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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, Fe3+-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. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies such as monoclonal antibodies (mAbs), bispecific and multispecific antibodies are pivotal in therapeutic protein development and have transformed disease treatments across various therapeutic areas. The integrity of therapeutic antibodies, however, is compromised by sequence liabilities, notably deamidation, where asparagine (N) and glutamine (Q) residues undergo chemical degradations. Deamidation negatively impacts the efficacy, stability, and safety of diverse classes of antibodies, thus necessitating the critical need for the early and accurate identification of vulnerable sites. In this article, a comprehensive antibody deamidation-specific dataset (n = 2285) of varied modalities was created by using high-throughput automated peptide mapping followed by supervised machine learning to predict the deamidation propensities, as well as the extents, throughout the entire antibody sequences. We propose a novel chimeric deep learning model, integrating protein language model (pLM)-derived embeddings with local sequence information for enhanced deamidation predictions. Remarkably, this model requires only sequence inputs, eliminating the need for laborious feature engineering. Our approach demonstrates state-of-the-art performance, offering a streamlined workflow for high-throughput automated peptide mapping and deamidation prediction, with the potential of broader applicability to other antibody sequence liabilities. [ABSTRACT FROM AUTHOR]

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40 °C, thermal stress at 55 °C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography (SEC) and cation exchange chromatography (CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC–MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies such as monoclonal antibodies (mAbs), bispecific and multispecific antibodies are pivotal in therapeutic protein development and have transformed disease treatments across various therapeutic areas. The integrity of therapeutic antibodies, however, is compromised by sequence liabilities, notably deamidation, where asparagine (N) and glutamine (Q) residues undergo chemical degradations. Deamidation negatively impacts the efficacy, stability, and safety of diverse classes of antibodies, thus necessitating the critical need for the early and accurate identification of vulnerable sites. In this article, a comprehensive antibody deamidation-specific dataset (n = 2285) of varied modalities was created by using high-throughput automated peptide mapping followed by supervised machine learning to predict the deamidation propensities, as well as the extents, throughout the entire antibody sequences. We propose a novel chimeric deep learning model, integrating protein language model (pLM)-derived embeddings with local sequence information for enhanced deamidation predictions. Remarkably, this model requires only sequence inputs, eliminating the need for laborious feature engineering. Our approach demonstrates state-of-the-art performance, offering a streamlined workflow for high-throughput automated peptide mapping and deamidation prediction, with the potential of broader applicability to other antibody sequence liabilities.

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ -immunoglobulin antibodies using liquid chromatography–mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100–1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine's accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Peptide mapping is a key method for studying the primary structure of proteins. With its sensitivity to the slightest changes in the covalent structure of a protein, this method is applicable both to medicinal product identification at the control stage and to production process stability monitoring.The aim of the study was to develop and validate a peptide-mapping procedure for the identification of a novel highly glycosylated recombinant C1 esterase inhibitor.Materials and methods. The authors studied recombinant human C1 esterase inhibitor products and trypsin. The study involved peptide mapping using reverse-phase high-performance liquid chromatography and high-resolution mass spectrometry. The following statistics were calculated to evaluate the results: mean, standard deviation, and coefficient of variation. The validation parameters included specificity, precision, and robustness.Results. The authors tested several variants of sample preparation for tryptic digests, including additional N-glycanase treatment and complete deglycosylation, and established the optimal conditions for sample preparation and chromatographic separation of C1 esterase inhibitor peptides to obtain consistent chromatographic profiles (peptide maps). The authors identified characteristic peaks and the corresponding relative retention time and area ranges. The absolute retention time of the second (characteristic) peak was approximately 16.5–16.9 minutes. The relative retention times were 2.14–2.21 for peak 9, 2.55–2.64 for peak 12, 2.97–3.14 for peak 14, 3.11–3.29 for peak 15, and 6.20–6.63 for peak 28.Conclusions. The authors developed a peptide-mapping procedure for C1 esterase inhibitors and optimised the conditions to achieve an over 18-hour reduction in sample preparation time. This procedure met the established acceptance criteria for specificity, precision, and robustness.

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

Introduction. Highly glycosylated proteins are the most abundant class of modern biopharmaceuticals. A majority of such therapeuticals produced by Russian biopharmaceutical companies is biosimilars. The foundation of biosimilar manufacturing is analytical assessment of structure equivalence to an original molecule. Fc-fusions present a challenge due to their structural properties. The only biosimilar of this kind registered in Russia is etanercept – a fusion of tumor necrosis factor receptor α and Fc-fragment of IgG1. Existing approaches widely used in protein analysis do not allow accurate and reliable description of glycoylation of these proteins. Development of new approaches and principles of such analysis is necessary, as the changes in biosimilar’s molecular structure can seriously affect its efficacy and safety.Aim. Development of double proteolysis approaches for glycopeptide mapping of Fc-fusion protein etanercept using Arg-C protease.Materials and methods. Etanercept was subjected to enzymatic hydrolysis using trypsin in combination with Arg-C or Asp-N. The resulting peptides were analyzed using HPLC-MS system Xevo G2-XS QTOF (Waters Corporation, USA). The conformation of glycan structure was performed via analysis of fragment spectra of glycopeptides, acquired with high collision energy mode (MS E ). UNIFI (version 1.8) with biopharmasuetical assessment setting (Waters Corporation, USA) was used to analyze the peptide maps.Results and discussion. It was found that using the combination of trypsin with protease Arg-C leads to reliable results Using the developed approach we successfully determined the majority of O-glycosylation sites and types of O-glycans. It was shown that for an effective O-glycopeptide maping N-deglycosylation stage is required. Most abundant N-glycan structures were identified for each of three N-glycosylation sites (N149, N171, N317). It was determined, that the combination of trypsin with Arg-C allows identification of three-antenna forms despite the presence of O-glycosylation site on the analyzed peptide. General N-glycosylation profile shows comparability of results for both approaches.Conclusion. As a result of this research we developed glycopeptide mapping approaches in which a combination of proteases is used. Using these methods sites of N- and O-glycosilation and glycofoms of etanercept were accurately and reproducibly determined. Developed procedures can be applied to other types of Fc-fusion proteins, making it of broader appeal and benefit to the overall biopharmaceutical industry. These approaches provide comprehensive information useful for structure-function studies and of potential value for product comparability measurements and possibly even future manufacturing control strategies.

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification. The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping. Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation. Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

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. • Opposite charge profile shifts observed for thermally stressed AAV8 and AAV1. • An optimized one-pot tryptic digestion protocol developed for AAV peptide mapping. • The location of modifications largely impacts the AAV retention on AEX. • A unique PTM specific to each AAV serotype is mainly responsible for the observed charge profile shift. [ABSTRACT FROM AUTHOR]

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. • Antibody is digested in non-reduced condition efficiently without buffer exchange. • Supplementing urea with guanidine hydrochloride denatures antibody but not enzyme. • Unique two-step digestion enhances digestion efficiency. • There are no elevated artifacts when comparing with a commercial digestion kit. • This is a generic method for disulfide bond mapping of antibody drugs. [ABSTRACT FROM AUTHOR]

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography–mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100–1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine’s accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65-21-92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (n = 44 cases and n = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development.

This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selenylation of cysteine and methionine thiols through selenate supplements increases the content of selenium-containing amino acids in various agricultural products. This modification results in numerous biological and health benefits. Despite their critical roles in human physiology, methods for high-coverage and efficient identification of selenylation peptides are limited. This study systematically developed a mass spectrometric method to identify selenylation peptides combined with in-gel trypsin proteolysis. In-gel proteolysis identified the two well-separated bands containing rice glutelin. We identified 11 rice glutelin subunits along with 42 selenylation peptides from the glutelin acidic subunits and 30 selenylation peptides from the glutelin basic subunits with high confidence. A comprehensive evaluation disclosed the mapping of selenium-containing rice glutelin subunits. Additionally, the selenylation modification of peptides coexisted with oxidation and iodoacetamide (IAM) alkylation. Moreover, the multidimensional MS criteria validated the results, while spectral statistics revealed the veritable Se/S substitution degree in Se-enriched rice. These findings collectively demonstrated the presence of numerous selenation sites in microheterogeneous glutelin subunits, thereby enhancing our understanding of the seleno-peptidomics of rice proteins. As significant bioactive organic compounds, the identified peptides in this study are promising candidates for a variety of bioactivities, including neuroprotective, anti-inflammatory, antioxidant, hepatoprotective, and immunomodulatory effects.

Biotherapeutics are subject to inherent heterogeneity due to the complex biomanufacturing processes. Numerous analytical techniques have been employed to identify, characterize, and monitor critical quality attributes (CQAs) to ensure product safety, and efficacy. Mass spectrometry (MS)-based multi-attribute method (MAM) has become increasingly popular in biopharmaceutical industry due to its potential to replace multiple traditional analytical methods. However, the correlation between MAM and conventional methods remains to be fully understood. Additionally, the complex analytical workflow and limited throughput of MAM restricts its implementation as a quality control (QC) release assay. Herein, we present a simple, robust, and rapid MAM workflow for monitoring CQAs. Our rapid approach allowed us to create a database from ~700 samples, including site-specific post-translational modifications (PTMs) quantitation results using MAM and data from traditional charge variant and oxidation characterization methods. To gain insights from this database, we employ multivariate data analysis (MVDA) to thoroughly exploit the data. By applying partial least squares regression (PLSR) models, we demonstrate the ability to quantitatively predict charge variants in ion exchange chromatography (IEX) assay and oxidation abundances in hydrophobic-interaction chromatography (HIC) assay using MAM data, highlighting the interconnectivity between MAM and traditional product quality assays. These findings help evaluate the suitability of MAM as a replacement for conventional methods for release, and more importantly, contribute to enhanced process and product understanding., Competing Interests: Declarations. Conflict of Interest: Authors confirm that there are no conflicts of interest or competing interests related to this work., (© 2024. Merck & Co., Inc., Rahway, NJ, USA and its affiliates, under exclusive licence to Springer Nature Switzerland AG.)

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5

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. [ABSTRACT FROM AUTHOR]

Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and immunogenicity, and in vivo transduction performance of the vector. It was reported that asparagine deamidation of AAV capsid proteins leads to charge variants/heterogeneity and altered vector function, reduction of stability, and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins and is mostly detected and quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based peptide mapping. However, deamidation can be spontaneously introduced during sample preparation before LC-MS/MS analysis. So far, no optimal sample preparations, instead, traditional sample preparation has been used for AAV VP peptide mapping, resulting in exaggerating the original deamidation levels. It is important to accurately monitor and provide true value of asparagine deamidation for development of AAV gene therapy products. In this study, we evaluated denaturation temperatures, digestion durations, and digestion temperatures using three different sample preparation formats for LC-MS/MS-based assessment of deamidation of AAV9 capsid proteins. The results demonstrated that the optimal sample preparation method for AAV9 VP peptide mapping minimized asparagine deamidation artifacts. Although AAV9 was used for method optimization, this study may also provide a guidance on how to control deamidation artifacts for other AAV serotypes. [ABSTRACT FROM AUTHOR]

The biopharmaceutical industry seeks to look for advanced analytical techniques to improve the quality of medicinal products. RP-UHPLC, or reverse phase ultra-high performance liquid chromatography, is a powerful tool for the precise detection of biological compounds. The objective of the study was to develop a precise and sensitive RP-UHPLC method for peptide mapping of semaglutide that resembles a glucagon-like peptide-1 receptor agonist used to treat type 2 diabetes mellitus. The method development phase involved the optimisation of fundamental parameters, including incubation time, column temperature, and enzyme volume to achieve reliable and precise peptide separation. The chromatographic setup comprised of Waters XBridge C18 ODS column. The RP-UHPLC method was established using a 1 mg/mL stock solution of Semaglutide. The results revealed that the optimal conditions established were 6 hours of incubation time with column temperature at 37°C and 20µL of Glu-C endopeptidase. Peptide mapping results demonstrate remarkable results with high accuracy of peak separation and retention time for six specific Semaglutide peptides. This robust analytical approach suggests valuable insights for the characterization of therapeutics in pharmaceutical formulations. Moreover, the potential applicability of this method for analyzing other therapeutic proteins reflects its importance in advancing the pharmaceutical research and development sector. [ABSTRACT FROM AUTHOR]

[Display omitted] Host cell proteins (HCPs) are process-related impurities generated during the production of biopharmaceuticals, which may contaminate the final product unless they are efficiently removed. Due to their potential impact on product safety, quality and efficacy, regulatory authorities require removal of HCPs during processing down to trace amounts in final manufactured biopharmaceuticals. The current standard method for detecting HCPs is enzyme-linked immunosorbent assay (ELISA), which should reveal the total amount of HCPs. A necessary orthogonal technique to get more granular information on HCPs is obtained by application of liquid chromatography-mass spectrometry (LC-MS) techniques that permit identification and quantification of individual HCPs. However, differences in sample preparation methods and MS acquisition techniques have led to discrepancies in detected HCPs between studies, which may compromise product safety, quality and efficacy. To address this issue, we have developed a novel and reproducible workflow for isolation, digestion, and mass spectrometry detection of HCPs that is applicable to downstream process characterization of therapeutic monoclonal antibodies (mAbs). This article describes a rapid and efficient workflow for the isolation, digestion and identification of HCPs. For the first time, Fc-receptor (FcγRIIIa) affinity chromatography is employed to isolate the HCP fraction from the mAb. Next, the HCPs are precipitated with acetone and digested using a newly developed "single-pot" method that improves digestion performance and prevents sample loss of problematic low-abundant HCPs. The new HCP isolation method outperforms protein A affinity chromatography for monitoring problematic HCPs. [ABSTRACT FROM AUTHOR]

Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33–40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies. [Display omitted] • Utilized silicon-based molecules as novel enzyme coupling agents. • Developed an automated liquid handling method for proteomics using an Agilent Bravo. • Trypsin IEPs achieved comparable results to in-gel while being faster and automatable. • Pepsin IEPs favored cleavage at Leu (>50%) over Phe, Tyr, Trp, and Met residues. • Demonstrated the potential of IMERs for 15-min high-throughput digestions. [ABSTRACT FROM AUTHOR]

The protruding domain (P-domain) of the murine norovirus (MNV) capsid protein VP1 is essential for infection. It mediates receptor binding and attachment of neutralizing antibodies. Protein NMR studies into interactions of the P-domain with ligands will yield insights not easily available from other biophysical techniques and will extend our understanding of MNV attachment to host cells. Such studies require at least partial NMR assignments. Here, we describe the assignment of about 70% of the Ala, Ile, LeuproS, Met, and ValproS methyl groups. An unfavorable distribution of methyl group resonance signals prevents complete assignment based exclusively on 4D HMQC-NOESY-HMQC experiments, yielding assignment of only 55 out of 100 methyl groups. Therefore, we created point mutants and measured pseudo contact shifts, extending and validating assignments based on methyl-methyl NOEs. Of note, the P-domains are present in two different forms caused by an approximate equal distribution of trans- and cis-configured proline residues in position 361. [ABSTRACT FROM AUTHOR]

Culture conditions have a profound impact on therapeutic protein production and glycosylation, a critical therapeutic-quality attribute, especially for monoclonal antibodies (mAbs). While the critical culture parameter of pH has been known since the early 1990s to affect protein glycosylation and production, detailed glycan and metabolic characterization and mechanistic understanding are critically lacking. Here, Chinese Hamster Ovary (CHO) cells were grown in bioreactors at pH 6.75, 7, and 7.25 (± 0.03) to examine how pH affects cell metabolism and site-specific N-linked glycosylation of the produced broadly neutralizing anti-HIV IgG1 mAb. VRC01 has N-linked glycosylation sites in both the Fc region and the Fab region, a situation not previously examined with respect to mAb glycosylation as affected by culture conditions. Using parsimonious Flux Balance Analysis (pFBA) and Flux Variability Analysis (FVA), we dissect and quantitate the impact of pH on cell growth, glucose/lactate metabolism, accumulation of the toxic metabolite ammonia, IgG production rates, and nonessential amino acid metabolism. pFBA revealed that beyond the established mechanism of glutamine conversion to glutamate, ammonia is also produced by the reaction converting serine to pyruvate, especially in the later phases of culture. pFBA also provided insights into the switch from ammonia production to consumption, notably due to depletion of glutamine, and consumption of glutamate and aspartate. We document that culture duration and pH alter the complex bimodal patterns (production/uptake) of several essential and non-essential amino acids. Site-specific N-linked glycan analysis using glycopeptide mapping demonstrated that pH significantly affects the glycosylation profiles of the two IgG1 sites. Fc region glycans were completely fucosylated but did not contain any sialylation. The Fab region glycans were not completely fucosylated but contained sialylated glycans. Bioreactor pH affected both the fucosylation and sialylation indexes in the Fab region and the galactosylation index of the Fc region. However, fucosylation in the Fc region was unaffected thus demonstrating that the effect of pH on site-specific N-linked glycosylation is complex., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Proteogenomics enables the confirmation and refinement of gene models, the detection of new ones, and the proposition of alternative transcripts using support at the protein level. Such evidence is usually generated using mass spectrometry and subsequent result mapping to various sequence databases. This workflow entails several problems: (1) To speed up the analysis, only a small set of expected proteins is searched; (2) database search tools generally do not provide mapping to the genome; and (3) upon new releases of the sequence databases, expensive rerunning of all results would need to be performed. Therefore, fast and accurate peptide mapping is needed as part of proteogenomic pipelines. Unfortunately, some available tools have technical shortcomings. Thus, a set of test cases was developed to allow tool developers to test their implementations comprehensively. The need for comprehensive testing is exemplified by PGx and PGM, two published tools that could only solve a subset of test cases. Lelantos passed all test cases. A set of comprehensive test cases has been developed to overcome these issues. Many unpublished peptide mapping tools are part of proteogenomic workflows, and such tools would also benefit from comprehensive testing. Finally, peptide mapping may also be crucial for proteomics because sequence databases change over time. In response, peptide remapping should be performed to ensure that peptides identifying a protein are proteotypic in a larger sequence context., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

Although re-sequencing of gene panels and mRNA expression profiling are now firmly established in clinical laboratories, in-depth proteome analysis has remained a niche technology, better suited for studying model systems rather than challenging materials such as clinical trial samples. To address this limitation, we have developed a novel and optimized platform called SP3-Clinical Tissue Proteomics (SP3-CTP) for in-depth proteome profiling of practical quantities of tumour tissues, including formalin fixed and paraffin embedded (FFPE). Using single 10 μm scrolls of clinical tumour blocks, we performed in-depth quantitative analyses of individual sections from ovarian tumours covering the high-grade serous, clear cell, and endometrioid histotypes. This examination enabled the generation of a novel high-resolution proteome map of ovarian cancer histotypes from clinical tissues. Comparison of the obtained proteome data with large-scale genome and transcriptome analyses validated the observed proteome biology for previously validated hallmarks of this disease, and also identified novel protein features. A tissue microarray analysis validated cystathionine gamma-lyase (CTH) as a novel clear cell carcinoma feature with potential clinical relevance. In addition to providing a milestone in the understanding of ovarian cancer biology, these results show that in-depth proteomic analysis of clinically annotated FFPE materials can be effectively used as a biomarker discovery tool and perhaps ultimately as a diagnostic approach.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development. [ABSTRACT FROM AUTHOR]

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO.

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 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 MSE 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. [ABSTRACT FROM AUTHOR]

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

ProLuCID, a new algorithm for peptide identification using tandem mass spectrometry and protein sequence databases has been developed. This algorithm uses a three tier scoring scheme. First, a binomial probability is used as a preliminary scoring scheme to select candidate peptides. The binomial probability scores generated by ProLuCID minimize molecular weight bias and are independent of database size. A modified cross-correlation score is calculated for each candidate peptide identified by the binomial probability. This cross-correlation scoring function models the isotopic distributions of fragment ions of candidate peptides which ultimately results in higher sensitivity and specificity than that obtained with the SEQUEST XCorr. Finally, ProLuCID uses the distribution of XCorr values for all of the selected candidate peptides to compute a Z score for the peptide hit with the highest XCorr. The ProLuCID Z score combines the discriminative power of XCorr and DeltaCN, the standard parameters for assessing the quality of the peptide identification using SEQUEST, and displays significant improvement in specificity over ProLuCID XCorr alone. ProLuCID is also able to take advantage of high resolution MS/MS spectra leading to further improvements in specificity when compared to low resolution tandem MS data. A comparison of filtered data searched with SEQUEST and ProLuCID using the same false discovery rate as estimated by a target-decoy database strategy, shows that ProLuCID was able to identify as many as 25% more proteins than SEQUEST. ProLuCID is implemented in Java and can be easily installed on a single computer or a computer cluster. This article is part of a Special Issue entitled: Computational Proteomics.

Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein-protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable. This article is part of a Special Issue entitled: Computational Proteomics.