produced using a variety of enrichment techniques, including, but not limited to, liquid chromatography. A preferred liquid chromatography technique includes, but is not limited to, size exclusion chromatography. In some embodiments, the enriched dimer sample contains some protein aggregates that are not dimers. Thus, the enriched dimer sample need not be composed of dimers alone. In one embodiment, the enriched dimer sample contains greater than 50% dimer.

[0051] Fragmentation or digestion of a protein product, for example to preserve the non-covalent interactions that form a mAb dimer, can be performed using conventional techniques, often called limited digestion, including chemical and enzymatic techniques. In one embodiment, a mAb dimer sample is digested under native conditions to produce various Fab and Fc fragments, for example. a mixture of F(ab), F(ab) homodimer and Fc monomer, with the use of a protease. An exemplary protease is the LysC endoprotease. Other proteases include papain and ficain. In one embodiment, the mAb dimer can be diluted in 10 mM Tris-HCl (pH 7.5) and then incubated with Lys-C endoprotease (Protein:enzyme = 400:1). This limited digestion treatment specifically cleaves the heavy chain at a Lys residue that is the N-terminus of the two disulfide bonds of the hinge region, leading to the generation of Fab and Fc fragments. Any non-covalent interactions with dimers (most commonly F(ab) - F(ab)) of the dimer species are retained in the mixture.

[0052] In another embodiment, the mAb dimer sample is digested into F(ab')2 and Fc* fragments (i.e., two Fc polypeptides that interact by non-covalent interactions, referred to herein as Fc monomers) under native conditions with IdeS protease sold under the name FabRICATOR®. The mAb dimer can first be diluted in 10 mM Tris-HCl (pH 8.0) and then digested with FabRICATOR® (1 milliunit IUB per 1 µg protein). This limited digestion treatment specifically cleaves between two Gly residues, a site that is C-terminal to the two disulfide bonds of the hinge region. This treatment can result in the generation of an F(ab')2 fragment and two identical Fc/2 fragments that are linked through non-covalent interactions (Fc*). The digestion mix can then be incubated with a reducing agent, which reduces the interchain disulfide bonds while keeping the intrachain disulfide bonds intact. As a result, the F(ab')2 fragments are reduced into two F(ab') fragments, and the Fd and LC are linked via non-covalent interaction. The non-covalent dimer interactions (most commonly F(ab') - F(ab')) of the dimer species are also maintained in the mixture.

[0053] Examples of reducing agents that may be used include, but are not limited to, dithiothreitol (DTT, CAS 3483-12-3), beta-mercaptoethanol (BME, 2BME, 2-ME, b-mer, CAS 60- 24-2), 2-aminoethanethiol (2-MEA-HCl, also called cysteamine-HCl, CAS 15657-0), Tris(2-carboxyethyl) phosphine hydrochloride, (TCEP, CAS 5961-85-3), cysteine (Cys-HCl, CAS 52-89-1) or 2-mercaptoethanesulfonic acid sodium salt (MESNA). Other methods for reducing protein bonds are known in the art, such as an immobilized reducing column containing resin to which a thiol-based reducing agent has been immobilized to enable solid phase reduction of peptide and protein disulfide bonds. Also envisaged are reducing agents, including oxidizing agents, suitable for reducing chemical interaction between the polypeptides.

2. Labeling of Protein Fragments i. Carboxyl Group Marking

[0054] In one embodiment, the digested mAb sample is labeled using carboxyl group labeling. The carboxyl group footprint is a covalent labeling technique commonly used to study the conformation and interactions of proteins, by labeling the side chains of Asp and Glu residues with glycine ethyl ester hydrochloride (GEE). In the GHG labeling reaction, the carboxyl groups of Asp and Glu residues located on the surface of a protein are easily modified, while those buried within the protein structure undergo less or no modification at all. In one embodiment, the digested sample can be mixed with 0.2 M 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 2 M GEE and incubated at room temperature for 1 hour. The GEE labeling reaction can be quenched by adding 1M Tris-HCl (pH 8). In one embodiment, the sample solution can then be concentrated. Sample concentration methods are well known in the art. Exemplary methods include, but are not limited to, membrane dialysis, precipitation or salt precipitation, cellulose membrane concentrators, and centrifugation. In a preferred embodiment, the sample is concentrated by centrifugation using, for example, an Ultracel-10K centrifugal filter.

ii. Rapid Photochemical Oxidation of Proteins (FPOP)

[0055] In one embodiment, the digested mAb sample is labeled using FPOP. FPOP is a well-developed hydroxyl radical labeling method that labels the protein on the amino acid residue side chain on the µs time scale and has been widely applied in the study of protein-protein/ligand interactions. In one embodiment, the digested mAb dimer containing mAb fragments and the non-covalently linked fragment dimers are buffer exchanged into an appropriate buffer, for example PBS (pH 7.4). Buffer exchange may be necessary because inhibitors react quickly with OH radicals and should be avoided when labeling. Common inhibitors include, but are not limited to, buffer solutions such as Tris-HCl and chemicals such as glycerol and DTT.

[0056] • OH tagging can cause conformational changes in the native conformation of the protein. In one embodiment, scavengers can be used to control the lifetime of the OH radical so that the protein is not overlabeled, ensuring that the native conformation is captured and not an overlabeled protein with conformational changes. Exemplary scavengers that may be used include, but are not limited to, Cys, Trp, Tyr, Met, His, Phe, Arg, Ile, Leu, Val, Pro, Gln, Thr, and Lys. In a preferred embodiment, the hijacker is His. The buffer exchange mix can first be mixed with PBS and concentrated scavenger solution and H2O2 stock solution. His can be added at a concentration of 200 to 500 µM. The His concentration can be 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 µM. An example mix contains 1 mg/mL mAb digestion mix, 350 µM His and 20 mM H2O2 to produce a final 100 µL of solution. In one embodiment, H2O2 can be added just prior to infusing the solution into the POFP tube.

[0057] The oxidative labeling of the protein can be monitored by modeling the intensities of unmodified species, +16 Da, +32 Da and +48 Da by Poisson distribution. The oxygen addition state distribution values can be tested for the degree of fit to the Poisson distribution. In one embodiment, data that do not fit the predicted Poisson distribution indicate excessive labeling of the protein. Alternatively, if the data fit the Poisson model, there is sufficient labeling without excessive protein labeling.

[0058] In one embodiment, the sample can be loaded into a 100 μL gas-tight syringe and introduced through a syringe pump coupled to a 150 μm i.d. fused silica tube. The tube may have a clear window in the center facing a laser beam. The laser may be an Excimer laser, for example a KrF Excimer laser. Any commercially available Excimer lasers can be used, eg COMPex 50 laser (Coherent Inc.). The power of the KrF Excimer laser can be between about 100 and 150 mJ/pulse. In a preferred embodiment, the laser power can be between about 100 and 120 mJ/pulse. The laser pulse frequency can be adjusted to 6 Hz. The width of the laser beam on the transparent window of the sample tube can be about 1.5mm to 3.0mm. The laser width can be 1.5mm, 1.75mm, 2mm, 2.25mm, 2.5mm, 2.75mm or 3mm. Laser power can vary over time. Therefore, in one embodiment, the same set of labeling experiments can be performed on the same day for maximum labeling consistency. In another embodiment, dosimeters can be added to monitor efficiency fluctuations from run-to-run marking.

[0059] After the pulsed laser is turned on, the sample solution can be infused into the tube, for example, at a flow rate of 23.86 μL/min. The flow rate can be adjusted according to the i.d. of the tube, the laser frequency and the measured width of the laser beam. The flow rate must be adequate to ensure a 20% exclusion volume to avoid repeated exposure and reaction to OH. In one embodiment, the capillary output can be collected in a vial containing solutions to reduce residual H2O2. The reduction of residual H2O2 can be achieved with the use of reducing agents, antioxidants or a combination thereof. Exemplary non-enzymatic antioxidant agents include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, carotene, taurine, hypotaurine, glutathione, selenium, zinc, methionine, and ubiquinone. Enzymatic antioxidant agents include, but are not limited to, SOD, catalase, glutaredoxin, and glutathione reductase. In a preferred embodiment, the solution for reducing residual H2O2 contains methionine and catalase. The labeled sample output solution can be concentrated by centrifugation, for example using an ultracel-10K centrifugal filter.

iii. Isolation of Dimers and Monomers from Labeled mAb Fragments

[0060] In one embodiment, the covalently labeled sample mixture can be separated using size exclusion chromatography, for example, a SEC BEH® column. After SEC separation and UV detection (280 nm), dimeric and monomeric fragments can be collected for tryptic peptide mapping analysis.

[0061] It is understood that the methods described are not limited to any specific sequence of fractionation (limited digestion) and labeling steps as presented in this document.

iv. Peptide Mapping of Tagged Dimer Fragments

[0062] In one embodiment, the dimer and monomer fractions may be subjected to tryptic peptide mapping. Tryptic peptide mapping methods are known in the art. In one embodiment, the dimer and monomer fractions can be dried, for example, in a SpeedVac and reconstituted in a solution containing 8 M urea, 5 mM dithiothreitol (DTT) and 0.1 M Tris-HCl (pH 7.5) before tryptic peptide mapping. The denatured and reduced samples can be alkylated with 10 mM iodoacetamide (IAA) in the dark for 30 minutes. Sample solutions can then be diluted with 0.1 M Tris-HCl (pH 7.5) to lower the urea concentration and digested with trypsin at an enzyme to substrate ratio of 1:20 (w/w) at 37° C overnight. Digestion can be stopped by adding 10% formic acid (FA) to each digested protein sample. Samples can then be separated by reverse phase UPLC followed by online mass spectrometric analysis to determine peptide masses and confirm peptide identities. In one embodiment, the MS and MS/MS experiments are conducted on a Thermo Q Exactive Plus MS system with high-energy collisional dissociation (HCD) employed for peptide fragmentation during the MS/MS experiments.

v. Identification and Characterization of Dimming Interfaces

[0063] In one embodiment for carboxylic group labeling, to identify GEE-labeled tryptic peptides, the LC-MS/MS data are searched against a database or search engine that contains the corresponding mAb protein sequence with variable modifications of GEE1 (+85.0522) and GEE2 (+57.0209) restricted to Asp and Glu residues, for example, using software such as BYONIC™ (Protein Metrics). To quantify the extent of GEE labeling in each tryptic peptide containing Asp/Glu residue, extracted ion chromatograms (EICs) are generated, based on the m/z of the first isotopic peak of the GEE-labeled peptide and native peptide, and the extracted peak areas are merged. The percentage of each GEE-tagged peptide is calculated using the corresponding EIC peak area versus the sum of the peak areas of the labeled and native peptides. In case a tryptic peptide contains multiple Asp and Glu residues, GEE-labeled peptides eluting at different retention times are examined first for MS/MS spectra to confirm the labeling sites and then the areas of Corresponding EIC peaks are used to calculate the percentage of site-specific staining. Finally, the GEE tag extensions are compared between the dimer fraction and the monomer fraction for each peptide or tryptic residue. Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface.

[0064] In one embodiment for FPOP labeling, the LC-MS/MS data are first searched against a database containing the corresponding mAb protein sequence with all known oxidative labeling products (Table 1) to identify all the tagging products/peptides. Peptide modification sites are identified based on MS/MS information and confirmed by accurate m/z, and the extent of modification for a given peptide was calculated as: where Iox is the intensity of the modified peptide (excluding Met oxidation due to possible oxidation of Met introduced during handling of the unlabeled sample) and I is the intensity of the unmodified peptide. Quantitative analysis of oxidative labeling at the residue level is also performed for peptides that showed differences in the extent of modification at the peptide level between dimers and monomers and peptides containing Met residues. Modification sites on the peptide are indicated with MS2 information. In some embodiments, where localization of a modification to a single residue is not possible, due to limited MS2 fragmentation information or the presence of interference from coelution of peptide isomers, the modification is indicated to occur in a set of possible residues . Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface.

[0065] One embodiment provides a method for identifying non-covalent interaction sites or dimerization interfaces on a protein drug by digesting the dimer-enriched sample of a protein drug under native limited digestion conditions to form a sample drug mixture containing non-covalently linked dimeric subdomains (e.g., F(ab) dimers) and monomeric subdomains, and introducing detectable modifications to the sample dimers and monomers of the protein drug mixture to produce modified dimers and monomers. The method includes separating the modified dimers and modified monomers using native size exclusion chromatography into modified dimer and modified monomer fractions, and digesting a multitude of peptide bonds from the modified dimer and modified monomer fractions to form digested peptide samples. . The method also includes analyzing the digested peptide samples using liquid chromatography/mass spectrometry to obtain the mass spectrometry data from the digested peptide samples and determining the sites of modification on the digested peptides in the digested peptide sample using the data. of mass spectrometry of the digested peptides compared to the known mass spectrometry data of the protein drug with specific residue modifications introduced into the protein drug.

[0066] In one embodiment, the dimers and monomers are modified by rapid photochemical oxidation to form detectable oxidative modifications in the dimers and monomers. The modification may be an amino acid side chain modification.

[0067] As noted earlier, the protein medicine may be an antibody or antigen-binding fragment thereof, a recombinant protein, a fusion protein, or combinations thereof. A preferred protein drug is a monoclonal antibody or antigen-binding fragment thereof.

[0068] In one embodiment, native size exclusion chromatography is performed using a mobile phase comprising ammonium acetate and ammonium bicarbonate. In another embodiment, the digested protein sample is separated by reverse phase liquid chromatography.

[0069] In one embodiment, the modification is selected from the group consisting of oxidation, dioxidation, trioxidation, kynurenine, dithiomethyl, decarboxylation, hydroxykynurenine, and combinations thereof.

[0070] Generally, the protein medicine is digested with an enzyme, for example protease digested. Exemplary proteases used in limited digestion include, but are not limited to, papain, ficain, endoprotease Lys-C and IdeS. Exemplary proteases used in peptide bond digestion to form a variety of peptides include, but are not limited to, trypsin, chymotrypsin, pepsin, and rLys-C.

[0071] In some embodiments, the protein drug sample is from a fed batch culture.

B. Methods of Producing Protein Drugs with Reduced Non-Covalent Interaction or Aggregation

[0072] One embodiment provides a method of producing an antibody, including the steps of culturing cells that produce the antibody under conditions suitable for producing the antibody; purifying the antibody under conditions suitable for extracting the antibody; mixing the antibody with excipients under suitable conditions to stabilize the antibody; obtaining a sample of the antibody i) from the cell culture, ii) after purifying the antibody from the cell culture, or iii) after adding excipients to the purified antibody; characterizing the dimerization interfaces on the antibody using the systems and methods described herein and modifying the conditions for producing the antibody or modifying the antibody dimerization interfaces to have reduced dimerization and non-covalent interaction relative to an unmodified antibody. Modifying the conditions for producing the antibody is explained earlier in this document.

[0073] In one embodiment, the modification may be a conservative amino acid substitution or a chemical modification of the dimerization interfaces.

Examples Example 1: Unique labeling of dimer and monomer mixture by covalent labeling Methods Limited digestion of mAb-1

[0074] mAb-1, an IgG1 isotype, was digested into F(ab) and Fc fragments under native conditions with LysC endoprotease. A mAb-1 dimer was first diluted to 1 μg/μL in 10 mM Tris-HCl (pH 7.5) and then incubated with Lys-C endoprotease (protein:enzyme = 400:1) at 37°C for 1 hour . This limited digestion treatment specifically cleaves the heavy chain at a Lys residue that is the N-terminus of the two disulfide bonds of the hinge region, leading to the generation of F(ab) and Fc fragments. Any non-covalent dimer interactions (most commonly F(ab) - F(ab)) of the dimer species are retained in the mixture.

Limited digestion of mAb-2

[0075] The mAb-2 and the IgG4 isotype were digested into F(ab')2 and Fc* fragments under native conditions with FabRICATOR®, an IdS enzyme from Streptococcocus pyogenes. The mAb-2 dimer is first diluted to 1 μg/μL in 10 mM Tris-HCl (pH 8.0) and then digested with FabRICATOR® (1 milliunit IUB per 1 μg protein) at 37 °C for 1 hour . This limited digestion treatment specifically cleaves between two Gly residues, a site that is C-terminal to the two disulfide bonds of the hinge region. This treatment resulted in the generation of an F(ab')2 fragment and two identical Fc/2 fragments that linked via non-covalent interactions (Fc*). The digestion mix is then incubated with 5 mM dithiothreitol (DTT) at 37°C for 30 minutes, which reduces interchain disulfide bonds while keeping intrachain disulfide bonds intact. As a result, the Fab2 fragments are reduced into two F(ab') fragments and the Fd and LC are linked via non-covalent interaction. The non-covalent dimer interactions (most commonly F(ab') - F(ab')) of the dimer species are also maintained in the mixture.

[0076] The above generated mixture of mAb fragments and non-covalently linked fragment dimers is first buffer exchanged in IxPBS (pH 7.4) at ~2 mg/ml.

Marking of the carboxyl group

[0077] The carboxyl group footprint is a covalent labeling technique commonly used to study the conformation and interactions of proteins by labeling the side chains of Asp and Glu residues with GEE. In the GHG labeling reaction, the carboxyl groups of Asp and Glu residues located on the surface of a protein are easily modified, while those buried within the protein structure undergo less or no modification at all. To perform the labeling, 100 μL of the mAb-1 digested sample was mixed with 50 μL of 0.2 M 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 50 μL of ester hydrochloride 2M glycine ethyl acetate (GEE) and incubated at room temperature for 1 hour. After quenching the GHG labeling reaction by addition of 500 µL of 1M Tris-HCl (pH 8), the sample solution was concentrated by centrifugation using an Ultracel-10K centrifugal filter to approximately 40 µL.

Rapid Photochemical Oxidation of Proteins (FPOP)

[0078] FPPO is a well-developed hydroxyl radical labeling method that labels the protein on the side chain of the amino acid residue on the μs time scale and has been widely applied in the study of protein-protein/ligand interactions. In the labeling process, 50 µL of the above buffer exchange mixture was first mixed with 1 x PBS and concentrated His solution and H2O2 stock solution to produce a final 100 µL of solution containing 1 mg/mL of digestion mix with mAb-2, 350 µM His and 20 mM H2O2. The H2O2 was added immediately before infusing the solution into the FPOP tube. The sample was loaded into a 100 μL gas-tight syringe and introduced through a syringe pump attached to a 150 μm inner diameter fused silica tube with a transparent window in the center facing the laser beam. The KrF Excimer laser power was adjusted to 100 to 120 mJ/pulse, and its pulse frequency adjusted to 6 Hz. The width of the laser beam on the transparent window of the sample tube was measured to be 3.0 mm. After turning on the pulsed laser, the sample solution was infused into the tube at a flow rate of 23.86 μL/min (adjusted according to tube id, laser frequency, and measured laser beam width) to ensure a 20% cut-off volume to avoid repeated •OH exposure and reaction. Capillary output was collected in a vial containing 20 µL of 100 mM Met and 2 µL of 1 mg/mL catalase to reduce residual H2O2. Finally, the labeled sample solution was concentrated by centrifugation using an ultracel-10K centrifugal filter to about 40 µL.

SEC/MS

[0079] The covalently labeled sample mixture was injected onto a SEC BEH® column (300 mm x 4.6 mm, 200Â, 1.7 μm) that was pre-equilibrated with the mobile phase (140 mM ammonium acetate and 10 mM ammonium bicarbonate, pH 7.4) at a flow rate of 0.2 mL/min. After SEC separation and UV detection (280 nm), dimeric and monomeric fragments were collected for tryptic peptide mapping analysis.

Peptide Mapping

[0080] Prior to tryptic digestion, all dimer and monomer fractions were dried in a SpeedVac and reconstituted in a solution containing 8 M urea, 5 mM dithiothreitol (DTT) and 0.1 M Tris-HCl (pH 7.5) and kept at 50 °C for 30 minutes. The denatured and reduced samples were alkylated with 10 mM iodoacetamide (IAA) in the dark for 30 minutes. The sample solutions were then diluted tenfold with 0.1 M Tris-HCl (pH 7.5) to lower the urea concentration to 0.8 M and digested with trypsin at an enzyme to substrate ratio of 1:20 (p /p) at 37 °C overnight. Digestion was then stopped by adding 10% FA to yield a final 0.2% FA. Aliquots (~5 µg) of each digested protein sample were then separated by reversed-phase UPLC followed by online mass spectrometric analysis to determine peptide masses and confirm peptide identities. MS and MS/MS experiments were conducted on a Thermo Q Exactive Plus MS system with higher energy collisional dissociation (HCD) employed for fragmentation of peptides during MS/MS experiments.

Results Carboxyl Group Marking

[0081] For labeling the carboxyl group, to identify GEE-labeled tryptic peptides, LC-MS/MS data were searched in a database containing the corresponding mAb protein sequence with variable modifications of GEE1 (+85.0522 ) and GEE2 (+57.0209) restricted to Asp and Glu residues. To quantify the extent of GEE labeling in each tryptic peptide containing Asp/Glu residue, extracted ion chromatograms (EICs) were generated, based on the m/z of the first isotopic peak of the GEE-labeled peptide and native peptide, and the extracted peak areas were integrated. The percentage of each GEE-labelled peptide was calculated using the corresponding EIC peak area in relation to the sum of the peak areas of the labeled and native peptides. In the case of a tryptic peptide containing multiple Asp and Glu residues, GEE-labeled peptides eluting at different retention times were examined first for MS/MS spectra to confirm the labeling sites and then the peak areas of corresponding EIC were used to calculate the percentage of site-specific staining. Finally, the GEE tag extensions were compared between the dimer fraction and the monomer fraction for each peptide or tryptic residue. Regions showing decreased tag lengths in the dimer fraction than in the monomer fraction are likely involved or close to the dimerization interface (Figures 2, 3A and 3B). Marking with FPOP

[0082] For FPOP labeling, the LC-MS/MS data were first searched against a database containing the corresponding mAb protein sequence with all known oxidative labeling products (Table 1) to identify all FPOP labeling products. marking. Peptide modification sites were identified based on MS/MS information and confirmed by precise m/z, and the extent of modification for a given peptide was calculated as: where Iox is the intensity of the modified peptide (oxidation of Met excluded due to possible oxidation of Met introduced during handling of the unlabeled sample) and I is the intensity of the unmodified peptide. A quantitative analysis of oxidative labeling at the residue level was also performed for peptides that showed differences in the extent of modification at the peptide level between dimers and monomers and peptides containing Met residues. Modification sites on the peptide were indicated with MS2 information. In some cases, where localization of a modification to a single residue was not possible, due to limited fragmentation information from MS2 or the presence of interference from coelution of peptide isomers, the modification was indicated to occur in a set of possible residues. . The Poisson distribution of species labeled +0, +16, +32, +48 etc. at the subdomain level indicates that there was no excessive tagging (Figures 4A to 4C). Regions that show tag lengths in the dimer fraction smaller than in the monomer fraction are likely to be involved at or close to the dimerization interface (Figures 5A and 5B). Table 1. Possible oxidative labeling products.

Example 2: FPOP Optimization Methods:

[0083] The FPOP protocol of Example 1 was followed. Various protocol components, ie, laser position, scavenger concentration, and inclusion of inhibitors, were manipulated to determine their effect on the assay.

Results:

[0084] Table 2 shows the experimental parameters for Figures 6A and 6B. As seen in Figures 6A and 6B, the position and power of the laser affect the labeling efficiency of the lysozyme protein. The oxidized species in the sample that was analyzed using the parameters in Figure 6B, including a closer focus position and a laser power of 122 mJ, show a good fit of the product distribution at the +0, +16, + state 32... for a Poisson distribution. This is indicative of greater labeling efficiency.Table 2. Experimental parameters

[0085] As demonstrated by the Poisson distribution data in Figures 7A to 7F and 8A to 8D, laser power and scavenger concentration affect protein labeling efficiency to a much greater extent than protein concentration.

[0086] The deglycosylated mAb1 without buffer exchange shows decreased labeling extent (Figure 9B) compared to the mAb1 subjected to buffer exchange (Figure 9A). This suggests that inhibitors can interfere with the oxidative labeling of proteins.

[0087] Although in the foregoing specification this invention has been described in relation to certain embodiments thereof and many details have been presented for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that some of the details described herein can vary considerably without departing from the basic principles of the invention.

[0088] All references mentioned in this specification are hereby incorporated by reference in their entirety.

Claims (12)

1. Method for identifying non-covalent interaction sites or dimerization interfaces on a protein drug with reduced non-covalent interactions or aggregations, wherein the dimerization interfaces or non-covalent interaction sites identified on the protein drug have been altered using conservative amino acid substitutions to modifying the dimerization interfaces of the protein drug to have reduced dimerization and non-covalent interaction with an unmodified protein drug, the method characterized in that it comprises: employing limited enzymatic digestion of the protein drug under native conditions to form a sample mixture protein drug dimer comprising non-covalently linked monomers and dimers; incubating the protein drug dimer sample mixture with a reducing agent; introducing detectable oxidative modifications into the dimers and monomers of the protein drug dimer sample mixture to produce modified dimers and monomers, wherein the detectable modifications are introduced by rapid photochemical oxidation of antibodies; separating modified dimers and modified monomers using native size exclusion chromatography into modified dimer and modified monomer fractions; digesting the modified dimer and modified monomer fractions to form digested peptide samples; separating the digested peptide samples using liquid chromatography/mass spectrometry (LC-MS) to obtain mass spectrometry data from the digested peptide samples; and determine the modification sites on each peptide in the digested peptide samples using the mass spectrometry data of the peptides compared to the known mass data of the protein drug by calculating the mass of each peptide and modified peptide of that drug protein, thus characterizing dimerization interfaces or non-covalent interaction sites in the protein drug. 2. Method according to claim 1, characterized in that the limited enzymatic digestion uses an enzyme that is selected from the group consisting of papain,ficain, endoprotease Lys-C and IdeS or its modified form. 3. Method according to claim 1, characterized in that the protein medicine comprises an antibody or antigen-binding fragment thereof, a recombinant protein, a fusion protein or combinations thereof. 4. Method according to claim 3, characterized in that the antibody is a monoclonal antibody, a bispecific antibody, or an antigen-binding fragment. 5. Method according to claim 1, characterized in that the digested peptide sample is separated by reverse phase liquid chromatography. 6. Method according to claim 1, characterized in that the chromatography is performed using a mobile phase comprising ammonium acetate and ammonium bicarbonate. 7. Method according to claim 1, characterized in that the modified dimer and modified monomer fractions are digested by an enzyme that breaks the covalent chemical bonds that link two consecutive amino acid residues to form peptides. 8. Method according to claim 1, characterized in that the protein drug sample comes from a fed batch culture, a purification process step or a formulated pharmaceutical substance. 9. Method according to claim 1, characterized in that the protein drug is an antibody and the dimers in the mixture comprise two interacting F(ab') or F(ab) fragments. 10. Method according to claim 1, characterized in that the protein medicine is an antibody and the monomers in the mixture comprise F(ab) fragments and Fc fragments. 11. Method according to claim 1, characterized in that the reducing agent is selected from the group consisting of dithiothreitol (DTT), beta-mercaptoethanol (BME), 2-aminoethanethiol (2-MEA-HCl), sodium hydrochloride Tris (2-carboxyethyl) phosphine, (TCEP), cysteine hydrochloride and 2-mercaptoethanesulfonic acid sodium salt (MESNA). 12. Method according to claim 1, characterized in that the incubation step reduces interchain disulfide bonds, keeping intrachain disulfide bonds intact.