Peptide Mapping Research Articles

Simple Endoglycosidase-assisted Peptide Mapping Workflow for Characterizing Non-consensus N-glycosylation in Therapeutic Monoclonal Antibodies

N-linked glycosylation, an extensively studied protein post-translational modification, was conventionally understood to occur at asparagine (Asn or N) sites with the consensus motif NXS/T, where X can be any amino acid residue except for proline, followed by serine or threonine. However, with advancements in characterization techniques and bioinformatic tools, increasing evidence indicates that Asn residues that are not located in the NXS/T consensus motif can also undergo N-glycosylation, which is also known as non-consensus or noncanonical N-glycosylation. Characterizing non-consensus N-glycosylation remains challenging because of the unpredictable sequon and its relatively low abundance. Here, we report an endoglycosidase-assisted peptide mapping workflow for mass spectrometry (MS) characterization of non-consensus N-glycosylation in monoclonal antibodies (mAbs). The feasibility of the workflow was demonstrated by a challenging case study, in which an atypical glycosite located within an NPNNXN sequence in a 25-residue tryptic peptide was identified in the fragment antigen-binding (Fab) region of a mAb. With the aids of endoglycosidase treatment, the resulting truncated glycan structures improved peptide ionization efficiency in MS and hence facilitated reliable quantitation of glycosite occupancy. Meanwhile, the remaining mono-/di-saccharides served as a large mass tag enabling differentiation between the glycopeptide and deamidated peptide, thus allowing for database searching for glycosite localization and semi-automation of the data processing workflow. This workflow offers a simple solution for characterizing non-consensus N-glycosylation for the development of therapeutic mAbs.

Flux balance analysis and peptide mapping elucidate the impact of bioreactor pH on Chinese hamster ovary (CHO) cell metabolism and N-linked glycosylation in the fab and Fc regions of the produced IgG

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.

Structural insight into recognition of Clostridioides difficile toxin A by novel neutralizing nanobodies targeting QTIN-like motifs within its receptor-binding domain

Clostridioides difficile causes a large proportion of nosocomial colon infections by producing toxins TcdA and TcdB as key virulence factors. TcdA and TcdB have analogous domain structures with a receptor-binding domain containing C-terminal combined repetitive oligopeptides (CROPs), an attractive target for the development of therapeutic antibodies. Here, we identify and characterize two potent neutralizing single-domain camelid anti-CROPsA antibodies, C4.2 and H5.2, with distinct mechanisms of action. Peptide mapping, high-resolution crystal structures and site-directed mutagenesis revealed that C4.2 and H5.2 nanobodies target the same C-terminal epitope centered on a 2667QTIN2670 motif, yet utilize different paratopes. Only for C4.2 is the complex geometry compatible with multisite binding using QTIN-like repeats throughout the CROPsA domain, as supported by Western blotting, ELISA, and SEC-MALS analysis. H5.2 binding is stronger and more selective for the C-terminal epitope than C4.2, although both nanobodies are sufficient to neutralize TcdA individually. The described epitope does not overlap with previously described epitopes of anti-CROPs antibodies and provides new modalities for disease treatment and diagnostics.

Exactly defined molecular weight poly(ethylene glycol) allows for facile identification of PEGylation sites on proteins

PEGylation (the covalent attachment of one or more poly(ethylene glycol) (PEG) units to a therapeutic) is a well-established technique in the pharmaceutical industry to increase blood-residence time and decrease immunogenicity. A challenging aspect of PEGylation is the dispersity of PEGylation agents, which results in batch-to-batch variations and analytical limitations. Herein, we present an approach to overcome these limitations by manufacturing a defined molecular weight (dispersity-free) PEGylation agent. We synthesise a defined molecular weight (Mw), linear 5 kDa methoxy-PEG (mPEG) active ester in an efficient and scalable manner using an iterative liquid-phase approach based on Nanostar Sieving. We then perform a comparative study on the random PEGylation and subsequent characterisation of the protein bovine serum albumin (BSA), using both the defined Mw, dispersity-free mPEG active ester, and a commercially available disperse 5 kDa mPEG active ester. We demonstrate that the defined Mw PEG both allows for facile monitoring of chemical modification reactions during the synthesis of the PEGylation agents, and facilitates straightforward identification of the PEGylated fragments within a PEGylated protein via a simple peptide mapping approach using UPLC-MS.

A Systematic Workflow for Fragmentation Identification of Therapeutic Antibodies by Liquid Chromatography-Mass Spectrometry.

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: G105-R106 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.

Limitation of anion exchange chromatography and potential application of hydrophobic interaction chromatography for monitoring AAV9 capsid degradation upon thermal stress

Adeno-Associated Virus (AAV) is often selected as the vector of choice for gene therapy due to its superior clinical performance compared to other gene delivery systems. Currently the characterization of AAV degradation, especially the chemical degradation of capsid, has been limited due to lack of suitable methods. Our study using AAV9 as a model molecule shows that anion exchange chromatography (AEX) as a charge-based separation method has limitations in monitoring the chemical degradation of AAV9 capsid due to a confounding effect from DNA cargo ejection. We developed a hydrophobic interaction chromatography (HIC) method, free from DNA interference, that could serve as a quick and reliable alternative to resource-demanding peptide mapping method for monitoring AAV capsid chemical degradation. Compared with brief thermal stress at 75 °C, AAV9 capsid exhibited much higher levels of chemical degradation but slower capsid titer loss upon extended exposure for 4 weeks at 40 °C.

Multi-attribute method analysis of therapeutic monoclonal antibodies using an automated sample preparation system

The multi-attribute method (MAM) has attracted increased attention as an alternative strategy for evaluating structural heterogeneity using conventional separation techniques such as ion-exchange chromatography and capillary electrophoresis of therapeutic monoclonal antibodies (mAbs). One of the remaining challenges for the practical use of the MAM is reliable and robust continuous monitoring. In this study, we successfully established an automated sample preparation system as a solution to this issue. Through method optimization, we confirmed that the peptide purification step using a solid-phase extraction column, which is usually performed after the digestion step, was not mandatory and that the addition of methionine as an oxidation inhibitor was able to significantly reduce artificial oxidation. Importantly, the use of our system enabled high-precision analysis for targeted peptide monitoring without relying on the operator’s knowledge and experience with peptide mapping using liquid chromatography/mass spectrometry (LC/MS). Our system could also be useful as a platform approach for targeted peptide monitoring in MAM workflow of traditional IgG-type mAbs. Furthermore, using common samples that were prepared using the automated system, we assessed the compatibility of LC/MS system in the targeted peptide monitoring via a collaborative study with MS vendors. The results showed that there was no significant difference in the mass accuracy, repeatability of the peak retention time and variations of intermediate precision of the measurement values among the four LC/MS systems, suggesting sufficient compatibility among the LC/MS systems. MAM system using our automated sample preparation method, which had high intermediate precision, will be useful for release and stability testing as well as characterization and manufacturing process development.

Evaluating the potential of hydrophilic interaction liquid chromatography for collagen peptide mapping analysis

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.

MALDI MSI Protocol for Spatial Bottom-Up Proteomics at Single-Cell Resolution.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) started with spatial mapping of peptides and proteins. Since then, numerous bottom-up protocols have been developed. However, achievable spatial resolution and sample preparation with many wet steps hindered the development of single cell-level workflows for bottom-up spatial proteomics. This study presents a protocol optimized for MALDI-MSI measurements of single cells within the context of their 2D culture. Sublimation of CHCA, followed by a dip in ice-cold ammonium phosphate monobasic (AmP), produced peptide-rich mass spectra while maintaining matrix crystal sizes around 400 nm. This enables MALDI-MSI imaging of proteins in single cells grown on an ITO slide with a throughput of approximately 7800 cells per day. 89 peptide-like features corresponding to a single MDA-MB-231 breast cancer cell were detected. Furthermore, by combining the MALDI-MSI data with LC-MS/MS data obtained on cell pellets, we have successfully identified 24 peptides corresponding to 17 proteins, including actin, vimentin, and transgelin-2.

Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping.

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.

Biomineralized Nuclear Receptor Protein-Selection Mass Spectrometry for the Discovery of Drugs and Toxics.

Protein-selection mass spectrometry is cost-effective for the discovery of drugs and toxics. Nuclear receptors (NRs) are major targets for pharmaceuticals and endocrine-disrupting chemicals and are, thus, widely used as "bait" proteins. However, their application is limited due to the tendency to lose protein activity during cold storage. To address this problem, we introduced a novel biomineralization-based approach to preserve activity in NRs, exemplified by human retinoic acid receptor alpha (hRARα), a target for cancer and leucocythemia therapy. Since information on the coordination chemistry of metal ion and NR protein complexes is almost unavailable, we applied peptide mapping analysis for the first time for the rational design of his-hRARα-Co phosphate nanobiomaterial with high bioactivity. This nanobiomaterial successfully captured hRARα bioactive chemicals from a Chinese herb and environmental water and discovered an unsaturated fatty acid, (±)-(9Z,11E)-13-hydroxy-9,11-octadecadienoic acid ((±)13-HODE), which exhibited strong hRARα antagonistic activity.

Identification of a Novel Transasparaginase Activity of Bacillus subtilis (bTG) for Sequence-Specific Bioconjugation.

The ability of Bacillus subtilis transglutaminase (bTG) to functionalize BSA has been investigated using peptide mapping experiments. Interestingly, the conjugation was not detected on a glutamine but on an asparagine residue. A sequence determination study was further performed, and a sequence of 10 amino acids for site-specific conjugation was identified. A monobody showing no native reactivity with the bTG enzyme was produced with the identified peptide sequences and successfully conjugated to various types of substrates in very high yields (>90%) with a 1/1/1.5 ratio of protein/amine/enzyme. Direct conjugation to the amino linker of a small interfering RNA (siRNA) was achieved in good yield, and no impact on the siRNA activity was observed following the conjugation. The identified sequences were further engineered in VHH and IgG scaffolds, and successful conjugation could also be observed with both small molecules and siRNA, confirming the potential of bTG for site-specific enzymatic bioconjugation.

An Integrated Strategy to Identify Tyrosine Sulfation from the Therapeutic Proteins.

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.

Human milk vs. Infant formula digestive fate: In vitro dynamic digestion and in vivo mini-piglet models lead to similar conclusions

Infant formula (IF), the only nutritionally adequate substitute for human milk (HM), still needs to be improved to be more biomimetic with HM, including in terms of digestive fate. The latter can be explored using different digestion models. The present study aimed to compare IF and HM digestion using in vivo (mini-piglet) and in vitro (dynamic system, DIDGI®) models. Fresh mature HM was collected and compared with a standard bovine IF. In vivo, 18 Yucatan mini-piglets (24-day-old) received HM or IF and were euthanized 30 min after the last meal. The entire digestive content was collected from the stomach to the colon. In vitro, the same meals were fed to an in vitro dynamic digestion model simulating the term infant at four weeks of age. Digesta were sampled regularly in the gastric and intestinal compartments. Structure (confocal microscopy and laser light scattering) and proteolysis (SDS-PAGE for residual intact proteins, OPA for hydrolysis degree, LC-MS/MS for peptides) were investigated along digestion. The digesta microstructure differed between HM and IF in a similar way between in vitro and in vivo digestion. In vitro gastric proteolysis of caseins and α-lactalbumin was significantly slower for HM than for IF, such as for the early intestinal proteolysis degree. In vitro bioaccessibility of free AAs explained only 30 % of the true ileal digestibility of AAs. Peptide mapping of caseins differed between HM and IF along their digestion. The relative peptide mapping data over six proteins from HM and IF were highly correlated between in vitro and in vivo digestion, particularly at 80 and 120 min of in vitro gastric digestion vs. in vivo stomach data and at 20 and 40 min of in vitro intestinal digestion vs. in vivo proximal jejunum data (r = 0.7–0.9, p < 0.0001, n = 1604). 40 to 50 % of the bioactive peptides identified in vivo were also found in vitro, with a good correlation of their abundances (r = 0.5, p < 0.0001, n = 61). Overall, in vitro and in vivo digestion were in good agreement, both indicating a different digestive fate for HM and IF.

The Accurate Prediction of Antibody Deamidations by Combining High-Throughput Automated Peptide Mapping and Protein Language Model-Based Deep Learning.

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.

In-depth characterization of a cysteine-linked ADC disitamab vedotin by multiple LC-MS analysis methods and cutting-edge imaged capillary isoelectric focusing coupled with native mass spectrometry

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.

Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues.

Mass spectrometry imaging (MSI) is a powerful technique for label-free spatial mapping of multiple classes of biomolecules in tissue sections. However, differences in desorption and ionization efficiency of different classes of molecules make it challenging to simultaneously map biomolecules at each omics layer in the same tissue sample. Herein, we present a correlative imaging method using nanospray desorption electrospray ionization (nano-DESI) MSI, which enables the spatial mapping of lipids, metabolites, peptides, and proteins with cellular-level spatial resolution in a single tissue section. We demonstrate the molecular profiling of specific cell types and identify truncated peptides in mouse pancreatic tissue. Distinct chemical gradients of peptides and lipids extending from endocrine cells to exocrine cells indicate their different roles in endocrine-exocrine crosstalk and intracellular signaling. The results underscore the power of the developed imaging approach for spatial multi-omics analysis that provides deep insights into cellular diversity and the intricate molecular interactions that occur within heterogenous biological tissues.

Degradomics defines proteolysis information flow from human knee osteoarthritis cartilage to matched synovial fluid and the contributions of secreted proteases ADAMTS5, MMP13 and CMA1 to articular cartilage breakdown

ObjectivesProteolytic cartilage extracellular matrix breakdown is a major mechanism of articular cartilage loss in osteoarthritis (OA) pathogenesis. We sought to determine the overlap of proteolytic peptides in matched knee OA cartilage and synovial fluid on a proteome-wide scale to increase the prospective biomarker repertoire and to attribute proteolytic cleavages to specific secreted proteases. DesignMatched human knee OA cartilage and synovial fluid (n=5) were analyzed by N-terminomics using Terminal Amine Isotopic Labeling of Substrates (TAILS), comprising labeling and enrichment of protein N-termini, high-resolution mass spectrometry and positional peptide mapping. Donor non-OA articular cartilage was digested with CMA1, MMP13 or ADAMTS5, and TAILS was used to identify cleavage sites, which were matched against cartilage and synovial fluid degradomes. ResultsOf over 20,000 cleaved peptides in the combined OA cartilage and synovial fluid degradomes, 677 peptides, originating from 153 proteins, were present in all cartilage and synovial fluid samples. CMA1, MMP13 and ADAMTS5 digestion of cartilage identified numerous cleavage sites for each protease and distinct cleavage site preferences. Peptides resulting from the activities of these proteases were detected in OA cartilage and synovial fluid. ConclusionsProteolytic fragments from both cartilage and circulating proteins are detectable by synovial fluid degradomics. CMA1, MMP13 and ADAMTS5 activity profiles in cartilage are distinct from each other and the previously determined HtrA1 profile. This work expands the proteolytic biomarker space for OA investigation, suggests that multiple, diverse proteases contribute to cartilage destruction, and demonstrates that their specific contributions can each be defined by multiple biomarkers.

Analytical Investigation of the Profile of Human Chorionic Gonadotropin in Highly Purified Human Menopausal Gonadotrophin Preparations.

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.