Multi-Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC.

The multi-attribute method (MAM) has emerged significantly in recent years to support biotherapeutic protein characterization from process development to the QC environment. MAM is a liquid chromatography mass spectrometry (LC-MS) based peptide mapping approach, which combines the benefits from liquid chromatography coupled to high resolution accurate mass mass spectrometry (LC-HRAM MS), enabling direct assessment of protein sequence and product quality attributes with site specificity. These product quality attributes may impact efficacy, safety, stability, and process robustness. MAM is intended to replace conventional analytical approaches as it offers a more streamlined strategy for parallel monitoring of multiple attributes in a single analysis with high sensitivity and confidence, and ultimately supports more robust Quality by Design (QbD) approaches and faster decision cycles for biotherapeutic development. MAM consists of three main stages. The first stage is sample digestion, which typically entails proteolytic digestion of the protein. The second stage is reversed-phase chromatographic separation of the generated peptides and detection by HRAM MS in two phases. During MAM Phase I (discovery phase), data-dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide workbook. During MAM Phase II (monitoring phase), full MS acquisition is only carried out for the monitoring of predefined product quality attributes (PQAs). The third stage is data processing, which entails analysis and reporting for each of the two phases including evaluation of sequence coverage, assessment of PQAs and peptide workbook creation during phase I, and targeted monitoring of predefined product attributes and new peak detection (NPD) during phase II. The latter is a comparative analysis that uses a base peak alignment algorithm to determine any non-monitored differences between the LC-MS chromatograms of a test sample and a reference standard. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In-solution sample digestion Alternate Protocol: Automated sample digestion Basic Protocol 2: Reversed-phase chromatographic separation and detection by HRAM-MS (RPLC-HRAM MS) Basic Protocol 3: Data processing and reporting.

Multi-Attribute Method (MAM): An Emerging Analytical Workflow for Biopharmaceutical Characterization, Batch Release and cGMP Purity Testing at the Peptide and Intact Protein Level.

The rapid growth of biotherapeutic industry, with more and more complex molecules entering the market, forces the need for advanced analytical platforms that can quickly and accurately identify and quantify product quality attributes. Mass spectrometry has the potential to provide more detailed information about the quality attributes of complex products, and MS methods are more sensitive than UV methods for detection of impurities. The multi-attribute method (MAM), a liquid chromatography-mass spectrometry based analytical approach is an emerging platform which supports biotherapeutic characterization and cGMP testing. The main advantage lies in the ability to monitor multiple quality attributes in a single assay, both at the peptide and the intact level, facilitating streamlined biopharmaceutical production, from research and development to the QC environment. This review highlights the current landscape of the MAM approach with special attention given to increased analytical throughput, general requirements for QC in terms of instrumentation and software, regulatory requirements, and industry acceptance of the MAM platform.

Exploring proteoforms of the IgG2 monoclonal antibody panitumumab using microchip capillary electrophoresis-mass spectrometry.

The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity. Microchip capillary electrophoresis-mass spectrometry (CE-MS) was subsequently utilised for a more detailed characterization. It was observed that panitumumab is subject to partial N-terminal pyroglutamate formation. Incomplete conversion is uncharacteristic for N-terminally exposed glutamines and in case of panitumumab gives rise to forms which show successive mass offsets of 17 Da, respectively. If not separated before mass spectrometric analysis, e.g. by capillary electrophoresis, such near isobaric species coalesce into single MS peaks, which subsequently hampers or prevents their assignment. With a total of 42 panitumumab isoforms assigned by CE-MS, these observations highlight a potential pitfall of commonly applied rapid identity testing workflows and demonstrate that even low complexity biopharmaceuticals can require separation strategies which offer high separation selectivity for species close in mass.

Multi-attribute method (MAM) to assess analytical comparability of adalimumab biosimilars.

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.

Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing.

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.

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. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.

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. A major obstacle for the analysis of proteins directly from cultures is the presence of impurities such as cell debris, host cell DNA, proteins and small-molecule compounds, which usually requires a series of capture and polishing steps using affinity and ion-exchange chromatography before characterization can be attempted. In the current study, we demonstrate direct coupling of protein A affinity chromatography with native mass spectrometry (ProA-MS) for development of a robust method that can be used to generate information on the PQA profile of mAbs and related products in as little as 5 min. The developed method was applied to several samples ranging in complexity and stability, such as simple and more complex monoclonal antibodies, as well as cysteine-conjugated antibody-drug conjugate mimics. Moreover, the method demonstrated suitability for the analysis of protein amounts of <1 µg, which suggests applicability during early-stage development activities.

Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow.

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.

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS.

Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies (mAbs) remains a challenge. Since residual HCPs might affect product stability or safety, constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg. The current standard analytical approach for this procedure is based on ELISA; however, this approach only measures the overall HCP content. Therefore, the use of orthogonal methods, such as liquid chromatography-mass spectrometry (LC-MS), has been established, as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present. In the present study, a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation, in combination with a data-independent acquisition (DIA) LC-MS analysis, was established. Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows, avoiding the need for dedicated instrumentation or personnel. Thereby, quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS / 药物分析学报

Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant pro-teins such as monoclonal antibodies (mAbs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry (LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the indi-vidual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisi-tion (DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes.

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. Graphical abstract.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry.

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

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. 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.

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. The presence of these extra glycosylation sites in the variable region of the molecule causes significant charge variant and glycan heterogeneity, which makes comprehensive analysis on the intact protein level challenging. Both pH gradient CEX-MS and CE-MS were found to be powerful for the separation of Cetuximab charge variants with eight major peaks being baseline resolved using both separation platforms. Informative native-like mass spectra were collected for each charge variant peak using both platforms that facilitated deconvolution and further analysis. The total proteoform coverage was exceptionally high with >100 isoforms identified and relatively quantified with CEX-MS, in case of CE-MS the proteoform coverage was >200. A deep insight into the heterogeneity of Cetuximab was unveiled, the high level of sensitivity achievable enables the implementation of the presented technologies even at early stages of the biopharmaceutical development platform, such as in developability assessment, process development and also for monitoring process consistency.

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 18O enriched water providing the possible quantification of site occupancy. 2-AA labelled glycan structures were annotated by CE-MS profiling in combination with exoglycosidase sequencing, revealing potential structures with terminal α-galactose and N-glycolyl-neuraminic acid (NGNA) mainly originating from the Fd fragment. Glycosylation analysis was also performed on different charge variants of Cetuximab that were separated using pH gradient cation-exchange chromatography to investigate the impact of glycosylation on the net charge of the protein.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies / 药物分析学报

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 ac-cording to application and the desired depth of data generated.