Biopharma Analysis

137592 4 8 6 Biopharmaceuticals come in many forms, from monoclonal antibodies (mAbs) and recombinant therapeutic proteins, to DNA vaccines and cell therapies. They are important for combatting a whole host of diseases and keeping the population healthy. However, in order for scientists to develop and refine these therapeutics and ensure their safety and efficacy, it is important that they are analyzed at every stage of their lifecycle. This infographic will explore why it is important to analyze biopharmaceuticals, some of the methods commonly used and what they can tell us. The analysis of biopharmaceuticals is imperative at every stage of their lifecycle, from development and testing, through scale up and manufacture to shelf-life determination and storage. This helps to ensure quality, efficacy and safety, providing scientists with information on factors such as composition, structure and function. Biopharmaceuticals can come with a unique set of analytical challenges compared to small molecule pharmaceuticals related to their size, physical characteristics, micro-heterogeneity and the fact that many are derived from biological processes that can introduce their own contaminants, such as HCPs. Analytical techniques for biopharmaceuticals may vary depending on the specific product and the information needed. A combination of methods is typically used to characterize and control the quality of biopharmaceuticals throughout their development and production lifecycle. Let’s consider some of the commonly used techniques. Characterization Biopharmaceuticals often have complex structures, alterations of which can impact activity, effectiveness and safety. Characterization of structural properties such as protein folding, binding and post-translational modification (PTMs) such as glycosylation, phosphorylation and lipidation, is therefore vital from the early stages of candidate selection through to the analysis of final products. Biosimilarity If a new biopharmaceutical product is intended to be a substitute for an existing one (biosimilar), extensive analytical testing is required to demonstrate that it is a suitable alternative in terms of its similarity, safety and efficacy. Stability testing Environmental factors such as light, temperature and pH can all contribute to physical and chemical changes in a product. Monitoring over time helps to ensure it remains effective and safe for the duration of its intended shelf life. Batch-to-batch consistency As many biopharmaceuticals are produced in batches, it is important to check that there is no variation in aspects such as quality, potency and composition from one batch to the next in order to produce consistent results. Post-market surveillance Even after product launch, ongoing testing is necessary to assess the long-term safety and efficacy to identify unexpected issues or changes in product quality or longevity. Mass spectrometry Mass spectrometry (MS) measures the mass-to-charge ratio (m/z) of atoms and/ or molecules in a sample and can be used to determine the exact molecular weight of the components present, both desirable and undesirable. Matrix-assisted laser desorption/ionization MS (MALDI-MS) can provide information on the intact mass and composition of proteins and peptides, enabling simultaneous detection and identification. MALDI-time-of-flight (TOF)/TOF MS can be used to assess isomerization in biotherapeutics such as mAbs. Isomerization is a critical quality attribute (CQA) that requires careful control and monitoring during the discovery and production process, as unwanted structural changes can negatively impact potency, safety and efficacy. NMR spectroscopy Nuclear magnetic resonance (NMR) spectroscopy helps to elucidate the 3D structure of proteins and peptides, as well as study conformational changes and interactions with ligands or other molecules. UV-Vis spectroscopy Ultraviolet-visible (UV-Vis) spectroscopy is a popular technique based on the optical properties of species within a sample. It can offer insights on aspects such as purity, concentration and intermolecular interactions at all stages of development and manufacturing. Capillary electrophoresis Capillary electrophoresis (CE) separates charged molecules using an electric field, enabling the quantitative determination of biopharmaceuticals and identification of impurities. Microscopy Aggregates of biotherapeutics such as mAbs can be detected using microscopy techniques including optical, electron and atomic force microscopy. Cryo electron microscopy (cryo EM) helps to examine a broad range of biological structures, facilitating the visualization otherwise difficult or complicated biologics relevant to biopharmaceutical targeting and development. Liquid chromatography Various forms of liquid chromatography (LC) are used to analyze biopharmaceuticals, to determine molecular weight, identity, homogeneity and purity. These include: • Size-exclusion chromatography (SEC) • Ion-exchange chromatography (IEC) • Affinity chromatography Compared to other sample types, the conditions required for LC of biopharmaceuticals can be harsh in terms of factors such as pH and salt content. Therefore, the instruments used need to be sufficiently robust. Surface interactions can also be potentially problematic and therefore bio-inert coatings can help to prevent this. LC paired with MS (LC-MS) has become one of the essential tools for in-depth characterization of protein biopharmaceuticals. Immunoassays Immunoassays like the enzyme-linked immunosorbent assay (ELISA) and western blot can be used to quantify and detect the presence of specific proteins and/or antibodies, both desirable and undesirable. HCP contamination is routinely tested for using an ELISA. Immunochemical procedures such as these can help to establish the homogeneity, purity, identity and quantity of a therapeutic protein. Binding assays with therapeutic antibodies can also help to determine avidity, affinity, immunoreactivity and identify potential cross-reactivity. Polyacrylamide gel electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE can provide information on the molecular weight and size of the proteins present, assess purity, abundance and identify aggregation issues. Two-dimensional (2D) electrophoresis can be used to separate out proteins that comigrate, identifying potentially contaminating HCPs that may otherwise be masked by the target. X-ray crystallography X-ray crystallography can provide helpful structural insights for protein biotherapeutics; however, protein purity must be high to resolve structures successfully. Other useful techniques include: • Circular dichroism (CD) spectroscopy • Differential scanning calorimetry (DSC) • Analytical ultracentrifugation • Potency assays • Biological assays • Protein sequencing Pharmacokinetics and pharmacodynamics In order to pick the best candidates and optimize treatment and dosing regimens, understanding how the body interacts with biopharmaceuticals (pharmacokinetics), including how they are absorbed, distributed, metabolized and eliminated (ADME) and their effects on the body (pharmacodynamics) requires extensive analyses. Quality control Biologics must meet specific quality requirements to ensure they are free from contaminants and impurities, such as host cell proteins (HCPs), isoforms, degraded product and aggregates, which may otherwise impact product safety, performance and dosing. They must also have the intended composition and activity. Process optimization By monitoring key parameters, adjustments can be made to improve yield, reduce production costs and enhance quality and consistency. Regulatory compliance As part of the drug approval process, extensive analytical data is required by authorities such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), demonstrating they are safe for use and meet required standards. Why do we analyze biopharmaceuticals? What methods are used to analyze biopharmaceuticals and what do they tell us?