Evaluating and characterising complicated protein structures and the therapeutic activity of advanced biotherapeutic molecules requires sensitive analytical technologies and informatics systems. As such, executing accurate, analytical assessments of complex biological molecules in a commercial laboratory setting can be challenging, explains Eliza Lee, Lead Scientist Analytics, Samsung Biologics
Today’s researchers require increasingly thorough assessments and analyses to bring their molecules to patients and markets in a compliant way. Fortunately, advanced analytical technologies now offer the insight and actionable data they need to ensure programme success.
To meet the demand for high-fidelity data, analytical technologies have advanced, making these assessments more effective and cost-efficient. In this article, these analytical technologies and their evolution will be examined.
Biopharmaceutical developers face many analytical challenges, including how to evaluate potential functionality of the molecule in vivo. At the cellular and immune system levels, complex biological molecules work within sophisticated, feedback-controlled chemical reactions. Analytical assessments therefore depend on a variety of functional assays to determine whether the molecule appropriately stimulates these systems (and relates to potency in vivo).
Best practice for researchers during early phase development is to begin assessments with an enzyme-linked immunosorbent assay (ELISA) to determine the binding potency of their biological molecule.1 ELISA is a plate-based technique that’s designed to detect and quantify soluble substances such as peptides, proteins, antibodies and hormones.
Commonly, the most critical aspect of this analysis is that it identifies a highly specific antibody/antigen interaction. The target macromolecule is immobilised on a microplate, complexed with an antibody then linked to a reporter enzyme.
Via incubation and with the appropriate substrate, the activity of the reporter enzyme can be observed and measured. Such an approach can help to accelerate an investigational new drug (IND) filing timeline because binding potency assays only usually require a short time for development and qualification.
ELISA is generally considered to be an early development tool because it yields fundamental data. However, because molecules often have multiple functional domains (that may interact with several molecules to produce the desired effect), a cell-based assay may be needed to accurately assess proper function.
Several different types of cell-based assays are available, including cell viability to determine the ratio of live and dead cells, and cell proliferation to assess the biological process of cells increasing with time through cell division.
Cell-based assays can also be used to measure anticancer drug effects and, depending on the type of analysis, can yield data to support chemistry, manufacturing and controls (CMC) development and technical transfer.2 Other key and highly revealing assays include cell signalling, cytotoxicity and cell apoptosis.
PCR assays are an effective, efficient means to copy or “amplify” small segments of DNA and are proving to be an essential analytical tool for manufacturing patient-safe biologics.3 Large amounts of DNA are necessary for many molecular and genetic analyses. Studies of isolated pieces of DNA are nearly impossible without PCR amplification as they often require high concentrations and volumes that are not commonly found in vivo. PCR-amplified DNA can be applied to a variety of laboratory and clinical procedures, including diagnosing genetic disorders, fingerprinting DNA and detecting bacteria or viruses.
To reduce the risk to patients, residual host cell DNA (HCD) must be analysed by quantitative PCR to meet appropriate WHO and US FDA guidelines. HCD contaminants introduced during the upstream process can elicit infectivity, oncogenicity and possibly immunomodulatory effects, and must therefore be regulated.
Similarly, viral clearance also needs to be demonstrated to ensure that process purification steps have removed contaminant viruses prior to clinical trials. To enhance the fidelity and accuracy of viral clearance studies, reverse-transcriptase PCR (RT-PCR) can be used to determine the presence of viruses and verify their identity.
Most finished biopharmaceuticals are formulated as liquids and aseptically filled and finished into primary containers for parenteral delivery. Therefore, throughout manufacturing, the actives and compounds in the formulation come into contact with a broad range of materials.
Extractables and leachables can both contribute to contamination and be toxic, carcinogenic or immunogenic.
By altering their physicochemical properties, some extractable and leachable compounds can even impact the quality of the therapeutic biological protein.
Biopharmaceutical process developers must therefore seek a full and comprehensive understanding of how extractables and leachables can interact with the drug substance and product. They must do this work early enough in development to mitigate potential stability and contamination risks before they disrupt the programme entirely.
Approximately 20 years ago, release and stability analyses for size variants were typically done using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Similarly, isoelectric focusing (IEF) gels were being used to assess and correlate charge variants.
However, both techniques are time-consuming and labour intensive, so are more expensive operationally. Furthermore, processing/imaging the gel slabs is material intensive, may result in a higher than necessary cost of goods (CoG) profile and be economically unsustainable in the long-term.
During the past decade or so, capillary electrophoresis (CE) has become more valuable in research and is central to accurate, cost-effective CE-SDS and i/CIEF analyses. Because capillary electrophoresis allows for the separation of a molecule based on size or charge, it can produce more robust and reproducible results.
Complex molecules such as bi/multispecific antibodies or fusion proteins are posing novel analytical challenges and often require innovative analytical techniques. For example, evaluating and assigning the critical quality attributes (CQAs) of complex molecules can be difficult and make characterisation extremely tricky. Characterising multispecific antibodies offers similar complexities and challenges.
For better analytical outcomes, platform methods require careful optimisation to ensure that all the results and data for complex molecules can be reproducible and determined in a robust manner. For example, CE methods can be more sensitive than size exclusion high-performance liquid chromatography (SE-HPLC) when resolving minor differences in protein product variants such as chain mispairings.
Surfactants such as polysorbate 80 (PS80) are frequently used in formulations. Regulators are becoming increasingly interested in providing more formulation characterisation information in their guidance. Although methods quantifying PS80 have improved, the reproducibility and repeatability for some assays still lack robustness and require thorough optimisation.
Glycan analysis, for example, can be challenging to achieve because glycosylation occurs heterogeneously (owing to branching and multiple glycan structures being present on a single site).
Quantitative glycosylation analysis can provide more reproducible and robust data.
Nevertheless, high-throughput, glycan analysis can save time, even if it only provides qualitative results. Best practice when choosing an appropriate glycan analysis method is to base the decision on the information needed, at which process stage the data is required and how glycan structure affects the molecule’s protein activity.
Equipped with robust platform methods, optimising analytical method development allows biologics CDMOs to assess product quality more effectively in an accelerated development environment. However, future developments, including technological advancement, the incorporation of analytical platform methods and the integration of high-throughput analysis will be increasingly more critical for CDMOs.
Without these advancements, biopharmaceuticals will not be able to keep up with increasing demand for shorter process timelines. Experience and expertise will remain of utmost importance for CDMOs when developing an array of fit-for-purpose analytical platforms that are ready to assay increasingly more complex biologic formulations and monoclonal antibodies.