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N-glycans represent the most common and abundant post-translational modification (PTM) in therapeutic antibodies, playing crucial roles in key functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Consequently, glycan profiling is regarded as a critical quality attribute (CQA) and is routinely performed to ensure antibody quality and consistency. The Rapi-Fluor method is a conventional standard for detailed glycan profiling, while intact mass analysis serves as a parallel CQA. However, the Rapi-Fluor method is a multi-step, time-consuming process that can limit high-throughput monitoring. In this study, we conducted a rigorous comparative validation of the Rapi-Fluor method and intact mass analysis for determining the glycan composition of ten therapeutic antibodies, comprising five original products and their biosimilars. Consistent with established findings, the biosimilars exhibited glycan compositions highly similar to their original counterparts. Furthermore, major glycans constituted over 85% of the total glycans across all samples. Crucially, the analytical comparison revealed highly congruent results between the Rapi-Fluor method and intact mass analysis, with quantitative differences in glycan composition being less than 10% across all ten therapeutic antibodies. This successfully demonstrates that intact mass analysis is a highly feasible, reliable, and significantly time-efficient alternative for rapidly and reliably assessing glycan composition, thereby accelerating quality control and process monitoring.

With the size of the biopharmaceutical market exponentially increasing, there is an aligned growth in the importance of data-rich analyses, not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships. In monoclonal antibodies, many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known. The importance of their function focuses analytical research efforts on the development of robust, accurate and fast methods to support drug development and quality control. Released N-glycan analysis is considered as the gold standard for glycosylation characterisation; however, it is not the only method for quantitative analysis of glycoform heterogeneity. In this study, ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies. While observing good comparability between the quantitative results generated, it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated. [Display omitted] •Comparative analysis of N-glycans present on four monoclonal antibodies using ten analytical methods reported.•Data presented to allow for impartial selection of appropriate analytical strategy depending on depth of information desired and ease of approach.•Methods included intact and subunit strategies and glycopeptide characterisation compared to released N-glycan as the current gold standard.

Microbial enzymes are increasingly finding applications as therapeutics due to their targeted activity and minimal side effects. Serratiopeptidase, also known as a miracle enzyme, has already proved its potential as an anti-inflammatory, mucolytic, fibrinolytic, analgesic in many studies. A cost effective, bioreactor level production process has been described here comprising of the fed-batch fermentation to produce recombinant serratiopeptidase protein expressed as a fusion construct. High yield of cell mass as well as protein was obtained by the optimization of bioreactor parameters. The downstream solubilization and purification processes were also optimized to achieve maximum yield of pure, active serratiopeptidase protein. A final yield of 2.5 ± 0.764 g L−1 of protein was obtained, having 8382 ± 291 U mg−1 of specific caseinolytic activity. Additionally, a novel, unexpected self-proteolytic activity of the enzyme that cleaves the N-terminal 6× His-SUMO fusion tag along with the enzyme propeptide, thus yielding a mature serratiopeptidase, was also found.

Diversity at the protein level accounts for much of our biological complexity. A proteoform family consists of all of the different forms of a protein (proteoforms) arising from a single gene, including sequence variants, splice variants, and posttranslationally modified forms. It is important to identify and quantify proteoforms to understand biological systems because different proteoforms from the same family can exhibit different functions. The established technique to identify proteoforms is top-down proteomics, where intact proteins are analyzed by mass spectrometry. Available top-down search software programs require quality fragmentation spectra to identify proteoforms, limiting the number of proteoforms that can be identified due to instrument time and spectral complexity. The subset of proteoforms identified is typically in the highly abundant and low molecular weight portion of the proteome. This dissertation describes the development of integrated proteomic strategies to address these challenges. We created a software program that constructs proteoform families by grouping together observed proteoforms based on differences in intact mass, enabling the identification of proteoforms by intact-mass analysis. Chapter 1 provides an overview of mass spectrometry-based proteomics and outlines current challenges in proteoform analysis. Chapter 2 describes the integration of proteoform family construction into a typical top-down proteomic workflow, resulting in a 40% increase in the number of proteoform identifications in an analysis of yeast lysate. Chapter 3 demonstrates the application of this integrated intact-mass and top-down strategy to identify and quantify murine mitochondrial proteoforms. Chapter 4 presents the construction of proteoform families in a human breast cancer cell line using data acquired on the 21 tesla FT-ICR mass spectrometry platform. Chapter 5 describes augmenting intact-mass analysis to determine candidate identifications for isotopically unresolved proteoforms, facilitating identification of human heart proteoforms > 50 kDa. Chapter 6 describes the integration of bottom-up peptide data and top-down proteoform data to improve proteoform identifications and to infer potential proteoform candidates with peptide-level evidence. Chapter 7 explains the research in this dissertation to a broader nonscientific audience. Finally, Chapter 8 discusses remaining challenges and future directions for integrated proteomic strategies towards the goal of comprehensive proteoform analysis.

This chapter contains sections titled: Introduction Comparative Sequence Analysis by MALDI‐TOF MS Applications of Nucleic Acid Analysis by MALDI‐TOF MS in Clinical Microbiology Conclusion References

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 μm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 μL at 1 mg mL−1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations.

This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC–MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC–MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird’s-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic. Graphical Abstract

Food poisoning outbreaks frequently involve staphylococcal enterotoxins (SEs). SEs include 33 distinct types and multiple sequence variants per SE type. Various mass spectrometry methods have been reported for the detection of SEs using a conventional bottom-up approach. However, the bottom-up approach cannot differentiate between all sequence variants due to partial sequence coverage, and it requires a long trypsin digestion time. While the alternative top-down approach can theoretically identify any sequence modifications, it generally provides lower sensitivity. In this study, we optimized top-down mass spectrometry conditions and incorporated a fully 15N-labeled SEA spiked early in the protocol to achieve sensitivity and repeatability comparable to bottom-up approaches. After robust immunoaffinity purification of the SEA, mass spectrometry signals were acquired on a Q-Orbitrap instrument operated in full-scan mode and targeted acquisition by parallel reaction monitoring (PRM), enabling the identification of sequence variants and precise quantification of SEA. The protocol was evaluated in liquid and solid dairy products and demonstrated detection limits of 0.5 ng/mL or ng/g in PRM and 1 ng/mL or ng/g in full-scan mode for milk and Roquefort cheese. The top-down method was successfully applied to various dairy products, allowing discrimination of contaminated versus non-contaminated food, quantification of SEA level and identification of the variant involved.

Glucosinolates (GSLs) are important precursor compounds with anticancer activities in Brassicaceae vegetables and are readily hydrolyzed by myrosinase. Given the diversity of these species, establishing an accurate and universal method to quantify intact GSLs in different plant tissues is necessary. Here, we compared and optimized three tissue disruption methods for sample preparation. After microwave treatment for 90 s, 13 GSLs in homogenized Chinese cabbage samples were recovered at 73–124%. However, a limitation of this method was that different tissues could not be processed under the same microwave conditions. Regarding universality, GSLs in Brassicaceae vegetables could be extracted from freeze-dried sample powder with 70% methanol (v/v) or frozen-fresh sample powder with 80% methanol (v/v). Moreover, heating extraction is necessary for GSLs extracted from frozen-fresh sample powder. Average recoveries of the two optimized methods were 74–119% with relative standard deviations ≤ 15%, with the limits of quantification 5.72–17.40 nmol/g dry weight and 0.80–1.43 nmol/g fresh weight, respectively. Notably, the method for analyzing intact GSLs was more efficient than that for desulfo-GSLs regarding operational complexity, detection speed and quantification accuracy. The developed method was applied to identify the characteristic GSLs in 15 Brassicaceae vegetables, providing a foundation for further research on GSLs.