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Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study presents a systematic approach for developing an innovative hydrophilic interaction liquid chromatography (HILIC) method for collagen peptide mapping analysis. The predominant post-translational modification (PTM) of collagen, proline hydroxylation, introduces polar hydroxyl groups throughout the collagen sequence, making HILIC a promising alternative to classical reversed-phase liquid chromatography (RPLC) approaches. This study employs sixteen model peptides, selected from in silico predicted tryptic peptides with zero missed cleavages and representing diverse physicochemical properties and structural motifs of collagen. The peptides were used as standards to conduct detailed chromatographic evaluation. Various HILIC stationary phases and mobile phases were systematically examined to identify optimal separation conditions for collagen peptides, contributing to a better understanding of peptide behavior in HILIC. The study also explores the effects of sample diluent and injection mode, comparing classical injection with the Performance Optimizing Injection Sequence (POISe), to determine their impact on HILIC performance. Introducing a plug of weak solvent (acetonitrile) prior to sample injection, effectively mitigates the mismatch in eluent strength between the fully aqueous sample diluent (resulting from tryptic digestion) and the mobile phase, addressing issues of peak distortion. Different injection volumes (from 0.5 to 8 µL) and acetonitrile ratios (1:1, 1:2, 1:5 and 1:10) were tested to optimize sample injection and increase sensitivity of collagen tryptic peptides. Following method optimization, HILIC was coupled with mass spectrometry (MS) to evaluate its effectiveness in analyzing collagen-digested samples. This evaluation included the assessment of peptide sequence coverage and the method ability to identify hydroxylation patterns, thereby demonstrating its potential for detailed peptide analysis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Caterina Temporini reports financial support was provided by Italian Ministry of University and Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Fragmentation is a phenomenon ubiquitously observed during research and development of therapeutic antibodies. The clear identification of the cleavage site is vital for comprehending fragmentation mechanisms and optimizing processes. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is now widely used to detect and quantify fragments, while its peak identification is hindered by immature capillary electrophoresis-sodium dodecyl sulfate coupled with mass spectrometry techniques. In this study, we developed a systematic workflow for fragment characterization, which integrated direct identification, fragment enrichment, and fragmentation confirmation using multiple techniques, such as microscale peptide mapping (PM), PM of N-termini labeled sample, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in-gel extraction for molecular weight (MW) and PM measurements. By employing this innovative workflow, we successfully identified the cleavage sites of two therapeutic antibodies. In the first case, through direct liquid chromatography-mass spectrometry (LC-MS) characterization, the cleavages leading to the loss of biological function were identified on the linker of a bispecific antibody. In the second case involving a tetravalent antibody, direct LC-MS analysis failed. However, more sophisticated analysis nailed down the critical cleavage at the LC/HC: G 105 -R 106 site in the CDR3 region of the antibody. Our systematic workflow provides a clear and accessible method for identifying cleavage sites with broad applicability across pharmaceutical laboratories.

Posttranslational modifications (PTMs) are potential critical quality attributes in biotherapeutic development, as they can affect drug efficacy and safety. Tyrosine sulfation plays a critical role in protein-protein interactions and has been found on many surface receptors as well as antibody complementarity-determining regions (CDR). However, the presence and function of tyrosine sulfation in therapeutic proteins have not been broadly investigated due to difficulties in detecting the modification. Here, we establish an integrated strategy to identify tyrosine sulfation in biotherapeutic proteins. In silico prediction was used to estimate possible modification sites, followed by the elucidation with intact LCMS and native SCX-MS. The combination of these three steps takes less than 1 h, which provides quick and confident preliminary detection of potential CQAs. Taking NB1 as an example, three +80 Da mass shifts were observed from intact mass analysis and three acidic peaks were monitored by SCX, allowing confirmation of modification as either phosphorylation or sulfation. Peptide mapping, Fe 3+ -IMAC enrichment, and dephosphorylation were further conducted to provide improved signal intensity and differentiation of modification such as sulfation or phosphorylation. With this integrated strategy, we were able to identify for the first time both tyrosine sulfation and serine phosphorylation in one therapeutic protein.

Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liqing Gu reports financial support was provided by Incyte Corporation. Tiger X. Hu reports financial support was provided by Incyte Corporation. Liqing Gu reports a relationship with Incyte Corporation that includes: employment and equity or stocks. Tiger X. Hu reports a relationship with Incyte Corporation that includes: employment and equity or stocks., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The characterization of cysteine-linked antibody‒drug conjugates (ADCs) can be more challenging than that of monoclonal antibodies (mAbs) and lysine-linked ADCs because the interchain disulfide bonds are reduced for payload conjugation, and the chains are noncovalently bonded to each other. Furthermore, payload conjugation and disulfide bond reduction/scrambling may introduce additional charge heterogeneity to biomolecules. This study illustrates an innovative workflow employing multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin, a recent-generation cysteine-linked ADC, including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF). RPLC was employed for reduced chains analysis, subunit analysis and peptide mapping. IEX and icIEF were used for charge heterogeneity analysis. The innovation of the integrated methodology emphasizes the importance of cutting-edge icIEF-MS online coupling under near-native conditions to reveal the heterogeneity of disitamab vedotin., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

After the successful sequencing of nucleic acids, nanopore technology has now been applied to proteins. Recently, it has been demonstrated that an electro-osmotic flow can be used to induce the transport of unraveled polypeptides across nanopores. Polypeptide translocation, however, is too fast for accurate reading its amino acid compositions. Here, we show that the introduction of hydrophobic residues into the lumen of the nanopore reduces the protein translocation speed. Importantly, the introduction of a tyrosine at the entry of the nanopore and an isoleucine at the entry of the β-barrel of the nanopore reduced the speed of translocation to ∼10 amino acids/millisecond while keeping a relatively large ionic current, a crucial component for protein identification. These nanopores showed unique features within their current signatures, which may pave the way toward protein fingerprinting using nanopores.

Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally., (© 2024 Wiley‐VCH GmbH.)

Background: Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required., Methods: In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity., Results: The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected., Conclusions: The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author Alois Jungbauer is an Editorial Board Member for Journal of Chromatography A and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Arnaud Delobel reports financial support was provided by Walloon Region. Sergei Saveliev reports a relationship with Promega Corporation that includes: employment. Alba Katiria Gonzalez Rivera reports a relationship with Promega Corporation that includes: employment. Marjeta Urh reports a relationship with Promega Corporation that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Stop codon readthroughs were examined in 48 recombinant therapeutic protein candidates produced from multiple clones of Chinese hamster ovary cells, using peptide mapping with LC-MS/MS detection. We found that stop codon readthrough is a common phenomenon occurring in most of these candidates, with levels varying from below the detection limit of ∼0.001 % to ∼1 %. The readthrough propensity depends on the stop codon being used, as well as the nucleotides surrounding it. The amino acids misincorporated into the stop position can be well-predicted by a third-base wobble mismatch and a first-base U/G mismatch during codon recognition, i.e., tyrosine or glutamine insertion for the UAA and UAG stop codons, and tryptophan, cysteine or arginine insertion for the UGA stop codon. Data shown in this report demonstrate the importance of optimizing the DNA sequence near the stop codon, and the importance of detecting stop codon readthroughs during the development of a therapeutic product., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paresh Tank reports financial support and article publishing charges were provided by Zelle biotechnology pvt. Ltd. Paresh Tank and co-authors reports a relationship with Zelle biotechnology pvt ltd that includes: employment., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Biologics are essential in cancer treatment because they stimulate the body's natural response to fight cancer, but they are expensive. Biosimilars are more affordable compared to patent biologicals, but it must be verified that they are as effective as their innovators. Characterization of biosimilars and assessment of interchangeability requires many data points for verification., Objective: The proposed study provides a quality assessment of two new bevacizumab (BVZ) biosimilars, produced by Amgen and Biocad, Inc., through the development and greenness assessment of an orthogonal testing protocol and purity indicating assay, including size-exclusion (SE-HPLC), reversed-phase (RP-HPLC), and cation exchange chromatography (CEX-HPLC) in addition to dynamic light scattering (DLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)., Methods: SE-HPLC method was performed and validated to screen the BVZ monomer and its aggregates and/or fragments. Peak purity and system suitability parameters were calculated. Results indicate that the orthogonal protocol is a useful tool for assessing monoclonal antibody stability. It is a key criterion for biosimilarity assessment. DLS and SDS-PAGE results were compared to each other to reveal close retention times and banding patterns between BVZ innovator and its biosimilars. These results showed that Avastin® and the investigated biosimilars have the same profile in terms of peak area of related compounds within the acceptance limit and apparent molecular weight, and the SDS-PAGE technique was found to be the most eco-friendly technique among others., Conclusions: The results obtained highlighted the importance of assessing similarities and differences in ensuring the biosimilarity and interchangeability of the studied products., Highlights: BVZ is one of the essential monoclonal antibodies in the treatment of colorectal cancer (CRC). BVZ biosimilars were evaluated by developing an orthogonal testing protocol and a purity-indicating assay. The size-exclusion (SE)-HPLC method was applied and validated to monitor the BVZ monomer and its aggregates. The results demonstrated the importance of assessing the stability and biosimilarity of BVZ., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate Chemical Reference Substance (CRS) as a replacement reference (batch 2) for the physicochemical methods in European Pharmacopoeia monograph 1316 Erythropoietin concentrated solution . Results from the study demonstrated that for the physicochemical methods described in the monograph - capillary zone electrophoresis, polyacrylamide gel electrophoresis and immunoblotting, peptide mapping and glycan mapping - the candidate CRS is essentially identical to CRS batch 1 and is suitable to be established as Erythropoietin for physicochemical tests CRS batch 2. CRS batch 2 is a freeze-dried preparation presented in vials. It was adopted at the 177th session of the European Pharmacopoeia Commission in November 2023 and is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM) under product code Y0001725., (© Council of Europe 2024.)

Non-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rearrangement of disulfide connections (disulfide scrambling), thus generating connectivity profiles that do not accurately represent the protein being analyzed. Common misconceptions (e.g. avoiding basic-pH digestion to prevent disulfide scrambling) have led to the development of alternative reagents and conditions that can alleviate this issue, but yield problematic digestion profiles. Herein, we systematically and comprehensively examine the primary considerations for accurate non-reduced peptide mapping, and provide effective, practical solutions to minimize undesired behavior while still yielding high-quality digests. Additionally, we present a method that exploits intentional disulfide scrambling as a reference tool to demonstrate the robustness of our proposed strategies. We also introduce maleimide as a cysteine-alkylating reagent and demonstrate its benefits over industry-leading analogs such as N-ethylmaleimide in terms of compatibility with regulatory reports.

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.

Establishing analytical and functional comparability serves as the foundation of biosimilar development. A critical part of this exercise is sequence similarity search and categorization of post-translational modifications (PTMs), often by peptide mapping using liquid chromatography-mass spectrometry (LC-MS). When performing bottom-up proteomic sample preparation, efficient digestion of the protein and extraction of peptides for subsequent mass spectrometric analysis can be a challenge. Conventional sample preparation strategies face the risk of allowing interference of chemicals which are essential for extraction but are likely to interfere with digestion, resulting in complex chromatographic profiles due to semi-cleavages, insufficient peptide cleavages, and other unwanted reactions. Further, peptide cleanup through commonly used immobilized C-18 pipette tips can cause significant peptide loss as well as variability in individual peptide yields, thereby causing artifacts of various product-related modifications. In this study, we proposed a simple enzymatic digestion technique by incorporating different molecular weight filters and protein precipitation, with the objective to minimize interference of denaturing, reducing, and alkylating agents throughout overnight digestion. As a result, the need for peptide cleanup is significantly reduced and results in higher peptide yield. The proposed FAPP approach outperformed the conventional method across multiple metrics including, 30% more peptides, 8.19% more fully digested peptides, 14% higher sequence coverage rate, and 11.82% more site-specific alterations. Quantitative and qualitative repeatability of the proposed approach have been demonstrated. It can be concluded that the filter-assisted protein precipitation (FAPP) protocol proposed in this study offers an effective substitute for the traditional approach., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anurag Singh Rathore reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology. Anurag Singh Rathore reports financial support was provided by Agilent Technologies., (Copyright © 2023 Elsevier B.V. All rights reserved.)

LC-MS analysis of therapeutic antibodies and other biotherapeutics from in-life studies (e.g., serum/plasma) has evolved from simple peptide digestion to peptide mapping and intact mass monitoring. From more advanced analytical approaches, a deeper understanding as to the fate of the biotherapeutic in vivo is gained. Here, we examine the next generation of approaches to facilitate the most comprehensive understanding of large molecule drug fate in circulation. Three case studies are presented: (1) use of relative and absolute calibration curves for biotherapeutic quantitation from the same sample set; (2) top-down mass spectrometry applied to bioanalytical assays; (3) biotherapeutic protein complexes from serum analyzed by native protein MS. We anticipate that these approaches will be further adapted and applied by other research groups.

The antiangiogenic drug bevacizumab is a blockbuster therapeutic pharmaceutical product that is used to treat many different types of cancer including kidney, colon, rectum, lung, and breast cancer. As a result, multiple biosimilars have been approved across the various regulatory jurisdictions in India (>20 in number till date). The rapidly growing market and acceptance of biosimilars was the motivation to perform comparability study of bevacizumab biosimilars that are presently available in the Indian market. A comprehensive analytical and functional biosimilarity assessment has been performed to examine and compare innovator product of bevacizumab (Avastin-innovator product, Roche Products (India) Pvt Ltd) and six biosimilars that are being marketed in India (Abevmy from Mylan Pharmaceuticals Pvt Ltd, Bevazza from Lupin Ltd, Bryxta from Zydus Cadila, Krabeva from Biocon, Ivzumab from RPG Life Sciences Ltd, and Advamab from Alkem Laboratories Ltd). Physiochemical characterization of drug products was performed with respect to their primary structure (intact mass, reduced mass, peptide mapping by LC-MS), higher order structure (secondary structure by FTIR, Far-UV-CD, and tertiary structure by Near-UV-CD, intrinsic fluorescence spectroscopy), impurity profile (SE-HPLC, SEC-MALS, extrinsic fluorescence: size heterogenicity, degradation, stability; DLS: hydrodynamic radius; WCX-HPLC: charge variants analysis) and post-translational modifications by measuring reduced glycans through fluorescence dye analysis. Functional characterization was performed by SPR and cell proliferation assay. Further, chemometrics based quantitative evaluation of biosimilarity has been performed by combining the data obtained from analytical characterization platform. The analysis of the analytical, functional and chemometric results revealed significant levels of similarity, with biosimilar4 being the sole exception. Despite being within product specifications, Biosimilar4 displayed significant deviations with respect to critical quality attributes, including a lower proportion of monomer content, a larger percentage of basic charge variant species, and a lower proportion of aglycosylated glycoform., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

LC-MS based multi-attribute methods (MAM) have drawn substantial attention due to their capability of simultaneously monitoring a large number of quality attributes of a biopharmaceutical product. For successful implementation of MAM, it is usually considered a requirement that the method is capable of detecting any new or missing peaks in the sample when compared to a control. Comparing a sample to a control for rare differences is also commonly practiced in many fields for investigational purpose. Because MS signal variability differs greatly between signals of different intensities, this type of comparison is often challenging, especially when the comparison is made without enough replicates. In this report we describe a statistical method for detecting rare differences between two very similar samples without replicate analyses. The method assumes that an overwhelming majority of components have equivalent abundance between the two samples, and signals with similar intensities have similar relative variability. By analyzing several monoclonal antibody peptide mapping datasets, we demonstrated that the method is suitable for new-peak detection for MAM as well as for other applications when rare differences between two samples need to be detected. The method greatly reduced false positive rate without a significant increase of false negative rate., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general., (© 2023. The Author(s).)

Enzymes are mainly extracted from the culture broth of microorganisms. Various commercially available enzyme preparations (EPs) are derived from different microorganisms, and the source of the EP should be the same as that mentioned in the manufacture's information. The development of analytical methods that can determine the origin of the final products is important for ensuring that the EPs are nontoxic, especially when used as food additives. In this study, various EPs were subjected to SDS-PAGE, and the main protein bands were excised. After in-gel digestion, the generated peptides were analysed using MALDI-TOF MS, and protein identification was performed by searching the set of peptide masses against protein databases. In total, 36 EPs including amylase, β-galactosidase, cellulase, hemicellulase and protease were analysed, and the information about the enzyme sources was obtained for 30 EPs. Among these, the biological sources determined for 25 EPs were consistent with the manufacturer's information; for the remaining five, enzymes produced by closely-related species were shown as matching proteins due to high sequence similarity. Six enzymes derived from four microorganisms could not be identified because their protein sequences were not registered in the database. As these databases are expanded, this approach of using SDS-PAGE and peptide mass fingerprinting (PMF) can determine the biological origin of enzymes rapidly and contribute to ensuring the safety of EPs.

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.

Recombinant therapeutic proteins have become the major class of drugs to treat various human diseases in recent years. Low levels of protein sequence variants (SVs) have been reported to be present in recombinant therapeutic proteins. The consequences of potential unwanted immune response from SVs of recombinant therapeutic proteins have increasingly drawn attention from regulatory authorities and the biopharmaceutical industry. It is highly desirable to detect low-level SVs during clone selection and early process development as part of the control strategy. Peptide mapping with LC-MS/MS analysis has been applied as a powerful tool to characterize post-translation modifications of therapeutic proteins. Despite the recent advancements in mass spectrometry hardware and software, it is still quite challenging and time-consuming to detect and identify low-level SVs. In this study, we present an optimized approach using new peak detection to detect and identify low level SVs with high confidence and high speed. The new approach makes sequence variants analysis by LC-MS/MS broadly applicable and practical in bioprocess development of therapeutic proteins.

Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co- and post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is labor-intensive, involving multiple manual steps. Recently, simple high-temperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories. Here, a modified workflow with an automatic digestion step is presented, which includes a two-step digestion at high and low temperatures, as opposed to the original one-step digestion at a high temperature. The new automatic digestion workflow significantly reduces the number of missed cleavages, obtaining a more complete digestion profile. In addition, we describe how chromatographic peak tailing and carry-over is dramatically reduced for hydrophobic peptides by switching from the traditional C18 reversed-phase (RP) column chemistry used for peptide mapping to a less retentive C4 column chemistry. No negative impact is observed on MS/MS-derived sequence coverage when switching to a C4 column chemistry. Overall, the new peptide mapping workflow significantly reduces the number of missed cleavages, yielding more robust and simple data interpretation, while providing dramatically reduced tailing and carry-over of hydrophobic peptides.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Fc-fusion proteins represent a successful class of biopharmaceutical products. They are considered highly heterogeneous products due to the common degradation of amino acids that occurs during their production in upstream and downstream processes (e.g., oxidation and deamidation) and, above all, their complex glycosylation profile. Multi-dimensional liquid chromatography-mass spectrometry (mD-LC-MS) has recently gained much interest for process analytical technology, enabling the integration of this analytical technology in production and purification environments. In this study, an online mD-LC-MS/MS peptide mapping method was developed for monitoring multiple quality attributes, including the N-glycosylation state of a complex Fc-fusion protein, which is made by combining two heavily glycosylated cytokines with an Fc domain. This fully automated workflow includes sample purification, reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis. Two immobilized enzyme cartridges based on trypsin and Lys-C protease were employed to generate a detailed glycosylation mapping, as trypsin allowed the identification of only one of four glycosylation sites, while Lys-C was more informative for two other sites. Site-specific glycosylation information such as antennarity, sialyation, and core fucosylation state was also determined. In addition to glycans, other post-translational modifications could be monitored simultaneously during the cell culture production processes by the mD-LC-MS/MS approach. In summary, the generated data demonstrate the applicability of mD-LC-MS for the monitoring and trending of multiple attributes for complex antibody formats over production processes in an automated and fast manner, compared to the complex and time-consuming traditional offline assays., (Published by Elsevier B.V.)

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., (© 2022 Wiley-VCH GmbH.)

The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in most tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using lysyl endopeptidase (Lys-C) for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using a streamlined Lys-C digestion without desalting with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins. In this work, we performed UV and visible light irradiation experiments on several therapeutic proteins, with or without the presence of a photosensitizer. Light-induced modifications were detected and characterized by tryptic digestion followed by LC-MS/MS analysis. A list of potential light-induced modifications, with their respective mass changes, was obtained. These modifications are primarily on methionine, tryptophan, histidine, cysteine, tyrosine and phenylalanine residues. Many of these modifications have not been previously reported on therapeutic proteins. Our findings therefore provide a database of potential light-induced modifications that would enable the routine characterization of light-induced modifications on therapeutic proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.

In the last 15 years, increasing evidence linking epigenetics to various aspects of cancer biology has prompted the investigation of histone post-translational modifications (PTMs) and histone variants in the context of clinical samples. The studies performed so far demonstrated the potential of this type of investigations for the discovery of both potential epigenetic biomarkers for patient stratification and novel epigenetic mechanisms potentially targetable for cancer therapy. Although traditionally the analysis of histones in clinical samples was performed through antibody-based methods, mass spectrometry (MS) has emerged as a more powerful tool for the unbiased, comprehensive, and quantitative investigation of histone PTMs and variants. MS has been extensively used for the analysis of epigenetic marks in cell lines and animal tissue and, thanks to recent technological advances, is now ready to be applied also to clinical samples. In this review, we will provide an overview on the quantitative MS-based analysis of histones, their PTMs and their variants in cancer clinical samples, highlighting current achievements and future perspectives for this novel field of research. Among the different MS-based approaches currently available for histone PTM profiling, we will focus on the 'bottom-up' strategy, namely the analysis of short proteolytic peptides, as it has been already successfully employed for the analysis of clinical samples., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MS E fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.