Characterization of cross-linking sites in antibody-drug conjugates

Abstract

The present invention relates, in general, to a method for characterizing crosslinking sites of a target protein. In particular, the present invention relates to identifying and quantifying crosslinking sites of a target protein and determining the contribution of crosslinking to the formation of high molecular weight species using size exclusion chromatography, peptide mapping analysis, and subunit analysis.

Description

Background Art

[0001] Cross-reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 457,829, filed Apr. 7, 2023, which is hereby incorporated by reference in its entirety.

[0002] Antibody-drug conjugates (ADCs) are an important class of biological therapeutics used in cancer treatment and can also be used for therapeutic applications beyond oncology. The antibody is covalently linked to the payload via either non-specific or site-specific conjugation. Conjugation via lysine (K) residues or reduced cysteine residues is the most common non-specific conjugation method, which can result in a highly heterogeneous mixture of ADCs with varying drug-to-antibody ratios (DARs) and payload conjugation sites. Given the complex nature of the product and the issues associated with lot-to-lot variability, non-specific conjugation often leads to uncertainties in pharmacokinetics, toxicity, and efficacy. In contrast, site-specific conjugation, which typically requires more advanced protein engineering, generally provides excellent control over DAR and conjugation site. Conjugation via engineered cysteine residues or non-natural amino acid residues are two major classes of site-specific conjugation, while conjugation via enzyme linkage is a new tool used to ensure control over DAR and conjugation site.

[0003] Microbial transglutaminases (mTGs) are enzymes that catalyze the formation of stable isopeptide bonds between glutamine (Q) side chains (i.e., γ-carboxamide groups) and primary amines. While most published studies have focused on mAb engineering to achieve higher site specificity and selectivity, how potential side effects during mTG-mediated conjugation affect the final ADC product quality has not been extensively studied. Target glutamine residues in mAbs incubated with mTG and its linker payload (LP) can undergo one of three potential reactions: (1) conjugate with the linker payload, (2) undergo deamidation by reaction with water, or (3) crosslink to a lysine (K) residue. Both of the latter two reactions are undesirable side effects that reduce the number of glutamine residues available for conjugation, resulting in a decrease in overall DAR.

[0004] Crosslinking to off-target amino acid residues also increases the level of high molecular weight (HMW) species in ADC products. HMW species are an important quality characteristic of therapeutic proteins due to their potential impact on both the efficacy and safety of the drug. Due to their size range from soluble oligomers to visible particles, HMW species can induce undesirable immunogenic reactions, potentially compromising the safety and efficacy of the drug.

[0005] Therefore, there is a need for methods to characterize high molecular weight species and crosslinking byproducts in ADCs, methods to produce ADCs with reduced HMW species, and methods to select antibodies for ADCs that are less susceptible to off-target crosslinking and HMW formation. [Overview of the Initiative]

[0006] Methods have been developed to produce ADCs with reduced HMW species. In exemplary embodiments, at least one off-target amino acid residue that forms a crosslink in the ADC can be identified by contacting the corresponding antibody with a linker and a crosslinking agent, digesting the crosslinked sample to form a peptide digest, and using liquid chromatography-mass spectrometry (LC-MS). The antibody may then be contacted with a protease to remove the off-target amino acid residue. In one embodiment, the protease may be a carboxypeptidase, such as carboxypeptidase B, and the off-target amino acid residue may be C-terminal lysine. The clipped antibody may then be contacted with a crosslinking agent and a linker-payload to produce an antibody-drug conjugate with reduced HMW species. In one embodiment, the crosslinking agent may be a microbial transglutaminase.

[0007] Methods have also been developed, such as ADC, to determine the contribution of site-specific crosslinking to HMW species of a target protein. In exemplary embodiments, the target protein can be subjected to conditions suitable for promoting site-specific crosslinking to produce the target crosslinked protein. In one embodiment, conditions suitable for promoting site-specific crosslinking may include contacting the target protein with a crosslinking agent, such as microbial transglutaminase. The target crosslinked protein can be subjected to analysis, such as SEC analysis, to quantify the proportion of HMW species. In parallel or sequentially, the target crosslinked protein can be subjected to analysis, such as peptide mapping analysis or subunit analysis, to quantify the proportion of site-specific crosslinked peptides. The proportion of site-specific crosslinked peptides can be compared with the proportion of HMW species to determine the contribution of site-specific crosslinking to HMW species. In one embodiment, the comparison uses a correlation formula that determines the proportion of site-specific crosslinked peptides that would be predicted if the HMW species were fully explained by site-specific crosslinking.

[0008] Further methods have been developed for selecting and / or manipulating antibodies for antibody-drug conjugates. In exemplary embodiments, a first antibody can be subjected to site-specific crosslinking to produce a crosslinked antibody. The crosslinked antibody can be subjected to analysis, such as peptide mapping analysis or subunit analysis, to quantify the site-specific crosslinking. This quantification can be compared to the same quantification for at least one additional antibody, and the comparison can be used to select antibodies for antibody-drug conjugates. In one embodiment, antibodies may be selected based on the formation of fewer site-specific crosslinks. In another embodiment, antibodies may be selected based on having fewer reactive off-target amino acid residues, such as reactive lysine. Additionally or alternatively, antibodies may be manipulated to remove reactive off-target amino acid residues identified using this method, thereby reducing the number of site-specific crosslinks formed by the manipulated antibody.

[0009] This disclosure provides a method for generating an antibody-drug conjugate with reduced high molecular weight (HMW) species. In some exemplary embodiments, the method may include (a) contacting an antibody containing C-terminal lysine with a carboxypeptidase to generate a clipped antibody, wherein the clipped antibody does not contain C-terminal lysine; and (b) contacting the clipped antibody with a crosslinking agent and a linker-payload to generate an antibody-drug conjugate with reduced HMW species.

[0010] In one embodiment, the carboxypeptidase is metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, or carboxypeptidase E. In another embodiment, the carboxypeptidase is carboxypeptidase B.

[0011] In one embodiment, the step of contacting the aforementioned antibody with the aforementioned carboxypeptidase is carried out at approximately 35°C to approximately 39°C for approximately 1 to 3 hours.

[0012] In one embodiment, the payload is a cytotoxic payload or a therapeutic payload.

[0013] In one embodiment, the antibody comprises glutamine that has been engineered for site-specific conjugation. In a particular embodiment, the crosslinking agent can crosslink the aforementioned glutamine and the aforementioned C-terminal lysine.

[0014] In one embodiment, the crosslinking agent is an enzyme. In a particular embodiment, the enzyme is a microbial transglutaminase.

[0015] This disclosure provides additional methods for generating antibody-drug conjugates with reduced high molecular weight (HMW) species. In some exemplary embodiments, the method is: (a) identifying at least one off-target amino acid residue that forms a crosslink in the antibody-drug conjugate, wherein the identifying step is (i) contacting a sample containing an antibody with a linker and a crosslinking agent to generate a crosslinked sample, wherein the crosslinking agent can crosslink the antibody with the linker at a target amino acid residue, and the crosslinking agent can crosslink the antibody at the target amino acid residue to the antibody at the off-target amino acid residue; (ii) contacting the crosslinked sample with at least one digestive enzyme to generate a peptide digest; and (iii) the aforementioned The process includes: (iv) identifying a crosslinked peptide by subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS); (b) identifying at least one off-target amino acid residue that forms a crosslink using the aforementioned identification; (c) contacting the aforementioned antibody with at least one protease to produce a clipped antibody, wherein the clipped antibody does not contain the aforementioned at least one identified off-target amino acid residue; and (d) contacting the aforementioned clipped antibody with a linker and the aforementioned crosslinking agent to produce an antibody-drug conjugate with reduced high molecular weight species.

[0016] In one embodiment, at least one off-target amino acid residue is lysine, and optionally, the lysine is C-terminal lysine. In another embodiment, the target amino acid residue is lysine, cysteine, a non-natural amino acid, or glutamine. In a further embodiment, the target amino acid residue is manipulated for site-directed conjugation.

[0017] In one embodiment, the linker is bound to a payload, which optionally is a cytotoxic payload or a therapeutic payload.

[0018] In one embodiment, the crosslinking agent is an enzyme. In a specific embodiment, the crosslinking agent is microbial transglutaminase (mTG).

[0019] In one embodiment, at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof. In a particular embodiment, at least one digestive enzyme is trypsin. In another specific embodiment, at least one digestive enzyme is IdeS or a variant thereof.

[0020] In one embodiment, liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography. In another embodiment, LC-MS analysis is RPLC-MS / MS analysis.

[0021] In one embodiment, at least one protease is a carboxypeptidase. In a particular embodiment, the carboxypeptidase is carboxypeptidase B.

[0022] In one embodiment, the step of contacting the aforementioned antibody with at least one of the aforementioned proteases is carried out at approximately 35°C to approximately 39°C for approximately 1 to approximately 3 hours.

[0023] In one aspect, the clipped antibody is an antibody lacking a C-terminal lysine.

[0024] The present disclosure also provides a method for characterizing cross-linking sites in a target protein. In some exemplary embodiments, the method comprises: (a) contacting a sample containing the target protein with a cross-linking agent to generate a cross-linked target protein, wherein the target protein contains at least one target amino acid residue that can be cross-linked by the cross-linking agent; (b) contacting the cross-linked target protein with at least one digestive enzyme to generate a peptide digest; (c) subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to characterize peptides containing cross-links at the at least one target amino acid residue; and (d) using the characterized peptides to characterize the cross-linking sites in the target protein.

[0025] In one aspect, the target protein is an antibody, bispecific antibody, antibody fragment, antibody-drug conjugate, fusion protein, or recombinant protein.

[0026] In one aspect, the cross-linking agent is an enzyme. In certain aspects, the cross-linking agent is microbial transglutaminase (mTG).

[0027] In one aspect, the target amino acid residue is lysine, cysteine, unnatural amino acid, or glutamine. In another aspect, the target amino acid residue is engineered for site-specific conjugation.

[0028] In one aspect, step (a) further comprises contacting the target protein and the cross-linking agent with a linker, wherein the cross-linking agent can cross-link the target protein to the linker.

[0029] In one aspect, the linker binds to a payload, and optionally, the payload is a cytotoxic payload or a therapeutic payload.

[0030] In one aspect, at least one digestive enzyme is selected from the group consisting of a protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulin-degrading enzyme (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, variants thereof, biologically active fragments thereof, homologs thereof, or combinations thereof. In certain aspects, at least one digestive enzyme is trypsin. In another specific aspect, at least one digestive enzyme is IdeS or a variant thereof.

[0031] In one aspect, liquid chromatography is selected from the group consisting of reverse-phase liquid chromatography, anion-exchange chromatography, cation-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography. In another aspect, the LC-MS analysis is RPLC-MS / MS analysis.

[0032] In one aspect, the cross-linking site contains lysine. In certain aspects, the lysine is a C-terminal lysine.

[0033] In one aspect, the step of characterizing the aforementioned cross-linking site includes identifying the amino acid residue that cross-links to the aforementioned target amino acid residue.

[0034] This disclosure further provides a method for identifying at least one reactive lysine in a protein of interest. In some exemplary embodiments, the method may include: (a) contacting a sample containing the protein of interest with a crosslinking agent to produce a crosslinked protein of interest, wherein the crosslinking agent can crosslink at least one amino acid residue in the protein of interest to at least one reactive lysine in the protein of interest; (b) contacting the crosslinked protein of interest with at least one digestive enzyme to produce a peptide digest; (c) subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to identify a peptide containing a crosslink between the at least one amino acid residue and at least one reactive lysine; and (d) using the identified peptide to identify the at least one reactive lysine in the protein of interest.

[0035] In one embodiment, the target protein is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein.

[0036] In one embodiment, the crosslinking agent is an enzyme. In a specific embodiment, the crosslinking agent is microbial transglutaminase (mTG).

[0037] In one embodiment, the reactive lysine is the C-terminal lysine.

[0038] In one embodiment, at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof. In a particular embodiment, at least one digestive enzyme is trypsin. In another specific embodiment, at least one digestive enzyme is IdeS or a variant thereof.

[0039] In one embodiment, liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography. In another embodiment, LC-MS analysis is RPLC-MS / MS analysis.

[0040] The Disclosure further provides a method for determining the contribution of site-directed crosslinking to high molecular weight species of a target protein. In some exemplary embodiments, the method may include: (a) subjecting the target protein to conditions suitable for promoting site-directed crosslinking to generate a target crosslinked protein; (b) subjecting the aforementioned target crosslinked protein to size exclusion chromatography (SEC) analysis to quantify the proportion of high molecular weight (HMW) species; (c) using the aforementioned quantification to determine a predicted proportion of site-directed crosslinked peptides that may contribute to the aforementioned HMW species using Formula 1; (d) subjecting the target crosslinked protein from step (a) to peptide mapping analysis to quantify the proportion of site-directed crosslinked peptides; and (e) comparing the quantified proportion of site-directed crosslinked peptides from step (d) with the predicted proportion of site-directed crosslinked peptides from step (c) to determine the contribution of site-directed crosslinking to the HMW species of the aforementioned target protein.

[0041] In one embodiment, the target protein is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein.

[0042] In one embodiment, site-specific crosslinking includes crosslinking of manipulated amino acid residues.

[0043] In one embodiment, providing the target protein under conditions suitable for promoting site-specific crosslinking includes contacting the aforementioned target protein with a crosslinking agent. In a particular embodiment, the crosslinking agent is an enzyme. In a more specific embodiment, the enzyme is microbial transglutaminase (mTG).

[0044] In one embodiment, peptide mapping analysis includes contacting the aforementioned cross-linked protein of interest with at least one digestive enzyme to produce a peptide digest, and then subjecting the peptide digest to RPLC-MS / MS analysis. In a particular embodiment, the at least one digestive enzyme is trypsin.

[0045] The Disclosure also provides a method for determining the contribution of C-terminal lysine to the formation of high molecular weight (HMW) species in an antibody-drug conjugate of interest. In some exemplary embodiments, the method may include: (a) contacting an antibody corresponding to an antibody-drug conjugate of interest with a carboxypeptidase to produce a clipped antibody, wherein the antibody contains C-terminal lysine and the clipped antibody does not; (b) contacting the antibody and the clipped antibody with a crosslinking agent to produce a crosslinked antibody and a crosslinked clipped antibody, wherein the crosslinking agent can crosslink at least one amino acid residue of the antibody and the clipped antibody to lysine; (c) subjecting the crosslinked antibody and the crosslinked clipped antibody to size exclusion chromatography (SEC) analysis to quantify the HMW species of the crosslinked antibody and the crosslinked clipped antibody; and (d) comparing the aforementioned quantification of the HMW species of the crosslinked antibody with the aforementioned quantification of the HMW species of the crosslinked clipped antibody to determine the contribution of C-terminal lysine to the formation of HMW species in the antibody-drug conjugate of interest.

[0046] In one embodiment, the carboxypeptidase is metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, or carboxypeptidase E. In another embodiment, the carboxypeptidase is carboxypeptidase B.

[0047] In one embodiment, the crosslinking agent is an enzyme. In a specific embodiment, the crosslinking agent is microbial transglutaminase (mTG).

[0048] The Disclosure further provides a method for selecting an antibody for an antibody-drug conjugate. In some exemplary embodiments, the method may include: (a) obtaining a sample comprising a first antibody wherein the first antibody comprises at least one target amino acid residue that can be crosslinked to at least one off-target amino acid residue by a crosslinking agent; (b) contacting the first antibody with the crosslinking agent to produce a crosslinked antibody; (c) contacting the crosslinked antibody with at least one digestive enzyme to produce a peptide digest; (d) subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to quantify, with respect to the first antibody, a peptide comprising at least one target amino acid residue crosslinked to at least one off-target amino acid residue; (e) repeating steps (a) to (d) with at least one additional antibody to quantify, with respect to at least one additional antibody, a peptide comprising at least one target amino acid residue crosslinked to at least one off-target amino acid residue; (f) comparing the quantifications from steps (d) and (e); and (g) selecting an antibody for an antibody-drug conjugate using the comparison described above.

[0049] In one embodiment, at least one target amino acid residue is lysine, cysteine, a non-natural amino acid, or glutamine. In another embodiment, at least one target amino acid residue is manipulated for site-directed conjugation.

[0050] In one embodiment, at least one off-target amino acid residue is lysine. In a particular embodiment, the lysine is C-terminal lysine.

[0051] In one embodiment, the crosslinking agent is an enzyme. In a specific embodiment, the crosslinking agent is microbial transglutaminase (mTG).

[0052] In one embodiment, step (b) further comprises contacting the aforementioned first antibody and the aforementioned crosslinking agent with the linker, wherein the aforementioned crosslinking agent can crosslink the aforementioned first antibody to the aforementioned linker. In a particular embodiment, the linker is bound to a payload. In a more specific embodiment, the payload is a cytotoxic payload or a therapeutic payload.

[0053] In one embodiment, at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof. In a particular embodiment, at least one digestive enzyme is trypsin. In another specific embodiment, at least one digestive enzyme is IdeS or a variant thereof.

[0054] In one embodiment, liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography. In another embodiment, LC-MS analysis is RPLC-MS / MS analysis.

[0055] These and other aspects of the present invention will be better recognized and understood when considered in conjunction with the following description and accompanying drawings. The following description illustrates various embodiments and numerous specific details thereof, but these are given as examples only and are not limiting. Many substitutions, modifications, additions, or reconfigurations may be made within the scope of the present invention. [Brief explanation of the drawing]

[0056] [Figure 1] Figure 1A shows high molecular weight (HMW) species formed when mAbs are incubated with microbial transglutaminase (mTG) according to an exemplary embodiment. Figure 1B shows the correlation between HMW species identified by size exclusion chromatography (SEC) and crosslinked peptides identified by reduced peptide mapping (RPM) according to an exemplary embodiment. [Figure 2] Figure 2A shows the crosslinking site of the lysine (K) residue on mAb1 according to an exemplary embodiment. Figure 2B shows the proportions of native peptide, deamidated peptide, and crosslinked peptide derived from mAb1 after various incubation periods with mTG according to an exemplary embodiment. Figure 2C shows the crosslinking site on mAb2 according to an exemplary embodiment. Figure 2D shows the proportions of native peptide, deamidated peptide, and crosslinked peptide derived from mAb2 after various incubation periods with mTG according to an exemplary embodiment. [Figure 3] Figure 3 shows the derivation of a correction factor that allows the proportion of HMW species to correlate with the proportion of cross-linked peptides, according to an exemplary embodiment. [Figure 4]Figure 4A shows the correlation between the percentage of crosslinked peptides measured by RPM and the percentage of crosslinked peptides predicted based on the percentage of HMW species measured by SEC for mAb1 incubated with mTG, according to an exemplary embodiment. Figure 4B shows the correlation between the percentage of crosslinked peptides measured by RPM and the percentage of crosslinked peptides predicted based on the percentage of HMW species measured by SEC for mAb1 incubated with mTG and linker payload (LP), according to an exemplary embodiment. [Figure 5] Figure 5 shows the proportion of HMW variants of mAb2 having and not having an intact heavy chain C-terminal lysine (K) residue, according to exemplary embodiments. [Figure 6] Figure 6A shows a subunit analysis of mAb2 having heavy chain C-terminal lysine according to an exemplary embodiment. Figure 6B shows a subunit analysis of mAb2 having heavy chain C-terminal lysine after incubation with mTG according to an exemplary embodiment. Figure 6C shows a subunit analysis of mAb2 without heavy chain C-terminal lysine according to an exemplary embodiment. Figure 6D shows a subunit analysis of mAb2 without heavy chain C-terminal lysine after incubation with mTG according to an exemplary embodiment. [Figure 7] Figure 7A shows the relative abundance of crosslinked peptides by reduced peptide mapping of mAb2 having C-terminal lysine on the heavy chain, according to an exemplary embodiment. Figure 7B shows the relative abundance of crosslinked peptides by reduced peptide mapping of mAb2 not having C-terminal lysine on the heavy chain, according to an exemplary embodiment. [Modes for carrying out the invention]

[0057] Antibody-drug conjugates (ADCs) are an important class of biological therapeutic agents used in cancer treatment. These drugs are designed to deliver cytotoxic payloads directly to cancer cells expressing target antigens by leveraging the specificity of monoclonal antibodies (mAbs). (Carter, PJ, and Senter, PD (2008) Antibody-drug conjugates for cancer therapy, Cancer Journal (Sudbury, Mass.) 14, 154-169; Perez, HL, et al. (2014) Antibody-drug conjugates: current status and future directions, Drug Discovery Today 19, 869-881; Alley, SC, et al. (2010) Antibody-drug conjugates: targeted drug delivery for cancer, Current opinion in chemical biology 14, 529-537). The concept of ADCs can also be expanded to address applications beyond the realm of oncology, and currently includes monoclonal antibodies conjugated with non-cytotoxic therapeutic payloads (McPherson, MJ, and Hobson, AD (2020) Pushing the Envelope: Advancement of ADCs Outside of Oncology, Methods in molecular biology (Clifton, NJ) 2078, 23-36; Yu, S., et al. (2018) Next Horizons: ADCs Beyond Oncology, In Innovations for Next-Generation Antibody-Drug Conjugates (Damelin, M., Ed.), pp 321-347, Springer International Publishing, Cham).

[0058] Regardless of the nature of the delivered payload, it is important for all ADCs to maintain a stable covalent bond between the antibody and its payload (Ducry, L., and Stump, B. (2010) Antibody-Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodies, Bioconjugate Chemistry 21, 5-13). Strategies for covalent conjugation of payloads to antibodies can be classified into two categories: nonspecific and site-specific conjugation (McCombs, JR, and Owen, SC (2015) Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry, AAPS J 17, 339-351).Conjugation via lysine (K) (Wisdom, GB (2005) Conjugation of antibodies to fluorescein or rhodamine, Methods in molecular biology (Clifton, NJ) 295, 131-134) or reduced cysteine ​​(Sun, MMC, et al. (2005) Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides, Bioconjugate chemistry 16, 1282-1290) residues is the most common nonspecific conjugation method, resulting in a highly heterogeneous mixture of ADCs with varying drug-to-antibody ratios (DARs) and payload conjugation sites (Gordon, MR, et al. (2015) Field Guide to Challenges and Opportunities in Antibody-Drug Conjugates for Chemists, Bioconjugate chemistry) 26, 2198-2215; Doronina, SO, et al. (2006) Enhanced Activity of Monomethylauristatin F through Monoclonal Antibody Delivery: Effects of Linker Technology on Efficacy and Toxicity, Bioconjugate chemistry 17, 114-124).Given the complex nature of the products and the issues associated with lot-to-lot variability, nonspecific conjugations often lead to uncertainties in pharmacokinetics, toxicity, and efficacy (Boylan, NJ, et al. (2013) Conjugation Site Heterogeneity Causes Variable Electrostatic Properties in Fc Conjugates, Bioconjugate Chemistry 24, 1008-1016; Wagh, A., et al. (2018) Challenges and new frontiers in analytical characterization of antibody-drug conjugates, mAbs 10, 222-243; Acchione, M., et al. (2012) Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates, mAbs 4, 362-372).

[0059] In contrast, site-specific conjugation generally provides superior control of DARs and conjugation sites, although it typically requires more advanced protein engineering (Behrens, CR, and Liu, B. (2014) Methods for site-specific drug conjugation to antibodies, mAbs 6, 46-53). Conjugation via manipulated cysteine ​​(Stimmel, JB, et al. (2000) Site-specific Conjugation on Serine → Cysteine ​​Variant Monoclonal Antibodies*, Journal of Biological Chemistry 275, 30445-30450; Junutula, JR, et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index, Nature Biotechnology 26, 925-932), or conjugation via non-natural amino acid residues (Young, TS, and Schultz, PG (2010) Beyond the canonical 20 amino acids: expanding the genetic lexicon, The Journal of Biological Chemistry 285, 11039-11044; Axup, JY, et al. (2012) Synthesis of site-specific antibody-drug conjugates using Unnatural amino acids (PNAS 109, 16101-16106) are one of the two major classes of site-directed conjugation, while enzyme-mediated conjugation is a novel tool used to ensure control of DAR and conjugation sites (Jeger, S., et al.).(2010)Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase,Angewandte Chemie(International ed.in English)49,9995-9997;Madej,M.P.,et al.(2012)Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by Sortase A-mediated protein ligation,Biotechnology and bioengineering 109,1461-1470)。.

[0060] Microbial transglutaminases (mTGs) are enzymes that catalyze the formation of stable isopeptide bonds between glutamine (Q) side chains (i.e., γ-carboxamide groups) and primary amines (Jeger 2010; Schneider, H., et al. (2020) Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates, Analytical biochemistry 595, 113615). Based on the specificity of mTGs, they recognize glutamine residues in consensus sequences and primary amines on protein ligands or in flexible regions. Therefore, most mTG-mediated ADCs are produced by conjugating specific glutamine residues in mAbs with primary amine-containing ligands (Anami, Y., and Tsuchikama, K. (2020) Transglutaminase-Mediated Conjugations, Methods in molecular biology (Clifton, NJ) 2078, 71-82).Examples of target glutamine conjugation sites in human IgG include Q295 adjacent to non-glycosylated N297 (Dennler, P., et al. (2014) Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates, Bioconjugate Chemistry 25, 569-578), both Q295 and Q297 when introducing the N295Q mutation (Lhospice, F., et al. (2015) Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models, Molecular Pharmaceutics 12, 1863-1871), and artificially incorporated glutamine-containing peptide tags (Q tags) (Ebenig, A., et al. One example is al. (2019) Efficient Site-Specific Antibody-Drug Conjugation by Engineering a Nature-Derived Recognition Tag for Microbial Transglutaminase, Chembiochem: a European journal of chemical biology 20, 2411-2419). In addition to its ability to react with primary amines, the glutamine side chain also reacts with water and can undergo deamidation if a primary amine is unavailable at the reaction site (Anami and Tsuchikama 2020).

[0061] While most published studies focus on mAb engineering to achieve higher site specificity and selectivity, how potential side effects during mTG-mediated conjugation affect the quality of the final ADC product has not been extensively studied (Farias, SE, et al. (2014) Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates, Bioconjugate Chemistry 25, 240-250). Target glutamine residues in mAbs incubated with mTG and its linker payload (LP) can undergo one of three potential reactions: (1) conjugate with the linker payload, (2) undergo deamidation by reaction with water, or (3) crosslink to a lysine (K) residue (Martins, IM, et al. (2014) Transglutaminases: recent achievements and new sources, Applied Microbiology and Biotechnology 98, 6957-6964). Both of the latter two reactions are undesirable side effects that reduce the number of glutamine residues available for conjugation, leading to a decrease in overall DAR. Furthermore, the formation of intermolecular glutamine-lysine (QK) crosslinks increases the level of high molecular weight (HMW) variants in the ADC product, which has potential implications for both drug efficacy and safety (Rosenberg, AS (2006) Effects of protein aggregates: An immunologic perspective, The AAPS Journal 8, E501-E507).Given that mTG-mediated QK crosslinking can directly affect product quality, this reaction should be closely monitored during the formation of all mTG-mediated ADCs and should be minimized by designing mAb sequences with lysine residues that are less accessible and therefore less likely to crosslink with target glutamine residues.

[0062] In this disclosure, the formation of HMW variants in mTG-mediated ADCs was investigated using size exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS). It was found that mTG-mediated QK crosslinking directly influenced the level of high molecular weight (HMW) variants in the final ADC product. The relationship between HMW variant formation and QK crosslinking was studied using a model system in which two mAbs containing glutamine engineered for site-specific conjugation were incubated with mTG in the absence of a linker payload. The level of HMW variants was determined using SEC, and the crosslinking sites were identified and crosslinking peptides quantified using reduced peptide mapping (RPM). By establishing correlations between these two assays, QK crosslinking between target glutamine residues and specific lysine residues was identified as the primary contributor to HMW size variants in the studied ADCs. More importantly, since the heavy chain (HC) C-terminal K was identified as a crosslinking site in both mAbs, it was shown that the level of the HMW variant was significantly reduced in ADCs in which the HC C-terminal K was completely removed before mTG-mediated conjugation. These results indicate that QK crosslinking and other side effects occur during mTG-mediated conjugation and need to be monitored and controlled to ensure the quality and consistency of the final ADC product.

[0063] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the tests, but specific methods and materials are described herein.

[0064] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation, as understood by those skilled in the art, and to include endpoints where a range is provided. As used herein, the terms "include," "includes," and "including" are intended to be non-limiting and should be understood to mean "comprise," "comprises," and "comprising," respectively.

[0065] As used herein, the terms “protein” or “protein of interest” may include any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains commonly known in the art as a “polypeptide.” A “polypeptide” refers to a polymer composed of amino acid residues, associated naturally occurring structural variants, and their synthetic non-natural analogs linked via peptide bonds. “Synthetic peptide or polypeptide” refers to a non-natural peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using automated polypeptide synthesizers. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein may comprise one or more polypeptides to form a single functional biomolecule. In another exemplary embodiment, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. A protein of interest may include any of the following: biological therapeutic proteins, recombinant proteins used in research or therapeutics, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. The proteins may be produced using recombinant cell lineage production systems, such as insect baculovirus lines, yeast lines (e.g., Pichia genus), and mammalian lines (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation” (the entire instruction is incorporated herein; Darius Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation,” 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012)). In some exemplary embodiments, the protein includes modifications, adducts, and other covalent moieties. Examples of these modifications, adducts, and parts include avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose-binding proteins (MBPs), chitin-binding proteins (CBPs), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes. Proteins can be classified based on their composition and solubility, and thus include simple proteins such as globular and fibrous proteins, conjugated proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins, as well as inducible proteins such as primary and secondary inducible proteins.

[0066] In some exemplary embodiments, the protein of interest may be a recombinant protein, an in vivo product of gene therapy, a therapeutic protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, an antigen-binding protein, a fusion protein, an scFv, a multi-subunit protein, an antibody-drug conjugate, a receptor, a receptor ligand, or a combination thereof.

[0067] As used herein, the term “recombinant protein” refers to a protein produced as a result of the transcription and translation of a gene supported on a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an isotype antibody selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule may be a full-length antibody (e.g., IgG1), or the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0068] As used herein, the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs. The term "antibody" as used herein also includes the antigen-binding fragment of a complete antibody molecule.

[0069] The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and similar terms, as used herein, include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding an antibody variable domain and optionally a constant domain. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA may be sequenced and engineered chemically or by using molecular biological techniques, for example, by arranging one or more variable domains and / or constant domains in a suitable configuration, or by introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids.

[0070] As used herein, “antibody fragment” includes, for example, a portion of an intact antibody, such as the antigen-binding region or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolation complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of the immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the immunoglobulin light and heavy chain variable regions are linked by a peptide linker. In some exemplary embodiments, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of the fragment, which is a fragment that binds to the same antigen as the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity equivalent to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments can be generated by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of intact antibodies and / or recombinantly from genes encoding partial antibody sequences. Alternatively, or in addition, antibody fragments may be produced entirely or partially synthetically. Antibody fragments may optionally include single-chain antibody fragments. Alternatively, or in addition, antibody fragments may include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, and more typically, at least about 200 amino acids.

[0071] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody typically contains two distinct heavy chains, each specifically binding to either two different molecules (e.g., antigens) or the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two orders of magnitude lower, or even three or four orders of magnitude lower, than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody can be on the same target or different targets (e.g., on the same protein or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable regions that recognize different epitopes of the same antigen can be fused with nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[0072] A typical bispecific antibody comprises two heavy chains, each having three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain; and an immunoglobulin light chain that does not confer antigen-binding specificity but is either associable with each heavy chain, or associable with each heavy chain and capable of binding one or more epitopes that bind to the heavy chain antigen-binding region, or associable with each heavy chain and capable of binding one or both of the heavy chains to one or both epitopes. BsAbs can be divided into two main classes: those possessing an Fc region (IgG-like) and those lacking an Fc region, the latter of which are usually smaller than IgG and IgG-like bispecific molecules that contain Fc. IgG-like bsAbs can take various forms, including but are not limited to triomab, knob-in-hole IgG (kih IgG), crossMab, orth-Fab IgG, dual-variable domain Ig (DVD-Ig), two-in-one or dual-acting Fab (DAF), single-stranded IgG Fv (IgG-scFv), or κλ bodies. Non-IgG-like variant formats include tandem scFv, diabody format, single-stranded diabody, tandem diabody (TandAb), biaffinity retargeting molecule (DART), DART-Fc, nanobody, or antibodies produced by the Dock-and-Lock (DNL) method (their entire teachings are incorporated herein; Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014)).Methods for generating bsAbs are not limited to quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation including chemical crosslinking agents, and genetic approaches utilizing recombinant DNA technology. Examples of bsAbs include those disclosed in the following patent applications, which are incorporated herein by reference: U.S. Patent Application No. 12 / 823838 filed June 25, 2010; U.S. Patent Application No. 13 / 488628 filed June 5, 2012; U.S. Patent Application No. 14 / 031075 filed September 19, 2013; U.S. Patent Application No. 14 / 808171 filed July 24, 2015; U.S. Patent Application No. 15 / 713574 filed September 22, 2017; and 201 U.S. Patent Application No. 15 / 713569 filed on September 22, 2017; U.S. Patent Application No. 15 / 386453 filed on December 21, 2016; U.S. Patent Application No. 15 / 386443 filed on December 21, 2016; U.S. Patent Application No. 15 / 22343 filed on July 29, 2016; and U.S. Patent Application No. 15814095 filed on November 15, 2017.

[0073] As used herein, “multispecific antibody” refers to an antibody that has binding specificity to at least two different antigens. Such molecules typically bind to only two antigens (i.e., bispecific antibodies, bsAb), but antibodies with further specificity, such as triplicate antibodies and KIH triplicate antibodies, can also be addressed by the systems and methods disclosed herein.

[0074] As used herein, the term “monoclonal antibody” is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies may be derived from a single clone, including any eukaryotic cell, prokaryotic cell, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in this disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, phage display technology, gene therapy, or a combination thereof.

[0075] As used herein, the term “conjugated peptide or protein” may refer to a peptide or protein conjugated to a biologically active drug by a linker, including “antibody-drug conjugate” or “ADC.” A conjugated peptide, conjugated protein, or antibody-drug conjugate may contain several molecules of a biologically active drug (or payload) that can be covalently bound to a conjugation site, such as the side chains of amino acid residues of the conjugated peptide, conjugated protein, or antibody (Siler Panowski et al., Site-specific antibody drug conjugates for cancer therapy, 6 mAbs 34-45 (2013)). Because antibodies can bind to specific sites on target cells, enabling efficient delivery of biologically active drugs to target cells, the development of ADCs is a strategy to improve drug efficacy. Antibodies used in ADCs can bind to the target site with sufficient affinity for selective accumulation and sustained retention. Most ADCs may have Kd values ​​in the nanomolar range. The payload can have a potency in the nanomolar / picomolar range and can reach achievable intracellular concentrations after the distribution of the ADC to the target tissue. The linker forming the binding between the payload and the antibody can be sufficiently stable in circulation to take advantage of the pharmacokinetic properties of the antibody portion (e.g., long half-life) and to ensure that the payload remains bound to the antibody as it is distributed within the tissue, but should allow for the efficient release of the biologically active drug once the ADC is taken up into the target cell. The linker may include a linker that is not cleavable during cell processing and a linker that is cleavable once the ADC reaches the target site. With a non-cleavable linker, the biologically active drug released intracellularly includes the payload and all elements of the linker that are still bound to amino acid residues of the antibody, e.g., lysine, cysteine, or glutamine residues, after complete proteolytic degradation of the ADC within the lysosome.A cleavable linker is one whose structure includes a cleavage site between the payload and the amino acid binding site on the antibody. The cleavage mechanism may include hydrolysis of acid-unstable bonds in acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by intracellular proteases or esterases, and reductive cleavage of disulfide bonds by an intracellular reducing environment.

[0076] The typical distribution profile of an ADC contains a mixture of the ADC, the unconjugated antibody, and the unconjugated drug payload. The amount of drug that can be delivered to target cells will be reduced in the presence of the unconjugated antibody because the unconjugated antibody competes with the drug-conjugated antibody for the target antigen. Generally, derived ADCs are highly heterogeneous species containing various ADC species with variable drug-to-antibody ratios (DARs), as well as various conjugation sites, including conjugated and unconjugated conjugation sites. In the case of ADCs produced using enzymatic conjugation reactions, such as mTG-mediated conjugation, the ADC species may include conjugated conjugation sites, cross-linked conjugation sites, and deamidated conjugation sites. The heterogeneity of ADCs can significantly affect the safety and efficacy of the drug due to the presence of undesirable ADC species. A desirable ADC formulation should contain a clearly defined DAR and a degree of homogeneity. Quantifying and characterizing site-specific drug conjugations of ADCs with variable DAR, such as site-specific quantification of drug conjugations, is an important process for controlling quality characteristics of ADC formulations that can directly affect the efficacy of ADCs.

[0077] A conjugated peptide or protein, such as an ADC, may be engineered to have a site-specific conjugation site, e.g., an engineered cysteine, an engineered non-native amino acid residue, or an engineered glutamine. The site-specific conjugation site may also be called a target site, a target conjugation site, or a target amino acid residue. The target amino acid residue may be an amino acid residue in a consensus sequence recognized by the relevant enzyme, such as a glutamine residue targeted by mTG. In contrast, an off-target amino acid residue may be an amino acid residue that forms an undesirable crosslink with the target amino acid residue and does not contribute to conjugation with a linker, e.g., a lysine residue that is crosslinked to the target glutamine residue by mTG. An off-target amino acid residue, e.g., a lysine residue, may be called a reactive amino acid residue, e.g., reactive lysine. Proteins such as antibodies may contain many amino acid residues that can theoretically form undesirable crosslinks, but in reality, only one or a few of those amino acid residues, such as lysine, may be reactive and participate in the crosslinking reaction. This disclosure provides herein methods for designing conjugated peptides or proteins and for selecting conjugated peptides or proteins, for example, by characterizing and reducing or removing potential crosslinking sites to avoid the formation of HMW species.

[0078] In some exemplary embodiments, the target protein may be produced from mammalian cells. Mammalian cells may be of human or non-human origin, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their lineages (e.g., HEK293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells).VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells, or derivatives thereof), Fibroblasts from any tissue or organ (including, but not limited to, the heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymphatic glands, pharyngeal tonsils, tonsils, bone marrow, and blood), and spleen), as well as fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells) Cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 This may include cells such as F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, mouse L cell line 2071, mouse L cell line LM, mouse L cell line, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof.

[0079] In some exemplary embodiments, a sample containing the protein of interest may be prepared before or after the concentration, separation, and / or analysis steps. Preparation steps may include alkylation, reduction, denaturation, digestion, and / or deglycosylation.

[0080] As used herein, the term “protein alkylating agent” refers to a drug used to alkylate specific free amino acid residues in a protein. Non-exclusive examples of protein alkylating agents include iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0081] As used herein, “protein denaturation” may refer to the process by which the three-dimensional shape of a molecule changes from its native state. Protein denaturation can be carried out using protein denaturants. Non-limiting examples of protein denaturants include heat, high or low pH, reducing agents such as DTT (see below), or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of a system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and their salts.

[0082] As used herein, the term “protein reducing agent” refers to a drug used to reduce disulfide crosslinks in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Elman’s reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof. Conventional methods of protein analysis, reduced peptide mapping, involve protein reduction before LC-MS analysis. In contrast, non-reduced peptide mapping omits the reduction sample preparation step to preserve endogenous disulfide bonds. In some exemplary embodiments, non-reduced preparations may be used, for example, to preserve endogenous disulfide bonds between Fab arms of an antibody or antibody-derived protein. In other exemplary embodiments, partially reduced preparations may be used, for example, to reduce disulfide bonds between Fab arms of an antibody or antibody-derived protein without completely reducing the protein.

[0083] As used herein, the term “digestion” refers to the hydrolysis of one or more peptide bonds in a protein. There are several approaches to carrying out the digestion of proteins in a sample using appropriate hydrolyzing agents, such as enzymatic digestion or non-enzymatic digestion.

[0084] As used herein, the term “digestive enzyme” refers to any of a number of different agents capable of performing the digestion of proteins. Non-limiting examples of hydrolysants capable of performing enzymatic digestion include proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or their biologically active fragments or homologs, or combinations thereof. For a recent review discussing available techniques for protein digestion, see Switazar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried MANiessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013)).

[0085] In some exemplary embodiments, IdeS or a variant thereof is used to cleave the antibody below the hinge region, generating Fc and Fab2 fragments. Digestion of the analyte may be advantageous because the size reduction can increase the sensitivity and specificity of analyte characterization and detection using LC-MS. When used for this purpose, digestion that separates the Fc fragment and retains the Fab2 fragment for analysis may be preferred. This is because the variable region of the target, such as the complementarity-determining region (CDR) of the antibody, is contained in the Fab2 fragment, while the Fc fragment is relatively uniform among antibodies and may therefore provide less relevant information. Alternatively, or additionally, digestion that separates the Fab2 fragment and retains the Fc fragment for analysis may be preferred because the Fc fragment contains the N-glycosylation site of the target. Analysis of digested antibody fragments (subunits), e.g., digestion with IdeS, followed by chromatographic analysis and / or mass spectrometry, is called subunit analysis.

[0086] IdeS digestion offers high efficiency and enables high analyte recovery. The digestion and elution processes may be carried out under natural conditions, allowing for simple integration into natural LC-MS systems. IdeS or its variants are commercially available, for example, as FabRICATOR® or FabRICATOR Z®.

[0087] In some exemplary embodiments, a carboxypeptidase is used to remove the carboxy-terminal (C-terminal) residue of the target protein. A carboxypeptidase is a protease that hydrolyzes peptide bonds at the C-terminus of a protein or peptide. Examples of carboxypeptidases include metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, and carboxypeptidase E. Carboxypeptidases may be useful for removing C-terminal lysine from the target protein. In other embodiments, other proteases may be used to remove protein or peptide residues that contribute to undesirable crosslinking, such as aminopeptidases. The protein subjected to proteolysis by a protease, such as a carboxypeptidase, such as an antibody, may be called a clipped protein, such as a clipped antibody. Clipped proteins may have substantially the same or improved structure, function, safety, and efficacy as the original protein.

[0088] As used herein, “sample” can be obtained from any step of a bioprocess, such as a cell culture medium (CCF), a harvested cell culture medium (HCCF), any step in downstream processing, a drug substance (DS), or a pharmaceutical product (DP) including the final formulation product. In some other specific exemplary embodiments, the sample may be selected from any step in downstream processes such as clarification, chromatographic generation, viral inactivation, or filtration. In some specific exemplary embodiments, the pharmaceutical product may be selected from a manufactured pharmaceutical product in a clinic, for shipment, storage, or handling.

[0089] In some exemplary embodiments, the sample is a biological sample. As used herein, the term “biological sample” refers to a sample taken from an organism, such as a human or a non-human mammal. A biological sample may include, or consist of, whole blood, plasma, serum, saliva, tears, semen, cheek tissue, organ tissue, urine, feces, skin, or hair. The sample may be taken from a patient, for example, a clinical sample. In some exemplary embodiments, the sample may be taken from a non-human animal, for example, a preclinical sample. In some exemplary embodiments, the sample may be taken from a non-human animal subjected to gene therapy to produce at least one target protein that may be contained in the sample. In some embodiments, the sample is a further processed form of any of the above-described embodiments of the sample.

[0090] As used herein, the term “impurity” may include any undesirable proteins present in a protein sample or protein biopharmaceutical. Impurities may include manufacturing process impurities and target substance impurities. Impurities may also be of known structure, partially characterized, or unspecified. Manufacturing process impurities may originate from the manufacturing process and may fall into three main categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vector, or total DNA) derived from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other culture medium components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leaches.

[0091] Impurities derived from the target substance (e.g., precursors, specific degradation products) may be molecular variants that arise during manufacturing and / or storage and do not possess properties comparable to the desired product in terms of activity, efficacy, and safety. Such variants may require considerable effort in isolation and characterization to identify the type of modification. Impurities derived from the target substance may include cleavage types, modified types, and aggregates. Cleavage types are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modification types include, but are not limited to, deamidation, isomerization, mismatched SS bonding, oxidation, or modified conjugate types (e.g., glycosylation, phosphorylation). Modification types may also include any post-translational modifications. Aggregates include dimers and higher multiples of the desired product. (Q6B standard: Test procedures and criteria for biotechnological / biological products, ICH August 1999, U.S. Department of Health and Human Services). In some exemplary embodiments, aggregates (HMW species) may be formed by off-target crosslinking of proteins, such as antibodies in antibody-drug conjugates.

[0092] As used herein, the term “liquid chromatography” refers to a process in which a biological / chemical mixture carried by a liquid can be separated into its components as a result of the differential distribution of its components as they flow through (or flow into) a fixed liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase (RP) liquid chromatography, ion exchange (IEX) chromatography, size exclusion chromatography (SEC), affinity chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), or mixed-mode chromatography (MMC).

[0093] In some exemplary embodiments, the methods of the present invention involve the use of size exclusion chromatography. Size exclusion chromatography or gel filtration relies on the separation of components according to their molecular size. Separation depends on the amount of time a substance spends in a porous stationary phase compared to the time it spends in a fluid. The probability of a molecule being present in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a substance to penetrate into a pore is determined by the diffusion mobility of higher macromolecules compared to smaller macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all, while for very small macromolecules, the probability of penetration is close to 1. Components with larger molecular sizes pass through the stationary phase more quickly, while components with smaller molecular sizes have longer pathway lengths through the pores of the stationary phase and are therefore retained in the stationary phase for longer. Variants of the target protein having a higher or lower molecular weight than the major species or the intended product may be called “size variants.”

[0094] Analytes eluted from an SEC column can be separated into fractions based on their elution time. For example, analytes that elute earlier than the functional form of the target protein, such as the monomeric form, can be broadly classified as high molecular weight (HMW) species. The HMW fraction may be further subdivided into, for example, very high molecular weight (vHMW) fractions and dimer fractions (representing the elution time of the dimer of the target protein). Analytes that elute later than the functional form of the target protein can be broadly classified as low molecular weight (LMW) species, and can be further subdivided into LMW fractions and later tail fractions.

[0095] HMW species, such as dimers, may be formed as products of crosslinking reactions. A crosslink is a bond or a short sequence of bonds that links one polypeptide chain to another. Crosslinks can occur spontaneously or synthetically. Examples of crosslinks include, for example, disulfide bonds, amine bonds, ester bonds, or imine bonds. Depending on the crosslinking mechanism, a crosslinking reaction may increase or decrease the total molecular weight of the crosslinked molecule compared to the unmodified molecule. In exemplary embodiments, the HMW species of a size variant of the protein in question, e.g., ADC, is a dimer formed by crosslinking. In some exemplary embodiments, the crosslink is formed between a glutamine residue and a lysine residue. In some specific embodiments, the crosslinked lysine is located at the C-terminus of the protein in question. In some exemplary embodiments, the crosslink is formed via an enzymatic reaction, for example, by the action of microbial transglutaminase. In some exemplary embodiments, crosslinking may result in the formation of dimeric, trimer, tetramer, or larger species of the protein in question, e.g., ADC.

[0096] As used herein, the term “crosslinking agent” refers to a molecule capable of forming crosslinks between two or more other molecules, or between two or more sites on one or more molecules. In some exemplary embodiments, the crosslinking agent is an enzyme ("crosslinking enzyme"), such as mTG.

[0097] Chromatographic materials for SEC may include size exclusion materials, which are resins or membranes. The matrix used for size exclusion is preferably an inert gel medium, which may be a crosslinked polysaccharide, for example, a complex of crosslinked agarose and / or dextran in the form of spherical beads. The degree of crosslinking determines the size of the pores present in the swollen gel beads. Molecules larger than a certain size do not enter the gel beads and therefore move most quickly through the chromatographic bed. Smaller molecules, such as surfactants, proteins, and DNA, enter the gel beads to varying degrees depending on their size and shape, and their passage through the bed is delayed. Thus, molecules are generally eluted in order of decreasing molecular size.

[0098] Porous chromatography resins suitable for viral size exclusion chromatography can be made from dextrose, agarose, polyacrylamide, or silica, each possessing different physical properties. Polymer combinations may also be used. The most commonly used is the one commercially available from Amersham Biosciences under the trademark name "SEPHADEX". Other size exclusion carriers from different structural materials are also suitable, e.g., Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery County, Pennsylvania) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, California).

[0099] In some exemplary embodiments, the mobile phase used to obtain the aforementioned elutes from size exclusion chromatography may contain volatile salts. In some specific embodiments, the mobile phase may contain ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0100] As used herein, the term “mass spectrometer” refers to an instrument capable of identifying specific molecular species and measuring their precise masses. This term implies the inclusion of any molecular detector by which polypeptides or peptides can be characterized. A mass spectrometer may comprise three main components: an ion source, a mass spectrometer, and a detector. The role of the ion source is to generate gas-phase ions. Analyte atoms, molecules, or clusters can be transferred into the gas phase and ionized simultaneously (as in electrospray ionization) or through another process. The choice of ion source depends on its application.

[0101] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes techniques for obtaining structural information of sample molecules by using multi-stage mass selection and mass separation. A prerequisite is that, after the initial mass selection step, the sample molecules are converted to the gas phase and ionized so that fragments are formed in a predictable and controllable manner. Multistage MS / MS, or MS n First, the precursor ion (MS 2 ) are selected and isolated, fragmented, and primary fragment ions (MS 3 ) is isolated, fragmented, and secondary fragments (MS 4This can be carried out by isolating the ions and obtaining meaningful information, or by continuing in the same manner as long as the fragment ion signal is detectable. Tandem MS has been successfully performed with a wide variety of analyzer combinations. The choice of analyzer combination for a particular application can be determined by many different factors, including size, cost, and availability, as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are spatial tandem and temporal tandem, although there are also hybrids in which a temporal tandem analyzer is coupled in space or with a spatial tandem analyzer. A spatial tandem mass spectrometer comprises an ion source, a precursor ion activator, and at least two non-trapped mass spectrometers. A specific m / z separation function can be designed so that ions are selected in one section of the instrument, dissociated in an intermediate region, and then the product ions are transmitted to another analyzer for m / z separation and data acquisition. In temporal tandem mass spectrometry, mass spectrometer ions generated by an ion source can be captured, isolated, fragmented, and m / z separated using the same physical apparatus. Peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications. These can be used for protein characterization by correlating experimental MS / MS data with theoretical MS / MS data, the latter being generated from potential peptides in protein sequence databases. Characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequencing, protein de novo sequencing, identification of post-translational modification sites, or identification of post-translational modifications, or intercomparability analysis, or a combination thereof.

[0102] As used herein, the term “mass spectrometer” includes any instrument capable of separating species, i.e., atoms, molecules, or clusters, according to their mass. Non-limiting examples of mass spectrometers that may be employed include time-of-flight (TOF), magnetoelectric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbit trap, Fourier transform ion cyclotron resonance (FTICR), and accelerator mass spectrometry (AMS) techniques.

[0103] In some exemplary embodiments, the mass spectrometer may operate with nanoelectrospray or nanospray. As used herein, the terms “nanoelectrospray” or “nanopray” refer to electrospray ionization in sample solution at very low solvent flow rates, typically hundreds of nanoliters per minute or less, often without the use of external solvent delivery. The electrospray injection setup forming the nanoelectrospray may use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small sample (analyte) solution volumes over a long period of time. A dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separation of the mixture before analysis by the mass spectrometer.

[0104] In some exemplary embodiments, a mass spectrometer may be coupled to a liquid chromatography-multi-reaction monitoring system. More generally, the mass spectrometer can be analyzed by selected reaction monitoring (SRM), including continuous reaction monitoring (CRM) and parallel reaction monitoring (PRM).

[0105] As used herein, “multiple reaction monitoring” or “MRM” refers to a mass spectrometry-based technique that can accurately quantify small molecules, peptides, and proteins in complex matrices with high sensitivity, specificity, and wide dynamic range (Paola Picotti & Ruedi Aebersold, Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions, 9 NATURE METHODS 555-566 (2012)). MRM can typically be performed using a triple quadrupole mass spectrometer, where the precursor ion corresponding to the selected small molecule / peptide is selected at the first quadrupole, and fragment ions of the precursor ion are selected for monitoring at the third quadrupole (Yong Seok Choi et al., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates, 930 JOURNAL OF CHROMATOGRAPHY B 129-135 (2013)).

[0106] In some exemplary embodiments, LC-MS may be performed under native conditions. As used herein, the term “native conditions” may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. Native mass spectrometry is an approach for studying intact biomolecular structures in their native or near-native state. The term “native” refers to the biological status of the analyte in solution before it is subjected to ionization. By controlling several parameters of the solution containing the biological analyte, such as pH and ionic strength, the native folded state of the biological analyte in solution can be maintained. Generally, native mass spectrometry is based on electrospray ionization, in which the biological analyte is sprayed from a non-denaturing solvent. Other terms such as non-covalent, native spray, electrospray ionization, non-denaturing, polymer, or supramolecular mass spectrometry can also describe native mass spectrometry. In exemplary embodiments, native MS allows for better spatial resolution compared to non-native MS and improves the detection of in vivo conversion products of therapeutic proteins. For a detailed review on native mass spectrometry, see Review: Elisabetta Boeri Erba & Carlo Pe-tosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0107] As used herein, the term “database” refers to an edited collection of protein sequences that may be present in a sample, for example, in the form of a file in FASTA format. The relevant protein sequences may be derived from the cDNA sequences of the species under study. Public databases that may be used to search for relevant protein sequences include, for example, databases hosted by Uniprot or Swiss-prot. Databases may be searched using what is referred herein as a “bioinformatics tool.” A bioinformatics tool has the ability to search for uninterpreted MS / MS spectra for all possible sequences in the database and to provide interpreted (annotated) MS / MS spectra as output. Non-exclusive examples of such tools include Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0108] The present invention is not limited to any of the aforementioned proteins, antibodies, monoclonal antibodies, bispecific antibodies, protein expression systems, antibody fragments, conjugated peptides or proteins, antibody-drug conjugates, conjugation sites, crosslinks, high molecular weight species, protein alkylating agents, protein denaturants, protein reducing agents, digestive enzymes, hydrolysants, samples, liquid chromatography systems, mobile phases, mass spectrometers, databases, or bioinformatics tools, and any protein, antibody, monoclonal antibody, bispecific antibody, protein expression system, antibody fragment, conjugated peptides or proteins, antibody-drug conjugates, conjugation sites, crosslinks, high molecular weight species, protein alkylating agents, protein denaturants, protein reducing agents, digestive enzymes, hydrolysants, samples, liquid chromatography systems, mobile phases, mass spectrometers, databases, or bioinformatics tools can be selected by any appropriate means.

[0109] The present invention will be better understood by referring to the following embodiments. However, they should not be construed as limiting the scope of the invention. [Examples]

[0110] Chemicals and reagents. All mAbs and ADCs were produced by Regeneron (Tarytown, New York, USA). Acetonitrile (ACN, LC-MS grade), trifluoroacetic acid, formic acid, dithiothreitol (DTT), iodoacetamide (IAA), tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), 8M guanidine-HCl solution, and Invitrogen UltraPure 1M Tris-HCl buffer, pH 7.5 were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

[0111] Microbial transglutaminase (mTG) was purchased from MilliporeSigma (Burlington, Massachusetts, USA). Urea and carboxypeptidase B (CpB) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, Wisconsin, USA). C18 (ACQUITY ultra-high performance LC peptide BEH C18 1.7 μm, 2.1 mm × 150 mm) and phenyl columns (ACQUITY ultra-high performance LC BEH phenyl 1.7 μm, 2.1 mm × 150 mm) were purchased from Waters (Milford, Massachusetts, USA). FabRICATOR was purchased from Genovis (Lund, Sweden). PNGase F was purchased from New England Biolabs (Ipsowich, Massachusetts, USA). Deionized water was supplied by a Milli-Q integrated water purification system fitted with a MilliPak Express 20 filter (MilliporeSigma, Burlington, Massachusetts, USA).

[0112] Preparation of mAb and ADC samples. To evaluate crosslinking in mAb samples, mAb1 and mAb2 were incubated with 1 U / mg Ab mTG in conjugation buffer at 37°C for 1, 3, or 6 hours. To generate ADCs, mAb1 and mAb2 were incubated with mTG and linker payload (LP) in conjugation buffer at 37°C for 6 hours. Control samples were incubated in conjugation buffer at 37°C for 6 hours. The HC C-terminal K in mAb2 was enzymatically removed by digestion with carboxypeptidase B (CpB) at 37°C for 2 hours. The CpB enzyme and baseline aggregates were then removed by SEC purification, followed by incubation in conjugation buffer at 37°C for 6 hours with mTG alone or with both mTG and LP. All reaction mixtures resulting from the above were subjected to SEC analysis immediately or frozen at -80°C for subsequent LC-MS peptide mapping or subunit analysis.

[0113] SEC analysis. A 5 μg mAb or ADC sample was loaded into an SEC column, and the reaction products were separated by size. Peaks eluted from the column were detected by UV absorption at 280 nm. Peak integration was performed using Empower software (Waters) to determine the peak area, and the relative abundance (area percentage) was calculated.

[0114] LC-MS / MS peptide mapping analysis. mAb and ADC samples were denatured, then reduced, and alkylated with TCEP and IAA, respectively. The reduced samples were digested with trypsin at 37°C for 4 hours, followed by quenching with TFA. The trypsin digest (5 μg sample) was loaded onto a C18 column.

[0115] Peptides eluted from a C18 column were analyzed by UV absorption at 214 nm and subjected to MS acquisition using a Q Exactive® Plus Hybrid Quadrupole-Orbitrap® mass spectrometer (Thermo). Source parameters were set as follows: spray voltage, 3.8 kV; auxiliary gas, 10; auxiliary gas temperature, 250 °C; capillary temperature, 320 °C; and S lens RF level, 50. Data-dependent acquisition (DDA) was performed using a single full MS scan from 300 m / z to 2000 m / z, followed by five consecutive MS / MS scans for five ions. The full MS scan was acquired at a resolution of 70,000 with an AGC target at 1E6. The MS / MS scan was acquired at a resolution of 17,500 with an AGC target at 1E5. The separation width was 4 m / z, and the normalized collision energy (NCE) was set to 27.

[0116] LC-MS subunit analysis. mAb and ADC samples were digested using FabRICATOR® and deglycosylated with PNGase F at 37°C for 2 hours. After digestion, the samples were denatured and reduced with guanidine HCl and DTT, respectively, and incubated at 60°C for 30 minutes. 20 μg samples of each preparation were injected into a phenyl column (reverse-phase column).

[0117] Subunits eluting from the phenyl column were detected by UV absorption at 280 nm and then subjected to MS acquisition on an X500B quadrupole time-of-flight (TOF) mass spectrometer (AB Sciex, Framingham, Massachusetts, USA). Complete MS scans were collected at 500 m / z to 5000 m / z with intact protein mode enabled. The ion source, curtain, and CAS gas were set to 60, 40, and 10 psi, respectively. The supply source temperature was set to 450°C, the spray voltage was 5500 V, and the declustering potential was 150 V.

[0118] Data Analysis. Peptides were identified by database search against mAb sequences using Byonic (version 4.6.1, Protein Metrics, San Carlos, California). Common mAb post-translational modifications (PTMs) were included as variable modifications in the search parameters, and cysteine ​​carbamide methylation was included as a fixed modification. Loss of NH3 (-17.0265 Da), glutamine deamidation (+0.9840 Da), and QK crosslinking associated with LP conjugation were also included as variable modifications. Peptide quantification was performed using Skyline software (version 21.2, MacCoss Lab Software, Seattle, Washington, USA). Subunit data analysis was performed using Sciex OS software (version 2.0 AB Sciex, Framingham, Massachusetts, USA). The Bio Tool Kit was used for protein deconvolution.

[0119] Example 1. Characterization of high molecular weight species of antibody-drug conjugates To study the relationship between HMW variant formation and mTG-mediated reactions, two mAbs (mAb1 and mAb2) containing target glutamine, each engineered for site-specific conjugation, were selected as model systems. mTG-mediated conjugation of mAb1 typically yielded high levels of HMW variants, while conjugation of mAb2 yielded relatively low levels. As shown in Figure 1A, both mAbs were incubated with mTG alone in the absence of LP to maximize HMW variant levels and facilitate crosslinking identification. After incubation of mAb1 with mTG alone for 1, 3, and 6 hours, the percentage of HMW variants determined by SEC increased from 6.1% in the control sample to 46.6%, 69.3%, and 74.3%, respectively, as shown in the left panel of Figure 1B. These high levels of HMW variants allow for reliable crosslinking identification by peptide mapping without the need for concentration or specialized workflows. As shown in the right panel of Figure 1B, changes in the abundance of native peptides, and the most significant changes in crosslinked peptides, can be readily observed in the UV profiles generated by reduced peptide mapping. Finally, the correlation between HMW variant formation and mTG-mediated crosslinking was established by comparing the increase in the proportion of HMW variants detected by SEC over time with the proportion of crosslinked peptides quantified from peptide mapping studies.

[0120] Example 2. Identification of off-target mTG crosslinking The reaction mixtures generated by incubation of mAb1 and mAb2 with mTG were analyzed by reduced peptide mapping to identify the cross-linked peptides. Database searches and manual data interpretation confirmed that both mAbs underwent mTG-mediated QK cross-linking. The cross-linking sites were successfully identified in samples generated from both mAb1 and mAb2.

[0121] As described in Example 1, analysis using SEC revealed that mTG incubation of mAb1 significantly increased the level of HMW variants. As shown in Figure 2A, the majority of crosslinks detected in mAb1 were between HC K56 or K446 (HC C-terminal K) and the manipulated target glutamine. After incubation with mTG, mAb1 target Q was found in one of three distinct forms: native, deamidated, or crosslinked. These observations are consistent with the current understanding of mTG-mediated reactions. The abundance of each form, including target Q, was determined based on their peak area and corresponding exact peptide mass, as shown in Table 1. As shown in Figure 2B, HC K56 was detected in a higher proportion of crosslinked peptides than HC K446. The total proportion of crosslinked peptides detected in target Q significantly increased with longer incubation times. This increase is mainly attributed to the increase in crosslinks between target Q and HC K56 over time. In addition to crosslinking, an increase in deamidation of target Q over time was also observed. This was as expected, considering that mTG catalyzes Q deamidation in the absence of primary amine-containing substrates such as primary amine-containing LP. [Table 1]

[0122] Based on these findings, the highly reactive HC K56 residue can be removed from the mAb1 backbone, for example, by mutations in the coding nucleic acid sequence. This small change can significantly reduce the level of HMW variants produced, potentially leading to an overall improvement in the quality of the ADC product. However, mutations introduced into HC K56 may have undesirable effects on other physiological and chemical properties and therefore require thorough evaluation.

[0123] The mAb2 amino acid sequence, lacking HC K56, yields a much lower increase in the level of HMW variants during mTG-mediated conjugation. In contrast to what was observed for mAb1, mAb2 HC K448 (C-terminal K) was identified as the primary crosslinking site, as shown in Figure 2C. Other minor crosslinking sites identified in the Fc and Fd regions of HC were detected at substantially low abundances, as shown in Table 2. Unlike mAb1, the total abundance of crosslinks detected in mAb2 containing target Q changed minimally over time, as shown in Figure 2D. On the other hand, the increase in mTG-mediated deamidation of target Q was far more significant in mAb2 compared to mAb1. These results suggest that deamidation is kinetically preferable to QK crosslinking in the absence of highly reactive lysine residues (e.g., HC K56 in mAb1). [Table 2]

[0124] The absence of multiple reactive lysine residues in these mAbs was unexpected. It has been previously reported that without specific lysine substitutions, only the HC C-terminal K can serve as an efficient site for conjugation with glutamine ligands (Spidel, JL, and Albone, EF (2019) Efficient Production of Homogeneous Lysine-Based Antibody Conjugates Using Microbial Transglutaminase, Methods in molecular biology (Clifton, NJ) 2033, 53-65; Spidel, JL, et al. (2017) Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase, Bioconjugate chemistry 28, 2471-2484). In this example, an additional lysine (HC K56) was identified in mAb1 that could form a crosslink with target Q. These results suggest that, although this phenomenon may not be commonly observed, certain native lysine residues in some mAbs may be effectively conjugated with glutamine ligands, and that these reactive lysines can be identified using the methods of the present invention.

[0125] Example 3. Correlation between HMW variant level and degree of mTG-mediated crosslinking. The relationship between the percentage of crosslinked peptides determined by peptide mapping (RPM-based crosslinking%) and the percentage of HMW variants determined by SEC (HMWSEC%) was evaluated to determine whether mTG-mediated crosslinking was the main contributing factor to the increase in HMW variant levels observed in mTG-mediated conjugation. By introducing a correction factor, the percentage of HMW variants determined by SEC, measured at intact levels, was converted to the percentage of crosslinked peptides, and as a result, the percentage of HMW variants determined by SEC could be correlated with the percentage of crosslinked peptides determined by RPM, measured at peptide levels. The model for converting HMWSEC% to crosslinking% is based on the observation that there was no increase in HMW% in the control sample (shown in Figure 1B), and therefore, it can be assumed that all HMW species are caused by QK crosslinking. As shown in Figure 3, one of the four target Q-containing peptides, two of the six target Q-containing peptides, and three of the eight target Q-containing peptides were crosslinked with dimers, trimers, and tetramers, respectively.

[0126] Based on this observation, the percentage of HMW dimers can be converted to the percentage of cross-linked peptides by dividing by a correction factor of 4, and similarly, the percentage of HMW trimers can be converted to the percentage of cross-linked peptides by dividing by 3. Similar conversions can be performed for all HMW species, and the correction factors for each HMW species up to 10mer are listed in Table 3. As a result, in order to convert the percentage of HMW variants determined by SEC to the percentage of cross-linked peptides, the dimer peaks identified by SEC were adjusted using the aforementioned correction factor of 4, and the peaks of all other HMW species were adjusted using a correction factor of 2.7. This is because the value of this correction factor changed minimally from the tetramer state to the 10mer state (see Equation 1). [Table 3] Equation 1: Calculation used to estimate the cross-linked peptide percentage from the HMW species percentage. All values ​​shown were determined by SEC.

number

[0127] The percentage of cross-linked peptides measured by peptide mapping, and the values ​​converted from SEC using Equation 1, are both included in Tables 4 and 5. The results shown in Figure 4A demonstrate a strong correlation between the measured and predicted percentages of cross-linked peptides in mAb1 after 1, 3, and 6-hour incubation with mTG. These results suggest that the increase in HMW variant levels in mAb1 observed after mTG-mediated conjugation may be primarily attributable to the formation of QK crosslinks. [Table 4] [Table 5]

[0128] Additional experiments were conducted in which mAb1 was incubated with both mTG and LP. All crosslinked peptides identified in incubations containing mTG alone were also detected, albeit at much lower abundances, in incubations containing both mTG and LP (producing ADCs). Furthermore, as shown in Figure 4B, there was a good correlation between the values ​​for the proportion of crosslinked peptides to these ADC samples, quantified using SEC and peptide mapping methods. These results also support the conclusion that the HMW variant in either the mAb or ADC is primarily due to mTG-mediated QK crosslinking.

[0129] Example 4. Removal of the C-terminal lysine of HC reduces the level of HMW variant detected in mTG-mediated ADCs. A good correlation was observed between the level of HMW variants in mAb1 and the degree of mTG-mediated QK crosslinking, but a similarly strong correlation was not identified in mAb2. mAb1 has two reactive K residues, but the HC C-terminal K is the only major crosslinking site in mAb2. Previous studies have shown that removal of the HC C-terminal K can completely disable the crosslinking between the two HCs that occurs during mTG-mediated conjugation (Siegmund, V., et al. (2015) Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation, Angewandte Chemie (International ed. in English) 54, 13420-13424). However, the relationship between the HC C-terminal K and the generation of crosslinked peptides and HMW variants has not been sufficiently investigated.

[0130] To determine how HC C-terminal K contributed to the formation of HMW species during mTG-mediated crosslinking, HC C-terminal K was removed from mAb2 using carboxypeptidase B (CpB). The resulting changes in the percentage of HMW variants formed during mTG-mediated conjugation were compared. As shown in Figure 5, after incubation with mTG alone, the percentage of HMW variants detected in mAb2 without HC C-terminal K was reduced by 50% compared to mAb2 with intact HC C-terminal K residues. This reduction was 40% in response to incubation with both mTG and LP. These results indicate that removal of HC C-terminal K can significantly reduce the level of HMW variants in the resulting ADC, suggesting that HC C-terminal K is a major contributor to mTG-mediated crosslinking in mAb2. Only 10% of mAb2 contained HC C-terminal K before CpB treatment, and these residues contributed only to an approximately 2% increase in the percentage of detected HMW variants, although this may vary from process to process. Therefore, HC C-terminal K represents a potential risk to HMW variant formation, and its removal should be considered in future drug development strategies.

[0131] The incubation samples described above were subjected to extensive characterization, including both subunit analysis and peptide mapping. Subunit analysis revealed that LC-Fc / 2(+HC C-terminal K) was the primary crosslinked fragment of the untreated mAb, as shown in Figure 6B. This observation was consistent with the peptide mapping results shown in Figure 7, which identified HC C-terminal K as the sole major site contributing to crosslinking. Relatively low levels of LC-Fc / 2(-HC C-terminal K) were observed with similar signal intensity in both the CpB-treated and untreated samples, as shown in Figures 6B and 6D. These results indicate that removal of HC C-terminal K does not provide any other lysine residues that are more sensitive to mTG-mediated crosslinking. Peptide mapping results also revealed low levels of crosslinking at small sites on HC. This suggests that K crosslinking sites can exist across the mAb backbone and may explain the observed increase in the proportion of HMW variants even after complete removal of HC C-terminal K.

[0132] This disclosure describes a method for determining the underlying mechanism of HMW variant formation in mTG-mediated ADCs using SEC and LC-MS techniques. Using the method of the present invention, it was found that mTG-mediated QK crosslinking is the primary cause of the increase in HMW variant levels in ADCs and therefore affects the quality of the final ADC product. QK crosslinking sites were identified in the two mAbs studied. A correlation was established between the percentage of HMW variants determined by SEC and the percentage of crosslinked peptides quantified by peptide mapping. More importantly, it was shown that the level of HMW variants was substantially reduced in ADCs conjugated from CpB-treated mAb2, in which the HC C-terminal K was completely removed. This result directly demonstrates that the HC C-terminal K was the primary contributor to crosslinking and HMW variant formation in mAb2.

[0133] Crosslinking reactions and other side effects occurring during mTG-mediated conjugation must be carefully monitored and controlled to maintain high quality and consistency of the ADC product. Identifying reactive lysine residues, including HC K56 in mAb1 and HC C-terminal K in both mAbs, provides insight into how the mAbs can be designed to minimize the formation of HMW variants in the resulting ADC. For example, highly reactive sites such as HC K56 in mAb1 should be removed during the early developmental stages by screening ADC candidates based on the level of their HMW variants. If localization and mutation of these residues prove necessary, extensive characterization can be performed using LC-MS techniques such as peptide mapping. Removal of HC C-terminal K may be a preferred route to minimize HMW variant formation and avoid future complications in ongoing product development.

Claims

1. A method for generating antibody-drug conjugates with reduced high molecular weight (HMW) species, (a) A step of producing a clipped antibody by contacting an antibody containing C-terminal lysine with a carboxypeptidase, wherein the clipped antibody does not contain C-terminal lysine. (b) A method comprising the step of contacting the clipped antibody with a crosslinking agent and a linker-payload to produce an antibody-drug conjugate with reduced HMW species.

2. The method according to claim 1, wherein the carboxypeptidase is metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, or carboxypeptidase E.

3. The method according to claim 1, wherein the carboxypeptidase is carboxypeptidase B.

4. The method according to claim 1, wherein the antibody is brought into contact with the carboxypeptidase at approximately 35°C to approximately 39°C for approximately 1 hour to approximately 3 hours.

5. The method according to claim 1, wherein the payload is a cytotoxic payload or a therapeutic payload.

6. The method according to claim 1, wherein the antibody comprises glutamine manipulated for site-specific conjugation, and optionally the crosslinking agent can crosslink the glutamine and the C-terminal lysine.

7. The method according to claim 1, wherein the crosslinking agent is an enzyme, and optionally the enzyme is a microbial transglutaminase.

8. A method for generating antibody-drug conjugates with reduced high molecular weight (HMW) species, (a) A step of identifying at least one off-target amino acid residue that forms a crosslink in an antibody-drug conjugate, (i) A sample containing an antibody is brought into contact with a linker and a crosslinking agent to produce a crosslinked sample, wherein the crosslinking agent can crosslink the antibody to the linker at target amino acid residues, and the crosslinking agent can crosslink the antibody at target amino acid residues to off-target amino acid residues. (ii) Contacting the crosslinked sample with at least one digestive enzyme to produce a peptide digest, (iii) The peptide digest is subjected to liquid chromatography-mass spectrometry (LC-MS) to identify the cross-linked peptide, (iv) The identifying step, which includes identifying at least one off-target amino acid residue that forms a crosslink using the identification, (b) A step of contacting the antibody with at least one protease to produce a clipped antibody, wherein the clipped antibody does not contain the at least one identified off-target amino acid residue, and (c) A method comprising the step of contacting the clipped antibody with a linker and the crosslinking agent to produce an antibody-drug conjugate with reduced high molecular weight species.

9. The method according to claim 8, wherein the at least one off-target amino acid residue is lysine, and optionally, the lysine is C-terminal lysine.

10. The method according to claim 8, wherein the target amino acid residue is lysine, cysteine, a non-natural amino acid, or glutamine.

11. The method according to claim 8, wherein the target amino acid residue is manipulated for site-directed conjugation.

12. The method according to claim 8, wherein the linker is bound to a payload, and optionally the payload is a cytotoxic payload or a therapeutic payload.

13. The method according to claim 8, wherein the crosslinking agent is an enzyme, and optionally, the crosslinking agent is microbial transglutaminase (mTG).

14. The method according to claim 8, wherein the at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof.

15. The method according to claim 8, wherein the at least one digestive enzyme is trypsin.

16. The method according to claim 8, wherein the at least one digestive enzyme is IdeS or a variant thereof.

17. The method according to claim 8, wherein the liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography.

18. The method according to claim 8, wherein the LC-MS analysis is RPLC-MS / MS analysis.

19. The method according to claim 8, wherein the at least one protease is a carboxypeptidase, and optionally the carboxypeptidase is carboxypeptidase B.

20. The method according to claim 8, wherein contact with the antibody is carried out at approximately 35°C to approximately 39°C for approximately 1 hour to approximately 3 hours.

21. The method according to claim 8, wherein the clipped antibody is an antibody lacking a C-terminal lysine.

22. A method for characterizing cross-linking sites in a target protein, (a) A step of producing a target crosslinked protein by contacting a sample containing a target protein with a crosslinking agent, wherein the target protein contains at least one target amino acid residue that can be crosslinked by the crosslinking agent. (b) A step of bringing the target cross-linked protein into contact with at least one digestive enzyme to produce a peptide digest, (c) A step of subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to characterize the peptide containing crosslinking at at least one target amino acid residue, (d) A method comprising the step of characterizing crosslinking sites in the target protein using the feature-analyzed peptide.

23. The method according to claim 22, wherein the target protein is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein.

24. The method according to claim 22, wherein the crosslinking agent is an enzyme, and optionally the enzyme is microbial transglutaminase (mTG).

25. The method according to claim 22, wherein the target amino acid residue is lysine, cysteine, a non-natural amino acid, or glutamine.

26. The method according to claim 22, wherein the target amino acid residue is manipulated for site-directed conjugation.

27. The method according to claim 22, wherein step (a) further comprises bringing the target protein and the crosslinking agent into contact with a linker, wherein the crosslinking agent can crosslink the target protein to the linker.

28. The method according to claim 27, wherein the linker is bound to a payload, and optionally the payload is a cytotoxic payload or a therapeutic payload.

29. The method according to claim 22, wherein the at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof.

30. The method according to claim 22, wherein the at least one digestive enzyme is trypsin.

31. The method according to claim 22, wherein the at least one digestive enzyme is IdeS or a variant thereof.

32. The method according to claim 22, wherein the liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography.

33. The method according to claim 22, wherein the LC-MS analysis is RPLC-MS / MS analysis.

34. The method according to claim 22, wherein the crosslinking site contains lysine, and optionally the lysine is C-terminal lysine.

35. The method according to claim 22, wherein characterizing the crosslinking site includes identifying an amino acid residue that crosslinks to the target amino acid residue.

36. A method for identifying at least one reactive lysine in a target protein, (a) A step of producing a target crosslinked protein by contacting a sample containing the target protein with a crosslinking agent, wherein the crosslinking agent can crosslink at least one amino acid residue in the target protein to at least one reactive lysine in the target protein; (b) A step of bringing the target cross-linked protein into contact with at least one digestive enzyme to produce a peptide digest, (c) A step of subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to identify a peptide containing a crosslink between at least one amino acid residue and at least one reactive lysine residue, (d) A method comprising the step of identifying the at least one reactive lysine in the protein of interest using the identified peptide.

37. The method according to claim 36, wherein the target protein is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein.

38. The method according to claim 36, wherein the crosslinking agent is an enzyme, and optionally the enzyme is microbial transglutaminase (mTG).

39. The method according to claim 36, wherein the reactive lysine is C-terminal lysine.

40. The method according to claim 36, wherein the at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof.

41. The method according to claim 36, wherein the at least one digestive enzyme is trypsin.

42. The method according to claim 36, wherein the at least one digestive enzyme is IdeS or a variant thereof.

43. The method according to claim 36, wherein the liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography.

44. The method according to claim 36, wherein the LC-MS analysis is RPLC-MS / MS analysis.

45. A method for determining the contribution of site-directed crosslinking to high molecular weight species of a target protein, (a) A step of subjecting the target protein to conditions suitable for promoting site-specific crosslinking to generate the target crosslinked protein, (b) A step of subjecting the target cross-linked protein to size exclusion chromatography (SEC) analysis to quantify the proportion of high molecular weight (HMW) species, (c) Using the quantification described above, a step of determining the predicted proportion of site-specific crosslinked peptides that can contribute to the HMW species using formula 1, (d) A step of subjecting the cross-linked protein of step (a) to peptide mapping analysis to quantify the proportion of site-specific cross-linked peptides, A method comprising: (e) a step of determining the contribution of site-specific crosslinking of the target protein to the HMW species by comparing the quantified proportion of site-specific crosslinked peptides in step (d) with the predicted proportion of site-specific crosslinked peptides in step (c).

46. The method according to claim 45, wherein the target protein is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein.

47. The method according to claim 45, wherein the site-specific crosslinking includes crosslinking of the manipulated amino acid residue.

48. The method according to claim 45, wherein providing the target protein under conditions suitable for promoting site-specific crosslinking includes contacting the target protein with a crosslinking agent.

49. The method according to claim 47, wherein the crosslinking agent is an enzyme, and optionally the enzyme is microbial transglutaminase (mTG).

50. The method according to claim 45, wherein the peptide mapping analysis comprises contacting the target cross-linked protein with at least one digestive enzyme to produce a peptide digest, and then subjecting the peptide digest to RPLC-MS / MS analysis.

51. The method according to claim 50, wherein the at least one digestive enzyme is trypsin.

52. A method for determining the contribution of C-terminal lysine to the formation of high molecular weight (HMW) species in a target antibody-drug conjugate, (a) A step of producing a clipped antibody by contacting an antibody corresponding to a target antibody-drug conjugate with a carboxypeptidase, wherein the antibody contains C-terminal lysine and the clipped antibody does not contain C-terminal lysine. (b) A step of producing a crosslinked antibody and a crosslinked clipped antibody by contacting the antibody and the clipped antibody with a crosslinking agent, wherein the crosslinking agent is capable of crosslinking at least one amino acid residue of the antibody and the clipped antibody to lysine. (c) A step of subjecting the crosslinked antibody and the crosslinked clipped antibody to size exclusion chromatography (SEC) analysis to quantify the HMW species of the crosslinked antibody and the crosslinked clipped antibody, and (d) A method comprising the step of determining the contribution of C-terminal lysine to the formation of HMW species in the target antibody-drug conjugate by comparing the quantification of HMW species of the crosslinked antibody with the quantification of HMW species of the crosslinked clipped antibody.

53. The method according to claim 52, wherein the carboxypeptidase is metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, or carboxypeptidase E.

54. The method according to claim 52, wherein the carboxypeptidase is carboxypeptidase B.

55. The method according to claim 52, wherein the crosslinking agent is an enzyme, and optionally, the crosslinking agent is microbial transglutaminase (mTG).

56. A method for selecting an antibody for an antibody-drug conjugate, (a) A step of obtaining a sample containing a first antibody, wherein the first antibody contains at least one target amino acid residue that can be crosslinked to at least one off-target amino acid residue by a crosslinking agent. (b) A step of bringing the first antibody into contact with the crosslinking agent to produce a crosslinked antibody, (c) A step of contacting the cross-linked antibody with at least one digestive enzyme to produce a peptide digest, (d) A step of subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) to quantify the peptide containing at least one target amino acid residue that is crosslinked to at least one off-target amino acid residue with respect to the first antibody. (e) Repeat steps (a) to (d) with at least one additional antibody to quantify a peptide containing at least one target amino acid residue that is crosslinked to at least one off-target amino acid residue with respect to at least one additional antibody. (f) A step of comparing the quantifications of steps (d) and (e), (g) A method comprising the step of selecting an antibody for an antibody-drug conjugate using the comparison described above.

57. The method according to claim 56, wherein the at least one target amino acid residue is lysine, cysteine, a non-natural amino acid, or glutamine.

58. The method according to claim 56, wherein the at least one target amino acid residue is manipulated for site-directed conjugation.

59. The method according to claim 56, wherein the at least one off-target amino acid residue is lysine, and optionally the lysine is C-terminal lysine.

60. The method according to claim 56, wherein the crosslinking agent is an enzyme, and optionally, the crosslinking agent is microbial transglutaminase (mTG).

61. The method according to claim 56, further comprising step (b) bringing the first antibody and the crosslinking agent into contact with a linker, wherein the crosslinking agent can crosslink the first antibody to the linker.

62. The method according to claim 61, wherein the linker is bound to a payload, and optionally the payload is a cytotoxic payload or a therapeutic payload.

63. The method according to claim 56, wherein the at least one digestive enzyme is selected from the group consisting of proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulinase (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, its variants, its biologically active fragments, its homologs, or combinations thereof.

64. The method according to claim 56, wherein the at least one digestive enzyme is trypsin.

65. The method according to claim 56, wherein the at least one digestive enzyme is IdeS or a variant thereof.

66. The method according to claim 56, wherein the liquid chromatography is selected from the group consisting of reversed-phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography.

67. The method according to claim 56, wherein the LC-MS analysis is RPLC-MS / MS analysis.

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