The multi-attribute method (MAM) is a liquid chromatography–mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on ...biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.
Accurate, reproducible, and fast quantification of N-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker ...discovery. Several methods exist for fluorescent labeling of N-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy, and expensive. Here we report an automated ultrafiltration-based N-glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 μg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform.
The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS)-based method that is used to directly characterize and monitor numerous product quality attributes (PQAs) at the ...amino acid level of a biopharmaceutical product. MAM enables identity testing based on primary sequence verification, detection and quantitation of post-translational modifications and impurities. This ability to simultaneously and directly determine PQAs of therapeutic proteins makes MAM a more informative, streamlined and productive workflow than conventional chromatographic and electrophoretic assays. MAM relies on proteolytic digestion of the sample followed by reversed-phase chromatographic separation and high-resolution LC-MS analysis in two phases. First, a discovery study to determine quality attributes for monitoring is followed by the creation of a targeted library based on high-resolution retention time plus accurate mass analysis. The second aspect of MAM is the monitoring phase based on the target peptide library and new peak detection using differential analysis of the data to determine the presence, absence or change of any species that might affect the activity or stability of the biotherapeutic. The sample preparation process takes between 90 and 120 min, whereas the time spent on instrumental and data analyses might vary from one to several days for different sample sizes, depending on the complexity of the molecule, the number of attributes to be monitored and the information to be detailed in the final report. MAM is developed to be used throughout the product life cycle, from process development through upstream and downstream processes to quality control release or under current good manufacturing practices regulations enforced by regulatory agencies.
•LC-MS monitoring of quality attributes in AAV-based gene therapy products.•Fast semi-automated peptide mapping method to obtain 100% sequence coverage of AAV5.•Monitoring PTMs of AAV5 using a 30 min ...pepsin digestion.•Challenging analysis of low concentrated AAV-samples are addressed by nanoflow LC-MS.•Automated workflow for studying sequence coverage and PTM in AAV serotypes.
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Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.
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. Incorporating these analytical strategies into rapid good manufacturing practice (GMP)-compliant workflows containing robust, but simplified, data processing methods is necessary to ensure effective product quality control (QC) during production. Here, we present a GMP-compliant LC–MS workflow for the rapid identification and in-depth characterization of AAVs. Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic acid as a mobile phase modifier is utilized to achieve the intact separation and identification of AAV VPs and their potential proteoforms. Peptide mapping is performed to confirm PTMs identified during intact VP analysis and for in-depth PTM characterization. The intact separations platform is then incorporated into a data processing workflow developed using GMP-compliant software capable of rapid AAV serotype identification and, if desired, specific serotype PTM monitoring and characterization. Such a platform provides product QC capabilities that are easily accessible in a regulatory setting.
Adalimumab drug product (Humira ®), the first fully human monoclonal antibody (mAb) approved by FDA in 2002, led the top ten list of best-selling mAbs in 2018 and has been the most profitable drug in ...the world. With the expiration of patent protection in Europe in 2018 and in United States by 2023, the landscape is changing as up to 10 adalimumab biosimilars are expected to enter the market in the US. Biosimilars offer the potential to lower costs on health care systems and increase patient accessibility. The analytical similarity of seven different adalimumab biosimilars was accomplished in the present study using the multi-attribute method (MAM), a LC-MS based peptide mapping technique that allows for primary sequence assessment and evaluation of multiple quality attributes including deamidation, oxidation, succinimide formation, N- and C- terminal composition and detailed N-glycosylation analysis. In the first step, characterization of the most relevant post-translational modifications of a reference product was attained during the discovery phase of MAM. During the second step, as part of the MAM targeted monitoring phase, adalimumab batch-to batch variability was evaluated to define statistical intervals for the establishment of similarity ranges. The third step describes biosimilarity evaluation of predefined quality attributes and new peak detection for the assessment of any new or modified peak compared to the reference product. This study highlights a new perspective of the MAM approach and its underlying power for biotherapeutic comparability exercises in addition to analytical characterization. MAM offers a streamlined comparability assessment workflow based on high-confidence quality attribute analysis using high-resolution accurate mass mass spectrometry (HRAM MS) and the capability to detect any new or modified peak compared to the reference product.
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•Adalimumab reference product patent expiration forces the need for a streamlined method to assess analytical comparability.•Multi-attribute method allows for the comparison of seven adalimumab biosimilars with high sensitivity and specificity.•Detailed N-glycosylation and 15 quality attributes are reported for adalimumab reference product in a single analysis.•The evaluation of batch-to-batch variability is the basis to establish the acceptance criteria for analytical comparability.
Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic ...digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.
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An operando dual-edge X-ray absorption spectroscopy on both transition-metal ordered and disordered LiNi0.5Mn1.5O4 during electrochemical delithiation and lithiation was carried out. The large data ...set was analyzed via a chemometric approach to gain reliable insights into the redox activity and the local structural changes of Ni and Mn throughout the electrochemical charge and discharge reaction. Our findings confirm that redox activity relies predominantly on the Ni2+/4+ redox couple involving a transient Ni3+ phase. Interestingly, a reversible minority contribution of Mn3+/4+ is also evinced in both LNMO materials. While the reaction steps and involved reactants of both ordered and disordered LNMO materials generally coincide, we highlight differences in terms of reaction dynamics as well as in local structural evolution induced by the TM ordering.
•Multiple chromatographic modes enable detailed correlative N-glycan analysis.•Cetuximab glycosylation strongly varies between murine, CHO and HEK expression systems.•Sialylation, galactosylation and ...high-mannose content among most striking differences.•CHO and HEK expression systems don’t express immunogenic glycan features.•Charge variant profiles strongly correlate with the degree of sialylation.
A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.
The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal ...immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5) were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.
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