Qualification of a LC-HRMS platform method for biosimilar development using NISTmab as a model.

Biosimilars are a cost-effective alternative to biopharmaceuticals, necessitating rigorous analytical methods for consistency and compliance. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is a versatile tool for assessing key attributes, encompassing molecular mass, primary structure, and post-translational modifications (PTMs). Adhering to ICH Q2R1, we validated an LC-HRMS based peptide mapping method using NISTmab as a reference. The method validation parameters, covering system suitability, specificity, accuracy, precision, robustness, and carryover, were comprehensively assessed. The method effectively differentiated the NISTmab from similar counterparts as well as from artificially introduced spiked conditions. Notably, the accuracy of mass error for NISTmab specific complementarity determining region peptides was within a maximum of 2.42 parts per million (ppm) from theoretical and the highest percent relative standard deviation (%RSD) observed for precision was 0.000219 %. It demonstrates precision in sequence coverage and PTM detection, with a visual inspection of total ion chromatogram approach for variability assessment. The method maintains robustness when subjected to diverse storage conditions, encompassing variations in column temperature and mobile phase composition. Negligible carryover was noted during the carryover analysis. In summary, this method serves as a versatile platform for multiple biosimilar development by effectively characterizing and identifying monoclonal antibodies, ultimately ensuring product quality.

Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Cutting-edge mass spectrometry strategy based on imaged capillary isoelectric focusing (icIEF) technology for characterizing charge heterogeneity of monoclonal antibody.

Imaging capillary isoelectric focusing (icIEF) technology has been becoming the gold criteria of monitoring monoclonal antibody (mAb) charge heterogeneity that is one of the major product-related variants in recombinant biopharmaceuticals, since the first commercial instrument developed twenty years ago. However, the protein identification in icIEF separation is just based on isoelectric point (pI) measurement of protein. Although high resolution mass spectrometry (HRMS) is currently the most powerful means of qualitative protein analysis, traditional icIEF cannot compatibly be used in conjunction with MS due to the use of less volatile reagents. In addition, protein heterogeneity characterization in depth such as peptide mapping by high performance liquid chromatography (HPLC) requires the focused protein bands to be collected as fractions after the icIEF separation, which is a great challenge in biopharmaceutical discovery. In this work, pembrolizumab was employed as targeting mAb (a highly selective anti-PD-1 humanized mAb), an integrated icIEF platform was developed including analytical profiling, MS coupling and fraction collections for charged variant preparation. Multiple operation modes can be rapidly and flexibly switched just by changing customized capillary separation cartridges without more configurations. Main component, four acidic variants (A1-A4) and three basic variants (B1-B3) were baseline separated then directly detected by icIEF-HRMS online coupling for rapid screening of intact protein heterogeneity where reliable and accurate molecular weight of protein charged variants were obtained. Next, by installing preparative capillary separation cartridge, fractions of major charge variants (A2-3 and B1-2) and main component were collected for following LC-MS peptide mapping characterization. The whole workflow of icIEF-based MS strategy for protein heterogeneity is straight forward, reliable and accurate, which provides a comprehensive and revolutionary technology for protein drug quality control (QC) monitoring, MS coupling for fingerprinting intact protein and HPLC-MS peptide mapping in depth.

Comprehensive investigation of a structural variant in a bi-specific, N-and C-terminal Fc-fusion molecule, and its monitoring with LC-MS based method.

There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.

Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping.

Tag-free protein modification has received considerable attention in the field of chemical biology owing to the versatility and simplicity of the reaction sequence. In 2021, a novel tag-free enzymatic modification of antibodies utilizing lipoate ligase A (LplA) was reported to reveal its potential in the production of site-specific antibody conjugates. Primary peptide mapping analysis revealed the biased site specificity of antibodies modified by LplA; however, quantitative analysis remains challenging because of the complicated heterogeneity derived from biased selective modification. In an effort to further understand the site occupancy of LplA-modified antibodies, this study employed numerous unconventional techniques and strategies. Optimization of HPLC conditions and utilization of enzymes such as trypsin, Glu-C, and chymotrypsin significantly increased sequence data coverage. The transition from traditional spectral counting to a more accurate peak area-based label-free quantification helped better analyze peptide modification levels. The results obtained indicate that LplA-induced modifications are specific lysines, particularly the light chain Lys188/190 site, which have an increased modification rate compared to chemically induced modifications. This study not only contributes to the understanding of peptide modification, but also presents an improved methodology that promises to stimulate further research in this field.

Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody.

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.

A Highly Efficient Workflow for Detecting and Identifying Sequence Variants in Therapeutic Proteins with a High Resolution LC-MS/MS Method.

Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow.

Longitudinal mapping of hepatitis B vaccine-induced B-cell linear epitopes in healthy individuals.

The elimination of hepatitis B virus (HBV) infection is partially facilitated by the prophylactic HB vaccine. As the loss of seroprotection over time remains a conundrum for long-lasting protection, a comprehensive dynamic analysis of immunogenic targets of the HB vaccine will provide novel insights into the improvement and design of potential targets. In this study, 36 healthy subjects without prior history of hepatitis B infection and negative for hepatitis B surface antibody (anti-HBs) were enrolled. Participants were given a series of three doses of HB vaccine on a 0-, 1-, and 6-month schedule and longitudinally followed up. We systematically mapped 55 overlapping 15-mer peptides covering the small S protein of hepatitis B virus (SHBs) of vaccinees' serum samples at seven time points by performing an ELISA assay. Additionally, the frequencies and function dynamics of adaptive immune response were assessed by flow cytometry. We found that the SHBs peptide coverage presented an overall upward trend along with the vaccination progress, and the individual subpartition recognition was strongly correlated with the anti-HBs titers. Moreover, we identified one dominant epitope (S29) located on "a determinant region" associated with effective vaccine response. Besides, significant correlations between the proportion of plasmablasts and proliferating B cells and levels of anti-HBs were ascertained. Taken together, our data characterized the dynamics of HB vaccine-induced neutralizing antibodies against B-cell linear epitopes on SHBs and adaptive immune response, which will be constructive to develop the next-generation vaccine.

Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies.

Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.

Elucidating chemical and disulfide heterogeneities in rituximab using reduced and non-reduced peptide mapping.

Peptide mapping by liquid chromatography-mass spectrometry is the gold standard to characterize post-translational modifications (PTMs) and disulfide bonds. The structural integrity, heterogeneity, and quality of biotherapeutic proteins are evaluated via reduced and non-reduced peptide mapping methods. However, non-enzymatic artifacts are often induced during sample preparation when alkaline pH conditions are used. To minimize these artifacts, methods using various acidic pH conditions have been developed by multiple researchers. However, these may lead to missed and non-specific cleavages during the analysis. In this study, an improved reduced and non-reduced peptide mapping method has been proposed to characterize endogenous chemical modifications and native disulfide bonds of monoclonal antibody-based products. Solubilization has been carried out at acidic pH conditions under high temperature, followed by the addition of tris (2-carboxyethyl) phosphine as a reducing agent and a low alkylating agent. It was observed that the non-enzymatic PTMs and non-native disulfide scrambled peptides significantly reduced under trypsin plus endoproteinase Lys-C digestion conditions at acidic pH as compared to the traditional methods. The results demonstrate that the proposed reduced and non-reduced peptide mapping method using trypsin plus Lys-C could identify and quantify various chemical and disulfide heterogeneities with minimal artifacts.

In-bone protein digestion followed by LC-MS/MS peptide analysis as a new way towards the routine proteomic characterization of human maxillary and mandibular bone tissue in oral surgery.

Proteomic characterization of alveolar bones in oral surgery represents an analytical challenge due to their insoluble character. The implementation of a straightforward technique could lead to the routine use of proteomics in this field. This work thus developed a simple technique for the characterization of bone tissue for human maxillary and mandibular bones. It is based on the direct in-bone tryptic digestion of proteins in both healthy and pathological human maxillary and mandibular bone samples. The released peptides were then identified by the LC-MS/MS. Using this approach, a total of 1120 proteins were identified in the maxillary bone and 1151 proteins in the mandibular bone. The subsequent partial least squares-discrimination analysis (PLS-DA) of protein data made it possible to reach 100% discrimination between the samples of healthy alveolar bones and those of the bone tissue surrounding the inflammatory focus. These results indicate that the in-bone protein digestion followed by the LC-MS/MS and subsequent statistical analysis can provide a deeper insight into the field of oral surgery at the molecular level. Furthermore, it could also have a diagnostic potential in the differentiation between the proteomic patterns of healthy and pathological alveolar bone tissue. Data are available via ProteomeXchange with the identifier PXD026775.

Extended characterization of unpaired cysteines in an IgG1 monoclonal antibody by LC-MS analysis.

The development of comprehensive methods to characterize unpaired cysteines in monoclonal antibodies (mAbs) is very important for understanding structural heterogeneity, impurity, and stability. In this paper, unpaired cysteines observed in a therapeutic antibody (mAb1) were thoroughly studied by Liquid Chromatography-Mass Spectrometry (LC-MS) methods at the intact mAb, domain, and peptide levels. Three cysteine variants were observed at the intact mAb level with each variant containing two unpaired cysteines. Variants containing four or six unpaired cysteines were not observed. Domain analysis indicated that two Fab variants, each containing two unpaired cysteines, were present while the third variant contained two unpaired cysteines on the Fc region. Peptide mapping analysis localized the six unpaired cysteines to Cys22/Cys96, Cys146/Cys202, and Cys369/Cys427 in the heavy chain. No significant changes were observed for these unpaired cysteines in mAb1 under high pH and heat-stressed conditions. Structural analysis and molecular modeling revealed that these unpaired cysteines were buried inside the three-dimensional structure. The integrated LC-MS methods together with stress studies and structural analysis may potentially be applied to the analysis of unpaired cysteines in other mAbs.

Lipoate-acid ligase a modification of native antibody: Synthesis and conjugation site analysis.

Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis.

Study of the geometry of open channels in a layer-bed-type microfluidic immobilized enzyme reactor.

This paper aims at studying open channel geometries in a layer-bed-type immobilized enzyme reactor with computer-aided simulations. The main properties of these reactors are their simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make these devices one of the simplest yet efficient enzymatic microreactors. The high surface-to-volume ratio of the reactor was achieved using narrow (25-75 µm wide) channels. The simulation demonstrated that curves support the mixing of solutions in the channel even in strong laminar flow conditions; thus, it is worth including several curves in the channel system. In the three different designs of microreactor proposed, the lengths of the channels were identical, but in two reactors, the liquid flow was split to 8 or 32 parallel streams at the inlet of the reactor. Despite their overall higher volumetric flow rate, the split-flow structures are advantageous due to the increased contact time. Saliva samples were used to test the efficiencies of the digestions in the microreactors.

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1-2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.

Dynamic pH barrage junction focusing of amino acids, peptides, and digested monoclonal antibodies in capillary electrophoresis-mass spectrometry.

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE-MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.

Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein.

PURPOSES: The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon. METHODS: A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques. RESULTS: The formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS. CONCLUSIONS: Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes.

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs. Graphical abstract.

Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography.

Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis due to the potential negative effect of these modifications on drug efficacy and stability. Succinimide has long been known to be an intermediate product of asparagine deamidation and aspartic acid isomerization, but despite the key role of succinimide in these reactions, its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site. These results are then used to establish a hydrophobic interaction chromatography (HIC)-based method that can be used for release and stability studies. In this method, unmodified protein, deamidated products, and succinimide are well separated and quantified. Good correlation was obtained between the data from low-pH protein digestion-based peptide mapping and the HIC-based method. Method qualification showed that the HIC-based method is robust, accurate, and precise and has excellent linearity.