Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins.

Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) have become a critical attribute monitored to evaluate product quality. Liquid chromatography-mass spectrometry (LC-MS) analysis of intact AAV VPs provides both quick and reliable serotype identification as well as proteoform information on each VP.

Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS.

Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5 min of analysis time.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc.

Mass spectrometry friendly pH-gradient anion exchange chromatography for the separation of full and empty adeno-associated virus (AAV) capsids.

The proportion of full and empty capsids represents a critical quality attribute of adeno-associated virus (AAV)-based therapeutics. In this study, pH-gradient anion exchange chromatography was utilized for the separation of full and empty capsid species. The developed method allowed for applicability to multiple AAV serotypes and facilitated subsequent mass spectrometric detection of intact AAVs.

Exploring Charge-Detection Mass Spectrometry on Chromatographic Time Scales.

Charge-detection mass spectrometry (CDMS) enables direct measurement of the charge of an ion alongside its mass-to-charge ratio. CDMS offers unique capabilities for the analysis of samples where isotopic resolution or the separation of charge states cannot be achieved, i.e.

SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS.

Residual host cell proteins (HCPs) represent a critical quality attribute of biotherapeutic drug products. Workflows enabling reliable HCP detection in monoclonal antibodies and recombinant proteins have been developed, which facilitated process optimization to improve product stability and safety, and allowed setting of acceptance limits for HCP content. However, the detection of HCPs in gene therapy products such as adeno-associated viral (AAV) vectors has been limited.

Biochemical and molecular characterization of sialylated cervical mucins in sheep†.

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway.

Rapid Analysis of Biotherapeutics Using Protein A Chromatography Coupled to Orbitrap Mass Spectrometry.

Monoclonal antibodies (mAbs) and related products undergo a wide range of modifications, many of which can often be directly associated to culture conditions during upstream processing. Ideally, such conditions should be monitored and fine-tuned based on real-time or close to real-time information obtained by the assessment of the product quality attribute (PQA) profile of the biopharmaceutical produced, which is the fundamental idea of process analytical technology. Therefore, methods that are simple, quick and robust, but sufficiently powerful, to allow for the generation of a comprehensive picture of the PQA profile of the protein of interest are required.

Inline electrochemical reduction of NISTmAb for middle-up subunit liquid chromatography-mass spectrometry analysis.

Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds.

2-Dimensional ultra-high performance liquid chromatography and DMT-MM derivatization paired with tandem mass spectrometry for comprehensive serum N-glycome characterization.

Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation.

Simultaneous monitoring of multiple attributes of pyrrolobenzodiazepine antibody-drug conjugates by size exclusion chromatography - high resolution mass spectrometry.

Antibody-drug conjugates (ADCs) are an emerging class of oncology treatments combining the unique specificity of monoclonal antibodies with the highly cytotoxic properties of small molecule compounds. Pyrrolobenzodiazepines (PBDs) are highly potent agents capable of inhibiting cellular DNA replication which leads to apoptosis. To ensure efficacy and patient safety upon administration of such toxic and heterogeneous molecules, their structure and quality attributes must be closely monitored.

Identification and characterization of O-linked glycans in cervical mucus as biomarkers of sperm transport: A novel sheep model.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen-thawed semen, which are due to reported differences in cervical sperm transport.

Native LC-MS for capturing quality attributes of biopharmaceuticals on the intact protein level.

Intact protein analysis by means of mass spectrometry has become a well-established method for the characterization of biotherapeutics. However, due to the highly complex nature of recombinant proteins, prior chromatographic separation is inevitable for a comprehensive analysis. In recent years, progress in coupling a variety of liquid chromatography-based native separation modes such as size exclusion, ion exchange and hydrophobic interaction chromatography to mass spectrometry (native LC-MS) has been reported, therefore allowing for rapid assessment of molecular mass and deep characterization of the heterogeneity of complex, recombinantly produced therapeutic proteins.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements.

Real-time characterization of mammalian cell culture bioprocesses by magnetic sector MS.

Mammalian cell culture processes were characterized upon the analysis of the exhaust-gas composition achieved through the on-line integration of a magnetic sector MS analyser with benchtop bioreactors. The non-invasive configuration of the magnetic sector MS provided continuous evaluation of the bioreactor's exhaust gas filter integrity and facilitated the accurate quantification of O2 and CO2 levels in the off-gas stream which ensured preserved bioreactor sterility prior to cell inoculation and provided evidence of the ongoing cellular respiratory activity throughout the cultures. Real-time determination of process parameters such as the Respiratory Quotient (RQ) allowed for precise pin-pointing of the occurrence of shifts in cellular metabolism which were correlated to depletion of key nutrients in the growth medium, demonstrating the suitability of this technology for tracking cell culture process performance.

Identification of additives in polymers from single-use bioprocessing bags by accelerated solvent extraction and ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry.

Single-use technologies are increasingly used in biopharmaceutical manufacturing. Despite their advantages, these plastic assemblies draw concern because they are a potential source of contamination due to extractable and leachable compounds (E&Ls). Characterising E&Ls from such materials is a necessary step in establishing their suitability for use.

Hypoxia Alters Epigenetic and -Glycosylation Profiles of Ovarian and Breast Cancer Cell Lines .

Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies.

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.

Comparative Elucidation of Cetuximab Heterogeneity on the Intact Protein Level by Cation Exchange Chromatography and Capillary Electrophoresis Coupled to Mass Spectrometry.

Charge sensitive separation methods such as ion exchange chromatography (CEX) and capillary electrophoresis (CE) have recently been coupled to mass spectrometry to facilitate high resolution profiling of proteoforms present within the charge variant profile of complex biopharmaceuticals. Here we apply pH gradient cation exchange chromatography and microfluidic capillary electrophoresis using the ZipChip platform for comparative characterization of the monoclonal antibody Cetuximab. Cetuximab harbors four glycans per molecule, two on each heavy chain, of which the Fab glycans have been reported to be complex and multiply sialylated.

Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry.

Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in O enriched water providing the possible quantification of site occupancy.

Sialic acid mediated mechanical activation of β adrenergic receptors by bacterial pili.

Meningococcus utilizes β-arrestin selective activation of endothelial cell β adrenergic receptor (βAR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that βAR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division.

Cracking Proteoform Complexity of Ovalbumin with Anion-Exchange Chromatography-High-Resolution Mass Spectrometry under Native Conditions.

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterization. Here, we report the development of a novel pH gradient-based anion-exchange chromatography method, which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterization of proteoforms at the intact protein level under native conditions.

Salivary N-glycosylation as a biomarker of oral cancer: A pilot study.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers.