Characterization of crosslinking sites in antibody-drug conjugates

Description

1 / 61 CHARACTERIZATION OF CROSSLINKING SITES IN ANTIBODY-DRUG CONJUGATES CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of the Provisional Application of U.S. No. 63 / 457,829, filed April 7, 2023, which is incorporated herein in its entirety by reference. BACKGROUND

[002] Antibody-drug conjugates (ADCs) are an important class of biotherapeutics used in cancer treatment and can also be used for therapeutic applications beyond oncology. Antibodies are covalently conjugated to payloads via non-specific or site-specific conjugations. Conjugations via lysine (K) or reduced cysteine ​​residues are the most common non-specific conjugation methods, which can result in a highly heterogeneous mixture of ADCs with different drug-to-antibody ratios (DARs) and payload conjugation sites. Given the complex nature of the product, as well as the problems associated with batch-to-batch variability, non-specific conjugation often leads to uncertainties in pharmacokinetics, toxicology, and efficacy. On the other hand, while generally requiring more sophisticated protein engineering, site-specific conjugation usually provides superior control of the DAR and conjugation site.Although conjugation via engineered cysteine ​​or non-natural amino acid residues are two main classes of site-specific conjugation, conjugation via enzymatic linkages is an emerging tool used to ensure control of DAR and conjugation sites.

[003] Microbial transglutaminase (mTG) is an enzyme that catalyzes the formation of stable isopeptide linkages between glutamine (Q) side chains (i.e., γ-carboxamide groups) and pri-amines. Petition 870250103116, dated 11 / 11 / 2025, p. 7 / 92 2 / 61 marias. Although most published studies focus on mAb engineering to achieve greater site specificity and selectivity, it has not been extensively studied how side reactions that can occur during mTG-mediated conjugation affect the final quality of the ADC product. The target glutamine residue in an mAb incubated with mTG and its ligand payload (LP) can undergo one of three potential reactions: (1) it can conjugate with the ligand payload, (2) it can undergo deamidation by reacting with water, or (3) it can become crosslinked to a lysine (K) residue. The latter two reactions are undesirable side reactions that will reduce the number of glutamine residues available for conjugation and lead to a lower overall DAR.

[004] Crosslinking with an off-target amino acid residue also increases the level of high molecular weight (HMW) species in ADC products. HMW species are a critical quality attribute of therapeutic proteins due to their potential impact on drug efficacy and safety. With sizes ranging from soluble oligomers to visible particles, HMW species can potentially trigger unwanted immunogenic responses, compromising the safety and efficacy of a drug.

[005] Therefore, there is a demand for methods to characterize high molecular weight species and crosslinking byproducts in ADCs, to produce ADCs with reduced HMW species, and to select antibodies for ADCs that are less susceptible to off-target crosslinking and HMW formation. SUMMARY

[006] A method has been developed to produce ADCs with reduced HMW species. In an exemplary embodiment, at least one off-target amino acid residue that forms cross-links in the ADC can be identified by placing the corresponding antibody in Petition 870250103116, dated 11 / 11 / 2025, p. 8 / 92 3 / 61 contact with a ligand and a crosslinking agent, digesting the crosslinked sample to form a peptide digest and using liquid chromatography-mass spectrometry (LC-MS) analysis. The antibody can then be contacted with a protease to remove the off-target amino acid residue. In one aspect, the protease can be a carboxypeptidase, for example, carboxypeptidase B, and the off-target amino acid residue can be a C-terminal lysine. The clipped antibody can then be contacted with a crosslinking agent and a binding payload to produce an antibody-drug conjugate with reduced HMW species. In one aspect, the crosslinking agent can be microbial transglutaminase.

[007] A method has also been developed to determine a contribution of site-specific crosslinking to HMW species of a protein of interest, for example, an ADC. In an exemplary embodiment, the protein of interest may be subjected to suitable conditions to promote site-specific crosslinking to produce a crosslinked protein of interest. In one aspect, the suitable conditions to promote site-specific crosslinking may include contact of the protein of interest with a crosslinking agent, for example, microbial transglutaminase. The crosslinked protein of interest may be subjected to analysis to quantify a percentage of HMW species, for example, SEC analysis. In parallel or sequentially, the crosslinked protein of interest may be subjected to analysis to quantify a percentage of site-specific crosslinked peptides, for example, peptide mapping analysis or subunit analysis.The percentage of site-specific crosslinked peptides can be compared to the percentage of HMW species to determine the contribution of site-specific crosslinking to HMW species. In one aspect, the comparison uses an equation of... Petition 870250103116, dated 11 / 11 / 2025, page 9 / 92 4 / 61 correlation that determines the percentage of site-specific crosslinked peptides that would be predicted if HMW species were entirely explained by site-specific crosslinking.

[008] A method has been further developed for selecting and / or manipulating an antibody for an antibody-drug conjugate. In an exemplary embodiment, a first antibody can be subjected to site-specific cross-linking to produce a cross-linked antibody. The cross-linked antibody can be subjected to analysis to quantify site-specific cross-linking, for example, peptide mapping analysis or subunit analysis. This quantification can be compared to the same quantification for at least one additional antibody, and the comparison can be used to select an antibody for an antibody-drug conjugate. In one aspect, an antibody can be selected based on forming less site-specific cross-linking. In another aspect, an antibody can be selected based on having fewer reactive amino acid residues outside the target, for example, reactive lysines.In addition, or alternatively, an antibody can be designed to remove reactive amino acid residues outside the target identified using this method, thereby reducing the number of site-specific cross-links formed by the designed antibody.

[009] This invention provides methods for producing an antibody-drug conjugate with reduced high molecular weight (HMW) species. In some exemplary embodiments, the methods may comprise: (a) contacting an antibody including a C-terminal lysine with a carboxypeptidase to produce a cut antibody, wherein the cut antibody does not include the C-terminal lysine; and (b) contacting the cut antibody with a crosslinking agent and a ligand payload to produce an antibody-drug conjugate with reduced HMW species. Petition 870250103116, dated 11 / 11 / 2025, page 10 / 92 5 / 61

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

[0011] In one aspect, the antibody-carboxypeptidase contact step is conducted for about 1 hour to about 3 hours at about 35°C to about 39°C.

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

[0013] In one aspect, the antibody includes a glutamine designed for site-specific conjugation. In a specific aspect, the crosslinking agent is capable of crosslinking glutamine and C-terminal lysine.

[0014] In one aspect, the crosslinking agent is an enzyme. In one specific aspect, the enzyme is microbial transglutaminase.

[0015] This invention provides additional methods for producing an antibody-drug conjugate with reduced high molecular weight (HMW) species. In some exemplary embodiments, the methods may comprise: (a) identifying at least one off-target amino acid residue that forms crosslinks in an antibody-drug conjugate, said identification comprising: (i) placing a sample including an antibody in contact with a linker and a crosslinking agent to produce a crosslinked sample, wherein said crosslinking agent is capable of crosslinking said antibody at a target amino acid residue to said linker, and wherein said crosslinking agent is capable of crosslinking said antibody at a target amino acid residue to said antibody at an off-target amino acid residue; (ii) placing said crosslinked sample in contact with at least one digestive enzyme to produce Petition 870250103116, dated 11 / 11 / 2025, page 11 / 92 6 / 61 a peptide digest; (iii) subject said peptide digest to liquid chromatography-mass spectrometry (LC-MS) analysis to identify crosslinked peptides; (iv) use said identification to identify at least one non-target amino acid residue that forms crosslinks; (b) bring the antibody into contact with at least one protease to produce a cut antibody, wherein the cut antibody does not include at least one identified non-target amino acid residue; and (c) bring the cut antibody into contact with a linker and the crosslinking agent to produce an antibody-drug conjugate with reduced high molecular weight species.

[0016] In one aspect, at least one non-target amino acid residue is a lysine, optionally wherein said lysine is a C-terminal lysine. In another aspect, the target amino acid residue is a lysine, a cysteine, a non-natural amino acid, or a glutamine. In a further aspect, the target amino acid residue is designed for site-specific conjugation.

[0017] In one aspect, the ligand is attached to a payload, optionally wherein said payload is a cytotoxic payload or a therapeutic payload.

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

[0019] In one aspect, at least one digestive enzyme is selected from the group consisting of Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (LysC), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin-degrading enzyme Petition 870250103116, dated 11 / 11 / 2025, p. 12 / 92 7 / 61 (IdeS), thermolysin, papain, pronase, protease V8, variants thereof, biologically active fragments thereof, homologs thereof, or combinations thereof. In one specific aspect, at least one digestive enzyme is trypsin. In another specific aspect, at least one digestive enzyme is IdeS or a variant thereof.

[0020] In one aspect, liquid chromatography is selected from a 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 aspect, LC-MS analysis is an RPLC-MS / MS analysis.

[0021] In one aspect, at least one protease is a carboxypeptidase. In one specific aspect, the carboxypeptidase is carboxypeptidase B.

[0022] In one aspect, the antibody contact step with at least one protease is conducted for about 1 hour to about 3 hours at about 35°C to about 39°C.

[0023] In one aspect, the cut antibody is an antibody without a C-terminal lysine.

[0024] This invention also provides methods for characterizing crosslinking sites in a protein of interest. In some exemplary embodiments, the methods may comprise: (a) placing a sample including a protein of interest in contact with a crosslinking agent to produce a crosslinked protein of interest, wherein said protein of interest includes at least one target amino acid residue that can be crosslinked by said crosslinking agent; (b) placing said crosslinked protein of interest in contact with at least one digestive enzyme to produce a peptide Petition 870250103116, dated 11 / 11 / 2025, p. 13 / 92 8 / 61 digested; (c) subject said digested peptide to liquid chromatography-mass spectrometry (LC-MS) analysis to characterize peptides that include a crosslinking in said at least one target amino acid residue; and (d) use said characterized peptides to characterize crosslinking sites in said protein of interest.

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

[0026] In one aspect, the crosslinking agent is an enzyme. In a specific aspect, the crosslinking agent is microbial transglutaminase (mTG).

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

[0028] In one aspect, step (a) further comprises bringing the protein of interest and the crosslinking agent into contact with a ligand, wherein the crosslinking agent is able to crosslink the protein of interest with the ligand.

[0029] In one aspect, the ligand is attached to a payload, optionally wherein said 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 Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (LysC), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin-degrading enzyme Petition 870250103116, dated 11 / 11 / 2025, p. 14 / 92 9 / 61 (IdeS), thermolysin, papain, pronase, protease V8, variants thereof, biologically active fragments thereof, homologs thereof, or combinations thereof. In one specific aspect, 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 a 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 aspect, LC-MS analysis is an RPLC-MS / MS analysis.

[0032] In one aspect, the crosslinking sites include a lysine. In one specific aspect, lysine is a C-terminal lysine.

[0033] In one aspect, the characterization step of said crosslinking sites includes the identification of amino acid residues that crosslink to said target amino acid residue.

[0034] This invention further provides methods for identifying at least one reactive lysine in a protein of interest. In some exemplary embodiments, the methods may comprise: (a) placing a sample including a protein of interest in contact with a crosslinking agent to produce a crosslinked protein of interest, wherein the crosslinking agent is capable of crosslinking at least one amino acid residue in the protein of interest to at least one reactive lysine in the protein of interest; (b) placing the crosslinked protein of interest in contact with at least one digestive enzyme to produce a digested peptide; (c) subjecting the digested peptide to liquid chromatography-mass spectrometry (LC-MS) analysis to identify peptides that include a crosslink between at least one amino acid residue and at least one reactive lysine; and (d) using Petition 870250103116, dated 11 / 11 / 2025, page 15 / 92 10 / 61 of the peptides identified were used to identify at least one reactive lysine in the protein of interest.

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

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

[0037] In one respect, reactive lysine is a C-terminal lysine.

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

[0039] In one aspect, liquid chromatography is selected from a 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 aspect, LC-MS analysis is an RPLC-MS / MS analysis.

[0040] This invention also provides methods for determining a contribution of site-specific crosslinking to high species Petition 870250103116, dated 11 / 11 / 2025, page 16 / 92 11 / 61 molecular weight of a protein of interest. In some exemplary embodiments, the methods may comprise: (a) subjecting a protein of interest to suitable conditions to promote site-specific crosslinking to produce a crosslinked protein of interest; (b) subjecting said crosslinked protein of interest to size exclusion chromatography (SEC) analysis to quantify a percentage of high molecular weight (HMW) species; (c) using said quantification to determine a predicted percentage of site-specific crosslinked peptides that may contribute to said HMW species, using Equation 1; (d) subjecting said crosslinked protein of interest from step (a) to peptide mapping analysis to quantify a percentage of site-specific crosslinked peptides;(e) compare the said quantified percentage of site-specific crosslinked peptides from step (d) with the said predicted percentage of site-specific crosslinked peptides from step (c) to determine a contribution of site-specific crosslinking to HMW species of said protein of interest.

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

[0042] In one aspect, site-specific crosslinking comprises crosslinking of a designed amino acid residue.

[0043] In one aspect, subjecting a protein of interest to suitable conditions to promote site-specific cross-linking involves bringing said protein of interest into contact with a cross-linking agent. In a specific aspect, the cross-linking agent is an enzyme. In a more specific aspect, the enzyme is microbial transglutaminase (mTG).

[0044] In one aspect, peptide mapping analysis in Petition 870250103116, dated 11 / 11 / 2025, p. 17 / 92 12 / 61 includes placing the cross-linked protein of interest in contact with at least one digestive enzyme to produce a digested peptide and then subjecting the digested peptide to RPLC-MS / MS analysis. In one specific aspect, the at least one digestive enzyme is trypsin.

[0045] This invention also provides methods for determining the contribution of a C-terminal lysine to the formation of high molecular weight (HMW) species in an antibody-drug conjugate of interest. In some exemplary embodiments, the methods may comprise: (a) contacting an antibody corresponding to an antibody-drug conjugate of interest with a carboxypeptidase to produce a cut antibody, wherein the antibody includes a C-terminal lysine and the cut antibody does not include a C-terminal lysine; (b) contacting the antibody and the cut antibody with a crosslinking agent to produce a crosslinked antibody and a crosslinked cut antibody, wherein the crosslinking agent is capable of crosslinking at least one amino acid residue of the antibody and the cut antibody with a lysine;(c) subject the crosslinked antibody and the crosslinked cut antibody to size exclusion chromatography (SEC) analysis to quantify HMW species of the crosslinked antibody and the crosslinked cut antibody; and (d) compare said quantification of HMW species of said crosslinked antibody with said quantification of HMW species of said cut crosslink antibody to determine a contribution of a C-terminal lysine to the formation of HMW species in said antibody-drug conjugate of interest.

[0046] In one aspect, carboxypeptidase is a metallocarboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, or carboxypeptidase E. In another aspect, carboxypeptidase is carboxypeptidase B. Petition 870250103116, dated 11 / 11 / 2025, p. 18 / 92 13 / 61

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

[0048] This invention further provides methods for selecting an antibody for an antibody-drug conjugate. In some exemplary embodiments, the methods may comprise: (a) obtaining a sample including a first antibody, wherein the first antibody comprises at least one target amino acid residue that can be crosslinked by a crosslinking agent to at least one non-target amino acid residue; (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) analysis to quantify peptides comprising at least one target amino acid residue crosslinked to at least one non-target amino acid residue for a first antibody;(e) repeat steps (a) to (d) with at least one additional antibody to quantify peptides comprising at least one target amino acid residue crosslinked to at least one non-target amino acid residue for at least one additional antibody; (f) compare the quantifications from steps (d) and (e); and (g) use said comparison to select an antibody for an antibody-drug conjugate.

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

[0050] In one aspect, at least one non-target amino acid residue is a lysine. In one specific aspect, the lysine is a C-terminal lysine. Petition 870250103116, dated 11 / 11 / 2025, p. 19 / 92 14 / 61

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

[0052] In one aspect, step (b) further comprises placing the first antibody and the crosslinking agent in contact with a ligand, wherein the crosslinking agent is able to crosslink the first antibody with the ligand. In a specific aspect, the ligand is attached to a payload. In a more specific aspect, the payload is a cytotoxic payload or a therapeutic payload.

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

[0054] In one aspect, liquid chromatography is selected from a 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 aspect, LC-MS analysis is an RPLC-MS / MS analysis.

[0055] These and other aspects of the invention will be better appreciated and understood when considered in conjunction with the following Petition 870250103116, dated 11 / 11 / 2025, page 20 / 92 15 / 61 description and accompanying drawings. The following description, although indicating various embodiments and specific details thereof, is provided by way of illustration and not limitation. Many substitutions, modifications, additions or rearrangements may be made within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1A illustrates high molecular weight (HMW) species formed when a mAb is incubated with microbial transglutaminase (mTG), according to an exemplary embodiment.

[0057] FIG. 1B shows a correlation of HMW species identified by size exclusion chromatography (SEC) and crosslinked peptides identified by reduced peptide mapping (RPM), according to an exemplary embodiment.

[0058] FIG. 2A shows crosslinked sites on lysine (K) residues in mAb1, according to an exemplary embodiment.

[0059] FIG. 2B shows a percentage of native, deamidated and crosslinked mAb1 peptides after varying durations of incubation with mTG, according to an exemplary embodiment.

[0060] FIG. 2C shows crosslinked sites in mAb2, according to an exemplary embodiment.

[0061] FIG. 2D shows a percentage of native, deamidated and crosslinked mAb2 peptides after varying durations of incubation with mTG, according to an exemplary embodiment.

[0062] FIG. 3 illustrates the derivation of the correction factor allowing a percentage of HMW species to be correlated to a percentage of cross-linked peptides, according to an exemplary embodiment.

[0063] FIG. 4A shows a correlation between the percentage of cross-linked peptides measured by RPM and the percentage of peptides Petition 870250103116, dated 11 / 11 / 2025, page 21 / 92 16 / 61 reticulated patterns predicted based on the percentage of HMW species measured by SEC for mAb1 incubated with mTG, according to an exemplary embodiment.

[0064] FIG. 4B shows a correlation between the percentage of cross-linked peptides measured by RPM and the percentage of cross-linked peptides predicted based on the percentage of HMW species measured by SEC for mAb1 incubated with mTG and a ligand payload (LP), according to an exemplary embodiment.

[0065] FIG. 5 shows a percentage of HMW variants for mAb2 with and without intact C-terminal lysine (K) heavy chain residues, according to an exemplary embodiment.

[0066] FIG. 6A shows the mAb2 subunit analysis with the C-terminal lysine of the heavy chain, according to an exemplary embodiment.

[0067] FIG. 6B shows the analysis of the mAb2 subunit with the C-terminal lysine of the heavy chain after incubation with mTG, according to an exemplary embodiment.

[0068] FIG. 6C shows the analysis of mAb2 subunits without the C-terminal lysine of the heavy chain, according to an exemplary embodiment.

[0069] FIG. 6D shows the analysis of mAb2 subunits without the C-terminal lysine of the heavy chain after incubation with mTG, according to an exemplary embodiment.

[0070] FIG. 7A shows a relative abundance of cross-linked peptides by mapping reduced peptide to mAb2 with the C-terminal lysine in the heavy chain, according to an exemplary embodiment.

[0071] FIG. 7B shows a relative abundance of cross-linked peptides by mapping reduced peptide to mAb2 without lysine. Petition 870250103116, dated 11 / 11 / 2025, page 22 / 92 17 / 61 C-terminal in the heavy chain, according to an exemplary embodiment. DETAILED DESCRIPTION

[0072] Antibody-drug conjugates (ADCs) are an important class of biotherapeutics used in cancer treatment. These drugs are designed to exploit the specificity of monoclonal antibodies (mAbs) to deliver cytotoxic payloads directly to cancer cells expressing antigens (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 oncology and now includes monoclonal antibodies that are 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).

[0073] Regardless of the nature of the payload to be delivered, it is fundamental that all ADCs maintain stable covalent bonds 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: non-specific and site-specific conjugations. Petition 870250103116, dated 11 / 11 / 2025, p. 23 / 92 18 / 61 (McCombs, JR, and Owen, SC (2015) Antibody drug conjugates: design and selection of binding chemistry, payload and conjugation, AAPS J 17, 339-351). Conjugations 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 ​​residues (Sun, MMC, et al. (2005) Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides, Bioconjugate Chemistry 16, 1282-1290) are the most common non-specific conjugation methods, which will result in a highly heterogeneous mixture of ADCs with different 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 product, as well as the problems associated with batch-to-batch variability, non-specific conjugation often leads to uncertainties in pharmacokinetics, toxicology, 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).

[0074] In contrast, although it generally requires more sophisticated protein engineering, site-specific conjugation usually provides superior control of the DAR and the conjugation site (Behrens, C. Petition 870250103116, dated 11 / 11 / 2025, p. 24 / 92 19 / 61 R., and Liu, B. (2014) Methods for site-specific drug conjugation to antibodies, mAbs 6, 46-53). While conjugation via engineered 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) Specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index, Nature Biotechnology 26, 925-932) or non-natural amino acid residues (Young, TS, and Schultz, PG (2010) Beyond the 20 canonical 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 non-natural amino acids, PNAS 109, 16101-16106) are two main classes of site-specific conjugation, conjugation via enzymatic linkages is an emerging 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, MP, et al. (2012) Engineering an anti-epidermal growth factor receptor antibody for single-chain format and labeling by Sortase A-mediated protein binding, Biotechnology and bioengineering 109, 1461-1470).

[0075] Microbial transglutaminase (mTG) is an enzyme that catalyzes 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 mTG, it recognizes glutamine residues in consensus sequences and primary amines that are in a ligand or in flexible regions of Petition 870250103116, dated 11 / 11 / 2025, p. 25 / 92 20 / 61 a protein. Thus, most mTG-mediated ADCs are generated by the conjugation of specific glutamine residues in mAbs with ligands containing primary amine (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 IgGs 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 an N295Q mutation (Lhospice, F., et al. (2015) Site-Specific Conjugation of Monomethyl Auristatin E Antibodies to AntiCD30 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.(2019) Site-Specific Efficient Antibody-Drug Conjugation by Engineering a Derivative Nature 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, glutamine side chains can also react with water and undergo deamidation if a primary amine is not available at the reaction site (Anami and Tsuchikama 2020).

[0076] Although most published studies focus on mAb engineering to achieve greater site specificity and selectivity, it has not been extensively studied how side reactions that may occur during mTG-mediated conjugation affect the final quality of the ADC product (Farias, SE, et al. (2014) Mass spectrometric characterization of transglutaminase-specific antibody-drug conjugates, Bioconjugate Chemistry 25, 240-250). The target glutamine residue in an mAb incubated with mTG and its payload of the li Petition 870250103116, dated 11 / 11 / 2025, p. 26 / 92 21 / 61 Glutamine (LP) can undergo one of three potential reactions: (1) it can conjugate with the payload of the ligand, (2) it can undergo deamidation by reacting with water, or (3) it can become crosslinked to a lysine (K) residue (Martins, IM, et al. (2014) Transglutaminases: recent achievements and new sources, Applied Microbiology and Biotechnology 98, 69576964). The latter two reactions are undesirable side reactions that will reduce the number of glutamine residues available for conjugation and lead to a lower overall DAR. Furthermore, the formation of intermolecular glutamine-lysine (QK) cross-links increases the level of high molecular weight (HMW) variants in ADC products, which has potential implications regarding drug potency and safety (Rosenberg, AS (2006) Effects of protein aggregates: an immunological perspective, The AAPS Journal 8, E501-E507).Given that mTG-mediated QK crosslinking can have a direct impact on product quality, this reaction should be closely monitored during the production 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.

[0077] In the present invention, the formation of HMW variants in mTG-mediated ADCs were investigated using size exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS). It was found that mTG-mediated QK crosslinking formation has a direct impact on the level of high molecular weight (HMW) variants in the final ADC product. A model system, in which two glutamine-containing mAbs designed for site-specific conjugation were incubated with mTG in the absence of ligand payload, was used to study the relationship between HMW variant formation and QK crosslinking. SEC was used to determine the level of HMW variants, and reduced peptide mapping (RPM) was used. Petition 870250103116, dated 11 / 11 / 2025, page 27 / 92 22 / 61 was used to identify crosslinked sites and quantify crosslinked peptides. By establishing a correlation between these two assays, QK crosslinking between the target glutamine and specific lysine residues was identified as the main contributor to HMW size variants in the studied ADCs. More importantly, as the K-C-terminal of the heavy chain (HC) was identified as a crosslinking site in both mAbs, it was demonstrated that the level of HMW variants was significantly reduced in ADCs where the K-C-terminal of the HC was completely removed before mTG-mediated conjugation. These results indicate that QK crosslinking and other side reactions occur during mTG-mediated conjugation and will need to be monitored and controlled to ensure the high quality and consistency of the final ADC product.

[0078] Unless otherwise described, all technical and scientific terms used herein have the same meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in practice or testing, specific methods and materials are now described.

[0079] The term “a / an” should be understood as “at least one” and the terms “about” and “approximately” should be understood as allowing standard variation, as would be understood by those skilled in the art, and where intervals are given, the endpoints are included. As used herein, the terms “include”, “includes” and “including” should not be limiting and should be understood as “comprise”, “comprises” and “comprising”, respectively.

[0080] As used herein, the term “protein” or “protein of interest” may include any amino acid polymer with covalently linked amide bonds. Proteins comprise one or more polymeric chains of amino acids, generally known in Petition 870250103116, dated 11 / 11 / 2025, p. 28 / 92 23 / 61 technique as “polypeptides”. “Polypeptide” refers to a polymer composed of amino acid residues, related natural structural variants, and non-natural synthetic analogs linked by peptide bonds. “Synthetic peptide or polypeptide” refers to a peptide or polypeptide that does not occur naturally. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Several methods of solid-phase peptide synthesis 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 aspect, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like.Proteins of interest may include any of the biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric Fc receptor fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins can be produced using recombinant cell-based production systems such as the insect baculovirus system, yeast systems (e.g., Pichia sp.), and mammalian systems (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” (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), whose complete teachings are incorporated herein). In Petition 870250103116, of 11 / 11 / 2025, p. 29 / 92. 24 / 61 In some exemplary forms, proteins comprise modifications, adducts, and other covalently linked fractions. These modifications, adducts, and fractions include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione-S-transferase (GST), myc epitope, fluorescent markers and other dyes and the like. Proteins can be classified based on composition and solubility and can therefore include simple proteins, such as globular proteins and fibrous proteins; Conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0081] In some exemplary embodiments, the protein of interest may be a recombinant protein, an in vivo gene therapy product, a therapeutic protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, an antigen-binding protein, a fusion protein, scFv, a multisubunit protein, an antibody-drug conjugate, a receptor, a receptor ligand, and combinations thereof.

[0082] As used herein, the term “recombinant protein” refers to a protein produced as a result of the transcription and translation of a gene carried in a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, for example, a chimeric, humanized, or fully human antibody.In certain exemplary embodiments, the recombinant protein may be an antibody of a selected isotype from the group. Petition 870250103116, dated 11 / 11 / 2025, p. 30 / 92 25 / 61 which consists of: IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., an IgG1) or, alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0083] The term “antibody,” as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as their multimers (e.g., IgM). Each heavy chain comprises a variable heavy chain region (abbreviated in this document as VH) and a constant heavy chain region. The constant heavy chain region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a variable light chain region (abbreviated herein as LCVR or VL) and a constant light chain region. The constant light chain region comprises one domain (CL1). The VH and VL regions can be subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs).Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A consensus amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of complete antibody molecules.

[0084] The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form an antibody. Petition 870250103116, dated 11 / 11 / 2025, page 31 / 92 26 / 61 complex. Antigen-binding fragments of an antibody can be derived, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and, optionally, constant antibody domains. This DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or it can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0085] As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a dAb fragment, an Fd' fragment, an Fd fragment, and an isolated complementarity-determining region (CDR), as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Fv fragments are the combination of the variable regions of the heavy and light chains of immunoglobulin, and ScFv proteins are recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains of immunoglobulin are connected by a peptide linker.In some exemplary embodiments, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody. Petition 870250103116, dated 11 / 11 / 2025, p. 32 / 92 27 / 61 such that is a fragment that binds to the same antigen as the parental antibody; in some exemplary embodiments, a fragment binds to the antigen with affinity comparable to that of the parental antibody and / or competes with the parental antibody for antigen binding. An antibody fragment may be produced by any means. For example, an antibody fragment may be produced enzymatically or chemically by fragmentation of an intact antibody and / or may be produced recombinantly from a gene encoding the partial antibody sequence. Alternatively, or additionally, an antibody fragment may be produced wholly or partially synthetically. An antibody fragment may optionally comprise a single-chain antibody fragment. Alternatively, or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide bonds.An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and, more typically, comprises at least about 200 amino acids.

[0086] The term “bispecific antibody” includes an antibody capable of selectively binding to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding to a different epitope – on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain. Petition 870250103116, dated 11 / 11 / 2025, page 33 / 92 28 / 61 heavy chain for the second epitope and vice versa. The epitopes recognized by the bispecific antibody may be on the same target or on a different target (e.g., on the same protein or on a different protein). Bispecific antibodies can be produced, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding variable heavy chain sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different constant regions of the heavy chain, and such sequences can be expressed on a cell expressing an immunoglobulin light chain.

[0087] A typical bispecific antibody has two heavy chains, each with 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 can associate with each heavy chain, or can associate with each heavy chain and bind to one or more of the epitopes linked by the antigen-binding regions of the heavy chain, or can associate with each heavy chain and allow binding of one or both heavy chains to one or both epitopes. BsAbs can be divided into two main classes: those that possess an Fc region (similar to IgG) and those that do not possess an Fc region, the latter being typically smaller than bispecific IgG molecules and IgG-like in comprising an Fc region.IgG-like bsAbs can have different formats, such as, but not limited to, triomab, IgG knobs into holes (kih IgG), crossMab, north-Fab IgG, dual variable domain Ig (DVD-Ig), dual-action or two-in-one Fab (DAF), single-chain IgG-Fv (IgG-scFv), or κλ bodies. Different non-IgG-like formats include tandem scFvs, diabody format, single-chain diabody, and diabodies. Petition 870250103116, dated 11 / 11 / 2025, page 34 / 92 29 / 61 in tandem (TandAbs), dual-affinity retargeting molecule (DART), DART-Fc, nanobodies or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific Antibodies and Their Applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGIA 130; Dafne Müller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), whose complete teachings are incorporated herein). The methods for producing bsAbs are not limited to quadroma technology based on somatic fusion of two different hybridoma cell lines, chemical conjugation involving chemical crosslinkers, and genetic approaches using recombinant DNA technology. Examples of bsAbs include those disclosed in the following patent applications, which are incorporated herein by reference: U.S. Ser. No. 12 / 823838, filed June 25, 2010; U.S. Ser. No. 13 / 488628, filed June 5, 2012; U.S. Ser.US Ser. No. 14 / 031075, filed September 19, 2013; US Ser. No. 14 / 808171, filed July 24, 2015; US Ser. No. 15 / 713574, filed September 22, 2017; US Ser. No. 15 / 713569, filed September 22, 2017; US Ser. No. 15 / 386453, filed December 21, 2016; US Ser. No. 15 / 386443, filed December 21, 2016; US Ser. No. 15 / 22343, filed July 29, 2016; and US Ser. No. 15814095, filed November 15, 2017.

[0088] As used herein, “multispecific antibody” refers to an antibody with binding specificities for at least two different antigens. While such molecules typically bind to only two antigens (i.e., bispecific antibodies, bsAbs), antibodies with additional specificities, such as trispecific antibody and trispecific KIH, can also be addressed by the system and method disclosed herein.

[0089] The term “monoclonal antibody”, as used here, does not Petition 870250103116, dated 11 / 11 / 2025, page 35 / 92 30 / 61 is limited to antibodies produced using hybridoma technology. A monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful for the present invention can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, phage display technologies, gene therapy, or a combination thereof.

[0090] As used herein, the term “peptide or protein conjugate” may refer to a peptide or protein linked to biologically active drug(s) by ligand(s), including “antibody-drug conjugate” or “ADC”. A peptide conjugate, a protein conjugate, or an antibody-drug conjugate may comprise multiple molecules of a biologically active drug (or payload) that can be covalently linked to conjugation sites, such as amino acid side chains of a peptide conjugate, a protein conjugate, or an antibody (Siler Panowski et al., Site-specific antibody drug assemblies for cancer therapy, 6 mAbs 34-45 (2013)). The development of ADCs is a strategy to improve drug efficacy, as antibodies can bind to specific sites on target cells, allowing for efficient delivery of biologically active drugs to target cells.An antibody used for an ADC may be able to bind with sufficient affinity for selective accumulation and long-lasting retention at a target site. Most ADCs may have Kd values ​​in the nanomolar range. The payload may have potency in the nanomolar / picomolar range and may be able to achieve attainable intracellular concentrations after distribution of the ADC into the target tissue. The ligand that forms the connection between the payload and the antibody may be able to be sufficiently stable in circulation to take advantage of the pharmacokinetic properties of the antibody fraction (e.g., Petition 870250103116, dated 11 / 11 / 2025, page 36 / 92 31 / 61 long half-life) and allow the payload to remain bound to the antibody as it distributes into tissues, but must allow for the efficient release of the biologically active drug as soon as the ADC can be absorbed by the target cells. The ligand may include those that are not cleavable during cellular processing and those that are cleavable when the ADC reaches the target site. With non-cleavable ligands, the biologically active drug released within the cell includes the payload and all elements of the ligand still bound to an amino acid residue of the antibody, for example, a lysine, cysteine, or glutamine residue, after complete proteolytic degradation of the ADC within the lysosome. Cleavable ligands are those whose structure includes a cleavage site between the payload and the amino acid binding site on the antibody.Cleavage mechanisms can include hydrolysis of acid-labile bonds in acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by an intracellular protease or esterase, and reductive cleavage of disulfide bonds by the reducing environment within cells.

[0091] The overall distribution profile of ADCs contains a mixture of ADCs, unconjugated antibodies, and unconjugated drug payloads. The amount of drug that can be delivered to target cells would decrease in the presence of unconjugated antibodies, since unconjugated antibodies compete with drug-conjugated antibodies for target antigens. Commonly, derived ADCs are highly heterogeneous species containing various ADC species with varying drug-to-antibody ratios (DARs) and varied conjugation sites, including conjugated conjugation sites and non-conjugated conjugation sites. In the case of ADCs generated using enzymatic conjugation reactions, for example, mTG-mediated conjugation, the ADC species may include conjugated conjugation sites, crosslinked conjugation sites, and non-conjugated conjugation sites. Petition 870250103116, dated 11 / 11 / 2025, page 37 / 92 32 / 61 deamidated conjugation. The heterogeneity of ADCs can have significant impacts on the safety and efficacy of medicines due to the presence of undesirable ADC species. Desirable ADC formulations should include well-defined DARs and a degree of homogeneity. Quantification and characterization of site-specific drug conjugations of ADCs with varying DARs, such as site-specific drug conjugation quantification, are critical processes for controlling the quality attributes of ADC formulations, which can directly affect the efficacy of ADCs.

[0092] A conjugated peptide or protein, for example, an ADC, may be designed to have a specific conjugation site, for example, a designed cysteine, a designed non-natural amino acid residue, or a designed glutamine. A specific conjugation site may also be called a target site, target conjugation site, or target amino acid residue. A target amino acid residue may be, for example, an amino acid residue within a consensus sequence that is recognized by a relevant enzyme, such as a target glutamine residue of mTG. In contrast, an off-target amino acid residue may be an amino acid residue that forms undesirable cross-links with a target amino acid residue and does not contribute to conjugation with a ligand, for example, a lysine residue that is cross-linked to a target glutamine residue by mTG.An off-target amino acid residue, for example, a lysine residue, can be called a reactive amino acid residue, for example, a reactive lysine. Although a protein, such as an antibody, may include many amino acid residues theoretically capable of forming undesirable cross-links, for example, lysines, in practice, only one or a few of these amino acid residues may be reactive and participate in cross-linking reactions. The invention herein provides methods. Petition 870250103116, dated 11 / 11 / 2025, page 38 / 92 33 / 61 to design a conjugated peptide or protein and methods for selecting a conjugated peptide or protein, for example, to avoid the formation of HMW species by characterizing and reducing or eliminating potential crosslinking sites.

[0093] In some exemplary embodiments, the protein of interest can be produced from mammalian cells. Mammalian cells can be of human or non-human origin and may include 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 strains (e.g., HEK293 embryonic renal 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 VA13 cells, etc.). 2RA, WISH cells, BS-CI cells, LLC-MK2 cells, M-3 clone cells, 1-10 cells, RAG cells, TCMK-1 cells,Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells and TH-I cells, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells or derivatives thereof), fibroblast cells from any tissue or organ (including, but not limited to, heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow and blood), spleen and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemic cells, Dempsey cells, Detroit cells). 551, Detroit cells 510, cells, Petition 870250103116, dated 11 / 11 / 2025, page 39 / 92 34 / 61 Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR90 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 cells, 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, strain 2071 cells (mouse L), strain LM cells (mouse L), L-MTK' cells (mouse L), NCTC 2472 and 2555 clones, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells and Jensen cells, Sp2 / 0, NS0, NS1 cells or derivatives thereof).

[0094] In some exemplary embodiments, the sample including the protein of interest may be prepared before or after the enrichment steps, separation steps and / or analysis steps. Preparation steps may include alkylation, reduction, denaturation, digestion and / or deglycosylation.

[0095] As used herein, the term “protein alkylating agent” refers to an agent used to alkylate certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0096] As used herein, “protein denaturation” can refer to a process in which the three-dimensional shape of a molecule is altered from its native state. Protein denaturation can be accomplished using a protein denaturing agent. Non-limiting examples of a protein denaturing agent include heat, high or low pH, reducing agents such as DTT (see below), or exposure to chaotropic agents. Various chaotropic agents can be used. Petition 870250103116, dated 11 / 11 / 2025, page 40 / 92 35 / 61 as protein denaturing agents. Chaotropic solutes increase the entropy of the 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-lauroylsarcosine, urea, and salts thereof.

[0097] As used herein, the term “protein reducing agent” refers to the agent used to reduce disulfide bridges in a protein. Non-limiting examples of protein reducing agents used to reduce a protein are dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof. A conventional method of protein analysis, reduced peptide mapping, involves reducing proteins prior to LC-MS analysis. In contrast, non-reduced peptide mapping omits the reduction sample preparation step to preserve endogenous disulfide bonds. In some exemplary embodiments, a non-reduced preparation may be used, for example, to preserve an endogenous disulfide bond between the Fab arms of an antibody or antibody-derived protein.In other exemplary embodiments, a partially reduced preparation may be used, for example, to reduce the disulfide bond between the Fab arms of an antibody or antibody-derived protein without fully reducing the protein.

[0098] As used herein, the term “digestion” refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to performing protein digestion in a sample. Petition 870250103116, dated 11 / 11 / 2025, page 41 / 92 36 / 61 using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion.

[0099] As used herein, the term “digestive enzyme” refers to any of a large number of different agents that can perform the digestion of a protein. Non-limiting examples of hydrolyzing agents that can perform enzymatic digestion include Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (LysN), LysC endoproteinase (Lys-C), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, V8 protease, or biologically active fragments or homologs thereof, or combinations thereof. For a recent review discussing the techniques available for protein digestion, see Switazar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried MA Niessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013)).

[00100] In some exemplary embodiments, IdeS or a variant thereof is used to cleave an antibody below the hinge region, producing an Fc fragment and a Fab2 fragment. Digestion of an analyte can be advantageous because size reduction can increase the sensitivity and specificity of analyte characterization and detection by LC-MS. When used for this purpose, digestion that separates an Fc fragment and retains a Fab2 fragment for analysis may be preferable. This is because variable regions of interest, such as the complementarity-determining region (CDR) of an antibody, are contained within the Fab2 fragment, while the Fc fragment may Petition 870250103116, dated 11 / 11 / 2025, page 42 / 92 37 / 61 being relatively uniform among antibodies and therefore providing less relevant information. Alternatively, or additionally, digestion that separates a Fab2e fragment and retains an Fc fragment for analysis may be preferable because the Fc fragment contains an N-glycosylation site of interest. The analysis of digested antibody fragments (subunits) of an antibody, for example, digestion with IdeS followed by chromatographic analysis and / or mass spectrometry, is called subunit analysis.

[00101] IdeS digestion has high efficiency, allowing high recovery of an analyte. The digestion and elution process can be performed under native conditions, allowing simple coupling to a native LC-MS system. IdeS or variants thereof are commercially available and can be marketed as, for example, FabRICATOR® or Fabricator Z®.

[00102] In some exemplary embodiments, a carboxypeptidase is used to remove a carboxy-terminal (C-terminal) residue from a protein of interest. A carboxypeptidase is a protease that hydrolyzes a peptide bond at the C-terminus of a protein or peptide. Carboxypeptidases include, for example, metallocarboxypeptidases, serine carboxypeptidases, cysteine ​​carboxypeptidases, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D, and carboxypeptidase E. A carboxypeptidase may be useful for removing a C-terminal lysine from a protein of interest. In other embodiments, other proteases may be used to remove residues from a protein or peptide that contribute to unwanted cross-linking, for example, aminopeptidases. A protein, for example an antibody, subjected to proteolysis by a protease, for example a carboxypeptidase, can be called a cleaved protein, for example, a cleaved antibody.A cut protein can have substantially the same or better structure, function, safety, and efficacy. Petition 870250103116, dated 11 / 11 / 2025, page 43 / 92 38 / 61 that the original protein.

[00103] As used herein, a “sample” may be obtained from any stage of the bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any stage in downstream processing, drug substance (DS), or a drug product (DP) comprising the final formulated product. In some other specific exemplary embodiments, the sample may be selected from any stage of the downstream process of clarification, chromatographic production, viral inactivation, or filtration. In some specific exemplary embodiments, the drug may be selected from a drug manufactured in the clinic, during transport, storage, or handling.

[00104] In some exemplary embodiments, the sample is a biological sample. As used herein, the term “biological sample” refers to a sample taken from a living organism, for example, a human or non-human mammal. A biological sample may comprise or consist of, for example, whole blood, plasma, serum, saliva, tears, semen, cheek tissue, organ tissue, urine, feces, skin, or hair. A sample may be taken from a patient, for example, a clinical sample. In some exemplary embodiments, a sample may be taken from a non-human animal, for example, a preclinical sample. In some exemplary embodiments, a sample may be taken from a non-human animal undergoing gene therapy to produce at least one protein of interest that may be included in the sample. In some embodiments, a sample is a further processed form of any of the sample examples mentioned above.

[00105] As used herein, the term “impurity” may include any undesirable protein present in a protein sample or pro Petition 870250103116, dated 11 / 11 / 2025, page 44 / 92 39 / 61 protein biopharmaceutical pipeline. Impurities may include process-related and product-related impurities. The impurity may also have a known structure, be partially characterized, or be unidentified. Process-related impurities may be derived from the manufacturing process and may include three main categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism and nucleic acid (genomic, vector, or total host cell DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other 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, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

[00106] Product-related impurities (e.g., precursors, certain degradation products) may be molecular variants that arise during manufacturing and / or storage and that do not have properties comparable to those of the desired product in terms of activity, efficacy, and safety. These variants may require considerable isolation and characterization effort to identify the type(s) of modification(s). Product-related impurities may include truncated forms, modified forms, and aggregates. Truncated forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modified forms include, but are not limited to, deamidated, isomerized, incompatible SS-linked, oxidized, or altered conjugated forms (by Petition 870250103116, dated 11 / 11 / 2025, page 45 / 92 40 / 61 example, glycosylation, phosphorylation). Modified forms may also include any form of post-translational modification. Aggregates include dimers and larger multiples of the desired product. (Specifications Q6B: Test Procedures and Acceptance 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 a protein, for example, an antibody in an antibody-drug conjugate.

[00107] 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 components as a result of the differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography (RP), ion-exchange chromatography (IEX), size-exclusion chromatography (SEC), affinity chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), or mixed-mode chromatography (MMC).

[00108] In some exemplary embodiments, the methods of the present invention include the use of size exclusion chromatography. Size exclusion chromatography or gel filtration relies on the separation of components based on their molecular size. The separation depends on the amount of time the substances spend in the porous stationary phase compared to the time in the fluid. The probability of a molecule residing in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a substance to permeate the pores is determined by the diffusion mobility of the macromolecules, which is greater for small macromolecules. Ma Petition 870250103116, dated 11 / 11 / 2025, page 46 / 92 41 / 61 Very large chromomolecules may not penetrate the pores of the stationary phase; and, for very small macromolecules, the probability of penetration is close to unity. Although larger molecular size components pass more quickly through the stationary phase, smaller molecular size components have a longer path through the pores of the stationary phase and are therefore retained longer in the stationary phase. Variants of a protein of interest that have a molecular weight greater or less than the parent species, or the intended product, may be called “size variants”.

[00109] Analytes eluted from an SEC column can be separated into fractions based on elution time. For example, analytes that elute before the functional form of a protein of interest, for example, the monomeric form, can be broadly categorized as high molecular weight (HMW) species. An HMW fraction can be subdivided into, for example, a very high molecular weight (vHMW) fraction and a dimer fraction (representing the elution time of a dimer of the protein of interest). Analytes that elute later than the functional form of a protein of interest can be broadly categorized as low molecular weight (LMW) species and can be subdivided into an LMW fraction and a trailing fraction.

[00110] A type of HMW, such as a dimer, can be formed as a product of a crosslinking reaction. A crosslink is a link or a short sequence of links that connects one polypeptide chain to another. Crosslinking can occur naturally or synthetically. Examples of crosslinks include, for example, disulfide bonds, amine bonds, ester bonds, or imine bonds. A crosslinking reaction can increase or decrease the total molecular weight of the crosslinked molecules relative to the unmodified molecules. Petition 870250103116, dated 11 / 11 / 2025, page 47 / 92 42 / 61 depending on the crosslinking mechanism. In one exemplary embodiment, a size variant of a protein of interest, for example, an HMW species of an ADC, is a dimer formed by crosslinking. In some exemplary embodiments, a crosslink is formed between a glutamine residue and a lysine residue. In some specific embodiments, a crosslinking lysine is located at the C-terminus of a protein of interest. In some exemplary embodiments, crosslinking is formed by means of an enzymatic reaction, for example, by the action of microbial transglutaminase. In some exemplary embodiments, crosslinking can cause the formation of a dimer species, a trimer species, a tetramer species, or larger species of a protein of interest, for example, an ADC.

[00111] As used herein, the term “crosslinking agent” refers to a molecule capable of forming a crosslink between two or more other molecules, or between two or more sites on one or more molecules. In some exemplary embodiments, a crosslinking agent is an enzyme (“crosslinking enzyme”), for example, mTG.

[00112] The chromatographic material for SEC may comprise a size-exclusion material, wherein the size-exclusion material is a resin or membrane. The matrix used for size exclusion is preferably an inert gel medium which may be a compound of cross-linked polysaccharides, for example, cross-linked agarose and / or dextran in the form of spheres. The degree of cross-linking determines the size of the pores present in the swollen gel spheres. Molecules larger than a certain size do not enter the gel spheres and therefore move more rapidly through the chromatographic bed. Smaller molecules, such as detergent, protein, DNA and the like, which enter the gel spheres to varying extent depending on their size and shape, are slowed down in their passage through the bed. The molecules are Petition 870250103116, dated 11 / 11 / 2025, page 48 / 92 43 / 61 generally eluted in decreasing order of molecular size.

[00113] Porous chromatographic resins suitable for virus size exclusion chromatography can be made from dextrose, agarose, polyacrylamide, or silica, which have different physical characteristics. Combinations of polymers can also be used. The most commonly used are those with the trade name “SEPHADEX”, available from Amersham Biosciences. Other size exclusion supports of different construction materials are also suitable, for example, Toyopearl 55F (polymethacrylate, from Tosoh Bioscience, Montgomery, Pennsylvania) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, California).

[00114] In some exemplary embodiments, the mobile phase used to obtain said eluate from size exclusion chromatography may comprise a volatile salt. In some specific embodiments, the mobile phase may comprise ammonium acetate, ammonium bicarbonate or ammonium formate, or combinations thereof.

[00115] As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their precise masses. The term is intended to include any molecular detector with which a polypeptide or peptide can be characterized. A mass spectrometer may include three main parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas-phase ions. Atoms, molecules, or clusters of analytes may be transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application.

[00116] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes a Petition 870250103116, dated 11 / 11 / 2025, page 49 / 92 44 / 61 technique in which structural information about sample molecules is obtained using multiple stages of mass selection and separation. A prerequisite is that the sample molecules are transformed into a gas phase and ionized so that fragments are formed in a predictable and controllable manner after the first mass selection step. Multistage MS / MS, or MSn, can be performed by first selecting and isolating a precursor ion (MS2), fragmenting it, isolating a primary fragment ion (MS3), fragmenting it, isolating a secondary fragment (MS4), and so on, as long as significant information can be obtained or the fragment ion signal is detectable. Tandem MS has been successfully performed with a wide variety of analyzer combinations.The choice of analyzers to be combined for a given application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also by size, cost, and availability. The two main categories of tandem MS methods are tandem in space and tandem in time, but there are also hybrids in which tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m / z separation functions can be designed so that, in one section of the instrument, ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m / z separation data acquisition.In tandem-in-time, mass spectrometer ions produced in the ion source can be captured, isolated, fragmented, and m / z separated in the same physical device. Peptides identified by the mass spectrometer can then be used. Petition 870250103116, dated 11 / 11 / 2025, pp. 50 / 92 45 / 61 as surrogate representatives of the intact protein and its post-translational modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter generated from possible peptides in a protein sequence database. Characterization includes, but is not limited to, sequencing amino acids of protein fragments, determining protein sequencing, determining de novo protein sequencing, locating or identifying post-translational modifications, or comparability analysis, or combinations thereof.

[00117] As used herein, the term “mass analyzer” includes a device that can separate species, i.e., atoms, molecules, or clusters, according to their mass. Non-limiting examples of mass analyzers that may be employed are time-of-flight (TOF), electromagnetic sector, quadrupole (Q) mass filter, quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and also the accelerator mass spectrometry (AMS) technique.

[00118] In some exemplary aspects, the mass spectrometer can work in nanoelectrospray or nanospray. The term “nanoelectrospray” or “nanospray,” as used herein, refers to ionization by electrospray at a very low solvent flow rate, typically hundreds of nanoliters per minute of sample solution or less, often without the use of an external solvent supply. The electrospray infusion setup that forms a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small volumes of sample solution (analyte) during Petition 870250103116, dated 11 / 11 / 2025, page 51 / 92 46 / 61 a long period of time. A dynamic nanospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separations in mixtures prior to mass spectrometry analysis.

[00119] In some exemplary embodiments, the mass spectrometer can be coupled to a multi-reaction monitoring system by liquid chromatography. More generally, a mass spectrometer may be capable of performing analyses by selected reaction monitoring (SRM), including consecutive reaction monitoring (CRM) and parallel reaction monitoring (PRM).

[00120] As used herein, “multiple reaction monitoring” or “MRM” refers to a mass spectrometry-based technique that can precisely quantify small molecules, peptides, and proteins within complex matrices with high sensitivity, specificity, and a 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 with triple quadrupole mass spectrometers, in which a precursor ion corresponding to the selected small molecules / peptides is selected in the first quadrupole and a fragment ion of the precursor ion is selected for monitoring in the third quadrupole (Yong Seok Choi et al., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimer's disease biomarker candidates, 930 JOURNAL OF CHROMATOGRAPHY B 129-135 (2013)).

[00121] In some exemplary embodiments, LC-MS can 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 a Petition 870250103116, dated 11 / 11 / 2025, p. 52 / 92 47 / 61 analyte. Native mass spectrometry is an approach to studying intact biomolecular structure in the native or near-native state. The term "native" refers to the biological state of the analyte in solution before it undergoes ionization. Several parameters, such as pH and ionic strength, of the solution containing the biological analytes can be controlled to maintain the folded native state of the biological analytes in solution. Commonly, native mass spectrometry is based on electrospray ionization, in which biological analytes are sprayed from a non-denaturing solvent. Other terms, such as non-covalent mass spectrometry, native spray, electrospray ionization, non-denaturing, macromolecular, or supramolecular, may also describe native mass spectrometry.In illustrative modalities, native MS allows for better spatial resolution compared to non-native MS, improving the detection of biotransformation products of a therapeutic protein. For a detailed analysis on native MS, see the analysis: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[00122] As used herein, the term “database” refers to a compiled collection of protein sequences that may exist in a sample, for example, in the form of a file in FASTA format. Relevant protein sequences may be derived from cDNA sequences of a species being studied. Public databases that can be used to search for relevant protein sequences include databases hosted, for example, by Uniprot or Swiss-prot. The databases can be searched using what is referred to here as “bioinformatics tools”. Bioinformatics tools provide the ability to search. Petition 870250103116, dated 11 / 11 / 2025, page 53 / 92 48 / 61 sar uninterpreted MS / MS spectra in all possible sequences in the database(s) and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools are 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).

[00123] It is understood that the present invention is not limited to any of the 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 denaturing agents, protein reducing agents, digestive enzymes, hydrolyzing agents, samples, liquid chromatography systems, mobile phases, mass spectrometers, bioinformatics databases or tools, and any 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 agent(s), protein denaturing agent(s),Protein reducing agent(s), digestive enzyme(s), hydrolyzing agent(s), sample(s), liquid chromatography system(s), mobile phase(s), mass spectrometer(s), bioinformatics database(s) or tool(s) may be selected by any suitable means. Petition 870250103116, dated 11 / 11 / 2025, page 54 / 92 49 / 61

[00124] The present invention will be more fully understood by reference to the following Examples. However, they should not be construed as limiting the scope of the invention. EXAMPLES

[00125] Chemicals and reagents. All mAbs and ADCs were produced at Regeneron (Tarrytown, NY, USA). Acetonitrile (ACN; LC-MS grade), trifluoroacetic acid, formic acid, dithiothreitol (DTT), iodoacetamide (IAA), tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), 8 M guanidine-HCl solution, and Invitrogen UltraPure 1 M Tris-HCl buffer, pH 7.5 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[00126] Microbial transglutaminase (mTG) was purchased from MilliporeSigma (Burlington, MA, USA). Urea and carboxypeptidase B (CpB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA). C18 (ACQUITY Ultra-performance LC peptide BEH C18 1.7 μm, 2.1 mm x 150 mm) and phenyl columns (ACQUITY Ultra-performance LC BEH Phenyl 1.7 μm, 2.1 mm x 150 mm) were purchased from Waters (Milford, MA, USA). FabRICATOR was purchased from Genovis (Lund, Sweden). PNGase F was purchased from New England Biolabs (Ipswich, MA, USA). Deionized water was supplied by a Milli-Q integral water purification system installed with a MilliPak Express 20 filter (MilliporeSigma, Burlington, MA, USA).

[00127] Preparation of mAb and ADC samples. To evaluate crosslinking in mAb samples, mAb1 and mAb2 were incubated with 1 U / mg Ab of mTG in conjugation buffer at 37°C for 1, 3, or 6 hours. To generate ADCs, mAb1 and mAb2 were incubated with mTG and ligand payload (LP) in conjugation buffer at 37°C for 6 hours. Control samples were incubated in conjugation buffer at Petition 870250103116, dated 11 / 11 / 2025, page 55 / 92 50 / 61 The OK C-terminal of the HC 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; this was followed by incubation with mTG alone or with mTG and LP in conjugation buffer at 37°C for 6 hours. All resulting reaction mixtures were subjected to SEC analysis immediately afterwards or frozen at -80°C for peptide mapping by LC-MS or subunit analysis at a later date.

[00128] SEC Analysis. Five μg of mAb or ADC samples were loaded onto an SEC column which separated the reaction products by size. Peaks eluted from the column were detected by UV absorption at 280 nm. Empower software (Waters) was used to perform peak integration to determine peak area and calculate relative abundance (percent area).

[00129] LC-MS / MS peptide mapping analysis. mAb and ADC samples were denatured, then reduced and alkylated with TCEP and IAA, respectively. Reduced samples were digested with trypsin for 4 hours at 37°C, followed by quenching with TFA. Tryptic digestions (5 μg samples) were loaded onto a C18 column.

[00130] Peptides eluted from the 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 with a full MS scan from 300 m / za to 2000 m / z followed by five sequential MS / MS scans for the five ions. Full scans of Petition 870250103116, dated 11 / 11 / 2025, page 56 / 92 51 / 61 MS scans were collected with a resolution of 70,000 with an AGC target of 1E6. MS / MS scans were collected with a resolution of 17,500 with an AGC target of 1E5. The isolation width was 4 m / ze and the normalized collision energy (NCE) was set at 27.

[00131] LC-MS subunit analysis. mAb and ADC samples were digested with 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. Twenty μg samples of each preparation were injected onto a phenyl column (reversed-phase column).

[00132] Subunits eluted 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, MA, USA). Complete MS scans were collected from 500 m / za to 5000 m / z with the intact protein mode activated. The ion source, curtain, and CAS gases were set at 60, 40, and 10 psi, respectively. The source temperature was set at 450°C with a spray voltage of 5,500 V and a disaggregation potential of 150 V.

[00133] Data Analysis. Peptides were identified by searching mAb sequence databases using Byonic (version 4.6.1, Protein Metrics, San Carlos, CA). Common post-translational modifications (PTMs) of mAbs were included in the search parameters as variable modifications; cysteine ​​carbamidomethylation was included as a fixed modification. QK crosslinking associated with NH3 loss (-17.0265 Da), glutamine deamidation (+0.9840 Da), and LP conjugation were also included as variable modifications. Peptide quantification was performed using Skyline software (version 21.2, MacCoss Lab Software, Seattle, WA, Petition 870250103116, dated 11 / 11 / 2025, page 57 / 92 52 / 61 (USA). Subunit data analysis was conducted using Sciex OS software (version 2.0 AB Sciex Framingham, MA, USA). The Bio Tool Kit was used for protein deconvolution. Example 1. Characterization of high molecular weight species of antibody-drug conjugates.

[00134] To study the relationship between HMW variant formation and mTG-mediated reactions, two mAbs (mAb1 and mAb2), each containing target glutamines designed for site-specific conjugation, were selected as model systems. Although mTG-mediated conjugation of mAb1 typically results in a high level of HMW variants, conjugation of mAb2 produced a comparatively low level of HMW variants. As shown in FIG. 1A, both mAbs were incubated with mTG alone in the absence of LP to maximize the level of HMW variants to facilitate cross-linking 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 FIG. 1B.These high levels of HMW variants allow for reliable identification of cross-links by peptide mapping without the need for enrichment or specialized workflows. As shown in the right panel of FIG. 1B, the change in abundance of the native peptide and the most prominent of the cross-linked peptides can be easily observed in UV profiles generated by reduced peptide mapping. Finally, the increase in the percentage of HMW variants detected by SEC over time was compared with the percentage of cross-linked peptides quantified from peptide mapping studies to establish a correlation between the formation of HMW variants and mTG-mediated cross-linking. Petition 870250103116, dated 11 / 11 / 2025, pp. 58 / 92 53 / 61 Example 2. Identification of off-target mTG crosslinking

[00135] Reaction mixtures generated by incubation of mAb1 and mAb2 with mTG were analyzed by reduced peptide mapping to identify crosslinked peptides. The results of the database search and manual interpretation of the data confirmed that both mAbs underwent mTG-mediated QK crosslinking. Crosslinked sites were successfully identified in samples generated from mAb1 and mAb2.

[00136] As described in Example 1, analysis using SEC revealed that mTG incubation of mAb1 resulted in a significant increase in the level of HMW variants. As shown in FIG. 2A, most of the crosslinks detected in mAb1 were between HC K56 or K446 (the K C-terminal of HC) and the designed target glutamine. After incubation with mTG, the Q target of mAb1 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 that included the Q target was determined based on peak area and the precise masses of the corresponding peptides, as shown in Table 1. As shown in FIG. 2B, HC K56 was detected in a larger fraction of the crosslinked peptides than HC K446.The total percentage of cross-linked peptides detected in the Q target increased significantly with longer incubation times; this increase was mainly due to increased cross-linking between the Q target and HC K56 over time. In addition to cross-linking, deamidation of the Q target also increased over time. This was expected, since mTG catalyzes Q deamidation in the absence of primary amine-containing substrates, such as a primary amine-containing LP. Table 1. Relative abundance of each mAb1 peptide that included the target glutamine (Q), including native, deamidated, and cross-linked forms. Petition 870250103116, dated 11 / 11 / 2025, p. 59 / 92 54 / 61 ND: Not detected. Peptide Form Control 1 hour 3 hours 6 hours Native 99.8% 73.0% 57.5% 45.3% Deamidated 0.2% 17.4% 27.8% 35.6% Crosslinking: HC K56 to target Q ND 8.3% 13.6% 18.2% Crosslinking: HC K446 to target Q ND 1.3% 1.0% 0.9% Total crosslinked ND 9.6% 14.6% 19.0%

[00137] Based on these findings, the highly reactive HC K56 residue can be eliminated from the mAb1 structure, for example, by mutating the coding nucleic acid sequence. This small change could result in a significant reduction in the level of HMW variants generated and could lead to an improvement in the overall quality of the ADC product. However, a mutation introduced into HC K56 could have an undesirable impact on other physicochemical properties and would therefore need to undergo a thorough evaluation.

[00138] The amino acid sequence of mAb2 does not include HC K56 and generates a much smaller increase in the level of HMW variants during mTG-mediated conjugation. In contrast to what was observed for mAb1, the HC K448 of mAb2 (C-terminal K) was identified as the predominant crosslinking site, as shown in FIG. 2C. Other smaller crosslinking sites identified in the Fc and Fd regions of HC were detected in substantially lower abundance, as shown in Table 2. Unlike mAb1, the abundance of total crosslinks detected in mAb2 that included the Q target changed minimally over time, as shown in FIG. 2D. On the other hand, the increase in mTG-mediated deamidation of the Q target was much more significant in mAb2 compared to mAb1. These results suggest that, in the absence of a highly reactive lysine residue (e.g., HC K56 in mAb1), deamidation is more kinetically favorable than QK crosslinking. Table 2. Relative abundance of each mAb2 peptide that included the target glutamine (Q), including native, deamidated, and cross-linked forms. Petition 870250103116, dated 11 / 11 / 2025, pp. 60 / 92 55 / 61 ND: Not detected. Peptide Form Control 1 hour 3 hours 6 hours Native 100.0% 49.8% 31.1% 17.4% Deamidated 0.0% 44.8% 63.9% 77.7% Crosslinking: HC K448 to target Q ND 4.6% 4.2% 4.0% Crosslinking: HC K222 to target Q ND 0.6% 0.5% 0.5% Crosslinking: HC K289 to target Q ND 0.1% 0.1% 0.2% Crosslinking: HC K327 to target Q ND 0.0% 0.0% 0.0% Crosslinking: HC K339 to target Q ND 0.1% 0.1% 0.1% Crosslinking: HC K341 to target Q ND 0.0% 0.0% 0.0% Total cross-linked ND 5.5% 5.1% 4.9%

[00139] The absence of multiple reactive lysine residues in these mAbs was not unexpected. It has been previously reported that, without specific lysine substitution, only the K C-terminal of HC can serve as an efficient site for conjugation with glutamine-based 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, 5365; 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 can cross-link with the Q target.These results suggest that, although this phenomenon may not be commonly observed, certain native lysine residues in some mAbs can be effectively conjugated with glutamine-based ligands, and the method of the present invention can be used to identify these reactive lysines. Example 3. Correlation between the level of HMW variants and the extent of mTG-mediated crosslinking.

[00140] The relationship between the percentage of cross-linked peptides determined by peptide mapping (% cross-linking by RPM) and the percentage of HMW variants determined by SEC (%HMWSEC) Petition 870250103116, dated 11 / 11 / 2025, pp. 61 / 92 56 / 61 was evaluated to determine if mTG-mediated crosslinking was a primary contributor to the elevated level of HMW variants observed in mTG-mediated conjugation. A correction factor was introduced to convert the percentage of HMW variants determined by SEC, which was measured at the intact level, into a percentage of crosslinked peptides, so that the percentage of HMW variants determined by SEC could be correlated with the percentage of crosslinked peptides determined by RPM, which was measured at the peptide level. The model for converting %HMW SEC to % crosslinking is based on the observation that there was no increase in %HMW in the control sample (as shown in FIG. 1B) and therefore it can be assumed that all HMW species are caused by QK crosslinking. As shown in FIG. 3, one of the four, two of the six, and three of the eight Q-containing peptides were cross-linked into dimers, trimers, and tetramers, respectively.

[00141] Based on this observation, the %HMWdimer can be converted to % crosslinked peptide by dividing the previous value by a correction factor of four; similarly, the %HMWtrimer can be converted to % crosslinked peptide by dividing by three. Similar conversions can be performed for all HMW species; correction factors for each HMW species up to 10-mers are listed in Table 3. As a result, to convert the percentage of HMW variants determined by SEC into a percentage of crosslinked peptides, the peak of the dimer identified by SEC was adjusted using the correction factor of 4 mentioned earlier, while the peaks for all other HMW species were adjusted using the correction factor of 2.7, because the value of this correction factor changed minimally from tetramer to 10-mer states (see Equation 1). Table 3. Correction factors for calculating the percentage of cross-linked peptide from the percentage of HMW variants for different Petition 870250103116, dated 11 / 11 / 2025, pp. 62 / 92 57 / 61 forms of HMW. HMW Form Correction Factor Dimer 4 Trimer 3 Tetramer 2.7 Pentamer 2.5 Hexamer 2.4 10-mer 2.2 Equation 1: Calculation used to estimate the percentage of cross-linked peptides in the percentage of HMW species. All values ​​shown were determined by SEC. %HMWsec crosslinking « [%HMWDimer / 4] + [(%HMWTotal - %HMWDimer) / 2.7]

[00142] The percentage of crosslinked peptides measured by peptide mapping and the SEC-converted values ​​using Equation 1 are included in Table 4 and Table 5. The results shown in FIG. 4A demonstrate a strong correlation between the measured and predicted percentage of crosslinked peptides in mAb1 after incubation with mTG for 1, 3, and 6 hours. These results suggest that the increase in the level of HMW variants of mAb1 observed after mTG-mediated conjugation can be attributed primarily to the formation of QK crosslinks. Table 4. HMW species and cross-linked peptides for mAb1 incubated with mTG 1 hour 3 hours 6 hours SEC HMW Total 46.6% 69.3% 74.3% HMW Dimer 19.3% 20.0% 19.0% % peptide cross-linked by HMW sec 13.3% 21.6% 23.6% RPM % peptide cross-linked by RPM 9.6% 14.6% 19.0% Table 5. HMW species and cross-linked peptides for mAb1 incubated Petition 870250103116, dated 11 / 11 / 2025, pp. 63 / 92 58 / 61 with mTG and LP, by molar ratio of LP:mAb 5:1 10:1 15:1 20:1 SEC HMW Total 25.0% 17.4% 13.6% 11.3% HMW Dimer 17.2% 12.9% 10.3% 8.6% % peptide cross-linked by HMW sec 5.6% 3.3% 2.2% 1.5% RPM % peptide cross-linked by RPM 2.2% 1.3% 0.7% 0.5%

[00143] Further experiments were performed in which mAb1 was incubated with mTG and LP. All crosslinked peptides identified in incubations that included only mTG were also detected in incubations that included mTG and LP (producing ADCs), although in much lower abundances. There was also a good correlation between the percentage values ​​of crosslinked peptides for these ADC samples quantified using SEC and peptide mapping methods, as shown in FIG. 4B. These results also support the conclusion that HMW variants, whether in mAbs or ADCs, result primarily from mTG-mediated QK crosslinking. Example 4. Removal of the C-terminal lysine from the HC reduces the level of HMW variants detected in mTG-mediated ADCs.

[00144] Although a good correlation was observed between the level of HMW variants and the extent of mTG-mediated QK crosslinking in mAb1, an equally strong correlation was not identified in the case of mAb2. Although mAb1 has two reactive K residues, the K-terminal HC is the only important crosslinking site in mAb2. Results from a previous study revealed that removing the K-terminal HC can completely abolish 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, An Petition 870250103116, dated 11 / 11 / 2025, pp. 64 / 92 59 / 61 gewandte Chemie (International English Edition) 54, 13420-13424). However, the relationship between the K C-terminal of HC and the generation of cross-linked peptides and HMW variants has not been extensively investigated.

[00145] To determine how the K C-terminal of HC contributed to the formation of HMW species during mTG-mediated crosslinking, carboxypeptidase B (CpB) was used to remove the K C-terminal of HC from mAb2. The resulting changes in the percentage of HMW variants formed during mTG-mediated conjugation were compared. As shown in FIG. 5, after incubation with mTG alone, the percentage of HMW variants detected in mAb2 without the K C-terminal of HC decreased by 50% compared to mAb2 with intact K C-terminal HC residues. This decrease was 40% in response to incubation with mTG and LP. These results indicate that removing the K C-terminal from the HC can significantly reduce the level of HMW variants in the resulting ADCs and suggest that the K C-terminal of the HC is the main contributor to mTG-mediated crosslinking in mAb2.Although only 10% of mAb2 contains K C-terminal HC before treatment with CpB, and these residues are responsible for only an increase of about ~2% in the percentage of HMW variants detected, this may change from process to process. Therefore, K C-terminal HC represents a potential risk for the formation of HMW variants, and its removal should be considered in future drug development strategies.

[00146] The incubation samples described above were subjected to extended characterization, including subunit analysis and peptide mapping. Subunit analysis revealed that LC-Fc / 2 (+ HC C-terminal K) was the main crosslinked fragment in the untreated mAb, as shown in FIG. 6B. This observation was consistent with the peptide mapping results shown in FIG. 7, where the K C-terminal of HC was identified as the single main site. Petition 870250103116, dated 11 / 11 / 2025, pp. 65 / 92 60 / 61 which contributes to crosslinking. Comparatively low levels of LCFc / 2 (-HC C-terminal K) were observed in both CpB-treated and untreated samples, with signals of similar intensity, as shown in FIG. 6B and FIG. 6D. These results indicate that removal of the K C-terminal from the HC does not make any of the other lysine residues more susceptible to mTG-mediated crosslinking. Peptide mapping results also revealed low-level crosslinking at smaller sites in the HC. This result suggests that K-crosslinked sites may exist in the mAb structure and may explain why an increase in the percentage of HMW variants was observed even after complete removal of the K C-terminal from the HC.

[00147] This invention presents methods for determining the mechanisms underlying the formation of HMW variants in mTG-mediated ADCs using SEC and LC-MS techniques. Using the methods of the present invention, it was found that mTG-mediated QK crosslinking is the main source of an increased level of HMW variants in ADCs, thus affecting the quality of the final ADC products. 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 demonstrated that the level of HMW variants was substantially reduced in the CpB-treated mAb2-conjugated ADC, in which the K C-terminal of the HC was completely removed. This result directly demonstrates that the K C-terminal of the HC was the main contributor to crosslinking and the formation of HMW variants in mAb2.

[00148] Crosslinking and other side reactions that occur during mTG-mediated conjugation need to be carefully monitored and controlled to maintain high quality and consistency. Petition 870250103116, dated 11 / 11 / 2025, pp. 66 / 92 61 / 61 of the ADC products. The identification of reactive lysine residues, including HC K56 in mAb1 and the C-terminal K-HC in both mAbs, provides insights into how mAbs can be designed to minimize the formation of HMW variants in the resulting ADCs. For example, highly reactive sites such as HC K56 in mAb1 should be eliminated during the early stages of development by screening ADC candidates based on their level of HMW variants. Extended characterization using LC-MS techniques, such as peptide mapping, can be performed if the localization and mutation of these residues are needed. Removal of the C-terminal K-HC may be a preferred route to minimize the formation of HMW variants and avoid future complications in ongoing product development. Petition 870250103116, dated 11 / 11 / 2025, pp. 67 / 92

Claims

1 / 14 CLAIMS 1. Method for producing an antibody-drug conjugate with reduced high molecular weight (HMW) species, characterized in that it comprises: (a) placing an antibody including a C-terminal lysine in contact with a carboxypeptidase to produce a modified antibody, wherein said modified antibody does not include said C-terminal lysine; and (b) placing the modified antibody in contact with a crosslinking agent and a binding payload to produce an antibody-drug conjugate with reduced HMW species.

2. Method according to claim 1, characterized in that the carboxypeptidase is a metallo-carboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D or carboxypeptidase E.

3. Method according to claim 1, characterized in that the carboxypeptidase is carboxypeptidase B.

4. Method according to claim 1, characterized in that the contact of the antibody with the carboxypeptidase is conducted for about 1 hour to about 3 hours at about 35 °C to about 39 °C.

5. Method according to claim 1, characterized in that the payload is either a cytotoxic payload or a therapeutic payload.

6. Method according to claim 1, characterized in that the antibody includes a glutamine designed for site-specific conjugation, optionally wherein the crosslinking agent is capable of crosslinking the glutamine and C-terminal lysine. Petition 870250103116, dated 11 / 11 / 2025, p. 68 / 92 2 / 14 7. Method according to claim 1, characterized in that the crosslinking agent is an enzyme, optionally wherein the enzyme is microbial transglutaminase.

8. Method for producing an antibody-drug conjugate with reduced high molecular weight (HMW) species, characterized in that it comprises: (a) identifying at least one off-target amino acid residue that forms crosslinks in an antibody-drug conjugate, comprising identifying: (i) placing a sample including an antibody in contact with a ligand and a crosslinking agent to produce a crosslinked sample, wherein the crosslinking agent is capable of crosslinking the antibody at a target amino acid residue to the ligand, and wherein the crosslinking agent is capable of crosslinking the antibody at a target amino acid residue to the antibody at an off-target amino acid residue; (ii) placing the crosslinked sample in contact with at least one digestive enzyme to produce a peptide digest; (iii) subjecting said digested peptide to liquid chromatography-mass spectrometry (LC-MS) analysis to identify crosslinked peptides;(iv) use said identification to identify at least one non-target amino acid residue that forms cross-links; (b) contact said antibody with at least one protease to produce a modified antibody, wherein said modified antibody does not include at least one identified non-target amino acid residue; and (c) contact the modified antibody with a linker and crosslinking agent to produce an antibody-drug conjugate with reduced high molecular weight species. Petition 870250103116, 11 / 11 / 2025, p. 69 / 92 3 / 14; 9. A method according to claim 8, characterized in that at least one off-target amino acid residue is a lysine, optionally wherein the lysine is a C-terminal lysine.

10. Method according to claim 8, characterized in that the target amino acid residue is a lysine, a cysteine, a non-natural amino acid or a glutamine.

11. Method according to claim 8, characterized in that the target amino acid residue is designed for site-specific conjugation.

12. Method according to claim 8, characterized in that the ligand is attached to a payload, optionally wherein the payload is a cytotoxic payload or a therapeutic payload.

13. Method according to claim 8, characterized in that the crosslinking agent is an enzyme, optionally wherein the crosslinking agent is microbial transglutaminase (mTG).

14. Method according to claim 8, characterized in that at least one digestive enzyme is selected from the group consisting of Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, V8 protease, variants thereof, biologically active fragments thereof, homologs thereof or combinations thereof.

15. Method according to claim 8, characterized in that at least one digestive enzyme is trypsin. Petition 870250103116, dated 11 / 11 / 2025, pp. 70 / 92 4 / 14 16. Method according to claim 8, characterized in that at least one digestive enzyme is IdeS or a variant thereof.

17. Method according to claim 8, characterized in that the liquid chromatography is selected from a 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. Method according to claim 8, characterized in that the LC-MS analysis is an RPLC-MS / MS analysis.

19. Method according to claim 8, characterized in that at least one protease is a carboxypeptidase, optionally wherein the carboxypeptidase is carboxypeptidase B.

20. Method according to claim 8, characterized in that the contact of the antibody with at least one protease is conducted for about 1 hour to about 3 hours at about 35 °C to about 39 °C.

21. Method according to claim 8, characterized in that the cut antibody is an antibody without a C-terminal lysine.

22. Method for characterizing crosslinking sites in a protein of interest, characterized in that it comprises: (a) contacting a sample including a protein of interest with a crosslinking agent to produce a crosslinked protein of interest, wherein said protein of interest includes at least one target amino acid residue that can be crosslinked by said crosslinking agent; Petition 870250103116, 11 / 11 / 2025, p. 71 / 92 5 / 14 (b) contacting the crosslinked protein of interest with at least one digestive enzyme to produce a peptide digest; (c) subjecting said peptide digest to liquid chromatography-mass spectrometry (LC-MS) analysis for characterization of peptides that include a crosslink at said at least one target amino acid residue; and (d) using said characterized peptides for characterization of crosslinking sites in said protein of interest.

23. Method according to claim 22, characterized in that the protein of interest is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein or a recombinant protein.

24. Method according to claim 22, characterized in that the crosslinking agent is an enzyme, optionally wherein the enzyme is microbial transglutaminase (mTG).

25. Method according to claim 22, characterized in that the target amino acid residue is a lysine, a cysteine, a non-natural amino acid or a glutamine.

26. Method according to claim 22, characterized in that the target amino acid residue is designed for site-specific conjugation.

27. Method according to claim 22, characterized in that step (a) further comprises bringing the protein of interest and the crosslinking agent into contact with a linker, wherein the crosslinking agent is able to crosslink the protein of interest with the linker. Petition 870250103116, dated 11 / 11 / 2025, pp. 72 / 92 6 / 14 28. Method according to claim 27, characterized in that the ligand is attached to a payload, optionally wherein the payload is a cytotoxic payload or a therapeutic payload.

29. Method according to claim 22, characterized in that at least one digestive enzyme is selected from the group consisting of Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, V8 protease, variants thereof, biologically active fragments thereof, homologs thereof or combinations thereof.

30. Method according to claim 22, characterized in that at least one digestive enzyme is trypsin.

31. Method according to claim 22, characterized in that at least one digestive enzyme is IdeS or a variant thereof.

32. Method according to claim 22, characterized in that the liquid chromatography is selected from a 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. Method according to claim 22, characterized in that the LC-MS analysis is an RPLC-MS / MS analysis. Petition 870250103116, dated 11 / 11 / 2025, pp. 73 / 92 7 / 14 34. Method according to claim 22, characterized in that the crosslinking sites include a lysine, optionally wherein the lysine is a C-terminal lysine.

35. Method according to claim 22, characterized in that the characterization of the crosslinking sites includes the identification of amino acid residues that crosslink to the target amino acid residue.

36. Method for identifying at least one reactive lysine in a protein of interest, characterized in that it comprises: (a) bringing a sample including a protein of interest into contact with a crosslinking agent to produce a crosslinked protein of interest, wherein said crosslinking agent is capable of crosslinking at least one amino acid residue in said protein of interest to at least one reactive lysine in said protein of interest; (b) bringing the crosslinked protein of interest into contact with at least one digestive enzyme to produce a peptide digestion; (c) subjecting said peptide digestion to liquid chromatography-mass spectrometry (LC-MS) analysis to identify peptides that include a crosslink between said at least one amino acid residue and at least one reactive lysine; and (d) using said identified peptides to identify at least one reactive lysine in said protein of interest.

37. Method according to claim 36, characterized in that said protein of interest is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein or a recombinant protein. Petition 870250103116, dated 11 / 11 / 2025, p. 74 / 92 8 / 14 38. Method according to claim 36, characterized in that said crosslinking agent is an enzyme, optionally wherein said enzyme is microbial transglutaminase (mTG).

39. Method according to claim 36, characterized in that said reactive lysine is a C-terminal lysine.

40. Method according to claim 36, characterized in that said at least one digestive enzyme is selected from the group consisting of Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, protease V8, their variants, their biologically active fragments, their homologs, or combinations thereof.

41. Method according to claim 36, characterized in that at least one digestive enzyme is trypsin.

42. Method according to claim 36, characterized in that at least one digestive enzyme is IdeS or a variant thereof.

43. Method according to claim 36, characterized in that said liquid chromatography is selected from a 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. Petition 870250103116, dated 11 / 11 / 2025, pp. 75 / 92 9 / 14 44. Method according to claim 36, characterized in that said LC-MS analysis is RPLCMS / MS analysis.

45. Method for determining a site-specific crosslinking contribution to high molecular weight species of a protein of interest, characterized in that it comprises: (a) subjecting a protein of interest to suitable conditions to promote site-specific crosslinking to produce a crosslinked protein of interest; (b) subjecting said crosslinked protein of interest to size exclusion chromatography (SEC) analysis to quantify a percentage of high molecular weight (HMW) species; (c) using said quantification to determine a predicted percentage of site-specific crosslinked peptides that may contribute to said HMW species, using Equation 1; (d) subjecting said crosslinked protein of interest from step (a) to peptide mapping analysis to quantify a percentage of site-specific crosslinked peptides;and (e) compare the said quantified percentage of site-specific crosslinked peptides from step (d) with the said predicted percentage of site-specific crosslinked peptides from step (c) to determine a site-specific crosslinking contribution to HMW species of said protein of interest.

46. ​​Method according to claim 45, characterized in that said protein of interest is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein or a recombinant protein. Petition 870250103116, dated 11 / 11 / 2025, pp. 76 / 92 10 / 14 47. Method according to claim 45, characterized in that said site-specific crosslinking comprises the crosslinking of a manipulated amino acid residue.

48. A method according to claim 45, characterized in that subjecting a protein of interest to suitable conditions to promote site-specific crosslinking includes bringing said protein of interest into contact with a crosslinking agent.

49. Method according to claim 47, characterized in that said crosslinking agent is an enzyme, optionally wherein said enzyme is microbial transglutaminase (mTG).

50. Method according to claim 45, characterized in that said peptide mapping analysis includes contacting said cross-linked protein of interest with at least one digestive enzyme to produce a peptide digestion and then subjecting said peptide digestion to RPLC-MS / MS analysis.

51. Method according to claim 50, characterized in that at least one digestive enzyme is trypsin.

52. Method for determining the contribution of a C-terminal lysine to the formation of high molecular weight (HMW) species in an antibody-drug conjugate of interest, characterized in that it comprises: (a) placing an antibody corresponding to an antibody-drug conjugate of interest in contact with a carboxypeptidase to produce a modified antibody, wherein said antibody includes a C-terminal lysine and said modified antibody does not include a C-terminal lysine; Petition 870250103116, dated 11 / 11 / 2025, p.77 / 92 11 / 14 (b) bring said antibody and said modified antibody into contact with a crosslinking agent to produce a crosslinked antibody and a crosslinked modified antibody, wherein said crosslinking agent is capable of crosslinking at least one amino acid residue of said antibody and said modified antibody into a lysine; (c) subject said crosslinked antibody and said modified crosslinked antibody to size exclusion chromatography (SEC) analysis to quantify the HMW species of said crosslinked antibody and said modified crosslinked antibody; and (d) compare said quantification of HMW species of said crosslinked antibody with said quantification of HMW species of said modified crosslinked antibody to determine a contribution of a C-terminal lysine to the formation of HMW species in said antibody-drug conjugate of interest.

53. Method according to claim 52, characterized in that said carboxypeptidase is a metallo-carboxypeptidase, serine carboxypeptidase, cysteine ​​carboxypeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase D or carboxypeptidase E.

54. Method according to claim 52, characterized in that said carboxypeptidase is carboxypeptidase B.

55. Method according to claim 52, characterized in that said crosslinking agent is an enzyme, optionally wherein said crosslinking agent is microbial transglutaminase (mTG).

56. Method for selecting an antibody for an antibody-drug conjugate, characterized in that it comprises: (a) obtaining a sample including a first antibody, wherein said first antibody comprises at least one target amino acid residue that can be crosslinked by a crosslinking agent to at least one non-target amino acid residue; (b) contacting said first antibody with said crosslinking agent to produce a crosslinked antibody; (c) contacting said crosslinked antibody with at least one digestive enzyme to produce a peptide digestion; (d) subjecting said peptide digestion to liquid chromatography-mass spectrometry (LC-MS) analysis to quantify peptides comprising said at least one target amino acid residue crosslinked to at least one non-target amino acid residue for a first antibody;(e) repeat steps (a)-(d) with at least one additional antibody to quantify peptides comprising at least one target amino acid residue crosslinked to at least one non-target amino acid residue for at least one additional antibody; (f) compare the quantifications from steps (d) and (e); and (g) use said comparison to select an antibody for an antibody-drug conjugate.

57. Method according to claim 56, characterized in that said at least one target amino acid residue is a lysine, a cysteine, a non-natural amino acid or a glutamine.

58. Method according to claim 56, characterized in that said at least one target amino acid residue is manipulated for site-specific conjugation.

59. Method according to claim 56, characterized in that said at least one nonspecific amino acid residue is a lysine, optionally wherein said lysine is a C-terminal lysine. Petition 870250103116, dated 11 / 11 / 2025, p. 79 / 92 13 / 14 60. Method according to claim 56, characterized in that said crosslinking agent is an enzyme, optionally wherein said crosslinking agent is microbial transglutaminase (mTG).

61. Method according to claim 56, characterized in that step (b) further comprises contacting said first antibody and said crosslinking agent with a ligand, wherein said crosslinking agent is capable of crosslinking said first antibody to said ligand.

62. Method according to claim 61, characterized in that said ligand is attached to a payload, optionally wherein said payload is a cytotoxic payload or a therapeutic payload.

63. Method according to claim 56, characterized in that said at least one digestive enzyme is selected from the group consisting of Aspergillus Saitoi protease, elastase, subtilisin, protease XIII, pepsin, trypsin, tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), Asp-N endoproteinase (Asp-N), Arg-C endoproteinase (Arg-C), Glu-C endoproteinase (Glu-C), outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, protease V8, their variants, their biologically active fragments, their homologs, or combinations thereof.

64. Method according to claim 56, characterized in that at least one digestive enzyme is trypsin.

65. Method according to claim 56, characterized in that said at least one digestive enzyme is IdeS or a variant thereof. Petition 870250103116, dated 11 / 11 / 2025, pp. 80 / 92 14 / 14 66. Method according to claim 56, characterized in that said liquid chromatography is selected from a 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. Method according to claim 56, characterized in that the said LC-MS analysis is an RPLCMS / MS analysis. Petition 870250103116, dated 11 / 11 / 2025, pp. 81 / 92

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