Applications of Mass Spectrometry in Biopharmaceutical Analysis

Article 1 Applications of Mass Spectrometry in Biopharmaceutical Analysis Srividya Kailasam, PhD The complexity and heterogeneity of biopharmaceuticals make it challenging to manufacture, screen and develop them. Critical quality attributes (CQAs) such as post-translational modifications (PTMs) – an example being isomerization of aspartic acid to iso-aspartic acid – can impact product quality, and must be identified and characterized throughout the drug discovery and development pipeline. Analytical techniques such as mass spectrometry (MS) are employed to ensure the safety and efficacy of biologics. The sensitivity and specificity of MS has made it an indispensable tool for biopharmaceutical analysis, including the characterization of biologics and higher order structure (HOS) analysis. The introduction of soft ionization techniques and numerous advances in the instrumentation have led to development of methods for quantification, quality control, biosimilarity assessment and numerous other analyses of biologics.1 “MS provides accurate mass of the biomolecules with critical PTMs, including glycans, generated during manufacturing, processing or chemical modification during handling or storge,” says Dr. Anurag Rathore, professor in the Department of Chemical Engineering, Indian Institute of Technology. Discussing the utility of MS, Dr. Rathore shares a research example from his lab. Charge heterogeneity of monoclonal antibodies (mAbs) – therapeutics often used in cancer treatment – is classified as a CQA. “In a study, we have demonstrated a method in which charge heterogeneity of mAbs were identified by native MS,” he describes.2 Dr. Jared Auclair, director of Biotechnology and Informatics, and director of the Biopharmaceutical Analysis Training Laboratory at Northeastern University, in Boston, Massachusetts, echoes Rathore’s thoughts: “One of the most predominant advantages of using MS for biopharmaceutical analysis is being able to identify the primary amino acid structure of the product as well as the specific PTMs present and the sites of these PTMs (e.g., glycosylation, deamidation and oxidation),” he says. “The sensitivity and specificity that MS offers is a key advantage as well,” Auclair adds. “New methodologies such as the Multi-Attribute Method (MAM) allow for sequences, PTMs and New Peak Detection (NPD) to be uncovered. NPD can give insight into changes to the biopharmaceutical, as well as impurities present. We use MS in the lab to look at host cell protein profiles as well as to try to improve the MAM process.” Supporting Mass Spectrometry Analysis with Magnetic Bead Technology APPLICATIONS OF MASS SPECTROMETRY IN BIOPHARMACEUTICAL ANALYSIS 2 Article Sera-Mag™ Carboxylate-Modified Magnetic Particles can be used in protein and peptide single-pot solid-phase-enhanced sample preparation (SP3) - a novel technique that takes advantage of these features to enable the fast and efficient removal of a wide range of detergents, salts and other contaminants from protein and peptide samples prior to mass spectrometry analysis. Ubiquity of mass spectrometry in biopharmaceutical analysis Different types of MS such as QTOF, triple quadrupole (TQ), MALDI have been used for biopharmaceutical analysis during drug discovery, development, for ADME studies, quantification, impurity profiling, bioanalysis and even clinical analysis. Some examples of the applications of MS-based techniques for the analysis of biotherapeutics during the various stages of their life cycle are listed below: Drug discovery and development MS is a sensitive technique for studying the structure, quality, efficacy and stability of therapeutic proteins such as antibodies during the discovery phase. It is also an effective tool to understand the underlying disease mechanisms and identify putative drug targets by studying the interactions between bacterial or viral proteins with host cell proteins. Testing existing drugs, or repurposed drugs, against newly identified targets can also accelerate therapeutic development timelines and reduce costs.3 Another example of an application of MS for drug discovery would be the use of MALDI-TOF/TOF MS to identify the exact position of iso aspartic acid residues in peptide standards as well, as a therapeutic mAb that is known to contain an isoaspartic acid residue in its heavy chain.4 It is well known that aspartic acid isomerization to isoaspartic acid is a critical quality attribute (CQA) that is monitored and controlled during drug discovery. ADME studies MS can be used to understand absorption, distribution, metabolism and excretion (ADME) of drug candidates. Desorption electrospray ionization (DESI) followed by mass spectral imaging (MSI) and MALDI-MSI have been applied to study the spatial distribution and metabolism of the immunosuppressant cyclosporine (CsA) – cyclic undecapeptide – and its metabolites in whole-body mouse sections and rat organs. As MSI is a label-free technique, it is highly suitable for ADME studies as it can be used to simultaneously detect the parent drug and its metabolites.5 Bioanalysis of Small Molecule Pharmaceuticals Using Accurate Mass Spectrometry Download this technical note to learn more about a system that can perform sensitive quantification of small molecule pharmaceutical compounds, achieve outstanding accuracy, precision and linearity and increase productivity. Quantification Quadrupole-time of flight MS coupled to a liquid chromatograph has been used to quantitate 22-KDa somatropin a recombinant human growth hormone, down to 10 ppb level in 75 μL rat plasma. A good correlation was observed for plasma concentration measured by deconvolution approach as well as summation of the responses of the four most intense charge states (14+ to 17+) of somatropin.6 Multiplexed site-specific PTMs quantification of mAbs in forced degradation samples, comparability samples APPLICATIONS OF MASS SPECTROMETRY IN BIOPHARMACEUTICAL ANALYSIS 3 Article and trisulfide standards of mAbs has been achieved using tandem mass tag (TMT)-based approach and targeted MS.7 Quality analysis The high resolution and high mass accuracy of QTOF MS has been leveraged to develop a MAM to study the safety, efficacy and quality of mAbs. Using a single workflow to monitor glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation is more time and cost efficient as compared to performing multiple orthogonal assays.8 The application of capillary electrophoresis (CE) hyphenated to MS for intact mass analysis of mAbs and fusion proteins, and a biotransformation study of two Fc- FGF21 molecules in a single-dose pharmacokinetic mice study, has been described.9 Impurity profiling MS based methods are used for ascertaining the quality of biopharmaceuticals by determining PTMs, quantifying host cell protein impurities, ensuring glycosylation consistency etc.10 Shotgun MS analysis and ELISA have been used to evaluate upand down-stream process parameters of four different biopharmaceutical products, two different process variants and one mock fermentation by identifying and quantifying HCP.11 Structure–function relationship As glycosylation of therapeutic proteins impacts their safety and efficacy, it is necessary to characterize the glycoproteins. High-resolution native MS can not only uncover the unique features of complex glycan, but also help ascertain the correlation between the glycoprotein structure and their cellular functions. 12 An LC-MS/MS Toxicology Workflow for Screening Drugs Download this app note to discover a workflow that can be used to Separate isomers of interest in forensic toxicology, Screen, identify, confirm and quantitate with one instrument and analyze compounds of different drug classes with a wide range of hydrophobicities, positive and negative polarities. Bioanalysis A triple quadrupole tandem MS coupled with ultra-high-pressure liquid chromatography (UHPLC) has been applied to quantify pasireotide, a cyclic hexapeptide administered to treat Cushing’s disease and acromegaly. The developed method was applied for the bioanalysis of pasireotide in plasma and the lower limit of quantification (LLOQ) was found to be 5 pg/mL.13 Clinical analysis An example of the application of MS for clinical analysis would be a hybrid of ligand-binding assay (LBA)- with three multiplex LC-MS/MS that has been developed and validated for the analysis of total antibody and antibody-drug conjugate MEDI4276 in human plasma. This method has been applied for a first-inhuman clinical trial.14 APPLICATIONS OF MASS SPECTROMETRY IN BIOPHARMACEUTICAL ANALYSIS 4 Article Advantages and limitations of using MS Parts per billion or lower sensitivity, high specificity, high resolving power of certain mass spectrometers, dynamic range, availability in a variety of formats, and the ability to couple it with different separation techniques make MS a versatile tool for the analysis of biopharmaceuticals. Developments in MS hardware and techniques such as hydrogen/deuterium exchange (HDX)-MS, and fast photochemical oxidation of proteins (FPOP) have been harnessed to characterize the higher order structures of biotherapeutics, including epitope mapping, aggregation assessment and comparability studies.15 Yet, MS-based biopharma analysis can be challenging due to a variety of factors, including extensive sample preparation, limited throughput and the need for skilled operators.16 “When performing analysis of biomolecules under denaturing conditions, loss of information on native structure of proteins occurs due to disruption of the weak non-covalent interactions, which is a major concern,” Rathore describes. “Although, native MS has been introduced, there remains a challenge when using MScompatible buffers due to the poor ionization of biomolecules in these buffers.” In Auclair’s mind, the bottleneck associated with using MS-based biopharma analysis relate to its complexity. “The biggest challenge is the complexity of the instrumentation and the potential data analysis. Also, the cost can be a roadblock to MS use,” he says. ICP-MS Analysis of Elemental Impurities in Pharmaceutical Products Download this free compendium to learn more about the analytical procedures used to validate ICP-MS for elemental analysis, ICP-MS analysis for cutaneous and transcutaneous drug product and the impact of ICP-MS on heavy metal testing. Future opportunities Commenting on the technological developments that have had the biggest impact on biopharmaceutical analysis, Dr. Rathore says, “Major developments have occurred in ion-mobility MS and mass analyzers for structural elucidation of glycan isomers and complex protein using native MS.” He also describes MAM methods that allow analysis of various CQAs of biotherapeutics in a single analysis as the most promising development in the horizon for biopharma analysis using MS. Auclair expresses similar views: “I think that MAM has had a significant impact on biopharmaceutical analysis allowing for multiple CQAs to be monitored at once. I also think the new instrumentation is becoming more accessible and less complex,” he says. It has been suggested that MS-based approaches can support Quality by Design (QbD) implementation, as it provides greater product and process understanding in addition to enhancing the efficiency of analysis.17 When asked about how developments such as automation, the use of artificial intelligence (AI) and QbD support the application of MS for biopharma analysis, Rathore says, “These developments will lead to reduced cost, time and errors in the analysis and improved reproducibility and robustness. They will also help in real-time monitoring of significant deviation in the CQAs”. Auclair explains that QbD is a key tool in any drug development program: “Instituting quality practices at the beginning of a process will help in eventually commercializing the product (e.g., faster, cheaper but still safe, effective and of good quality). MS allows for multiple CQAs to be analyzed in a QbD process.” Novel bioinformatic tools with AI capabilities could also support future advancements in data handling for MS-based biopharmaceutical analysis. Auclair says: “Bioinformatics tools, essentially those with AI/ ML capabilities, could allow for real-time data analysis and for process changes to be implemented to APPLICATIONS OF MASS SPECTROMETRY IN BIOPHARMACEUTICAL ANALYSIS 5 Article ensure the best quality product is manufactured.” As to the unmet needs remaining in MS-based biopharmaceutical analysis, Auclair concludes, “I think additional needs exist around automation, flexibility and integration. There is a significant opportunity in the process analytical technologies (PAT) space to provide real-time data analysis and implementation of process changes. Lastly, I think biopharmaceutical analysis will evolve as more complex products come to market (e.g., cell and gene therapies).” References 1. Apostol I, Bondarenkon PV, Ren D, et al. Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry. Curr Opin in Biotech. 2021;71: 206-215. doi: 10.1016/j.copbio.2021.08.001 2. Sarin D, Kumar S, Rathore AS. Multi-attribute monitoring of charge-based heterogeneity of recombinant monoclonal antibodies using 2D HIC-WCX-MS. Anal Chem. 2022;94(43):15018-15026. doi: 10.1021/acs.analchem.2c02931 3. Terracciano R, Preianò M, Fregola A, Pelaia C, Montalcini T, Savino R. 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