How do we know the medicines we take are safe and effective from batch to batch? Medicinal products like erythropoietin and gene therapies can be very challenging to quality control. Dr Mark Lies, Global Business Manager of Capillary Electrophoresis, SCIEX describes how capillary electrophoresis is being applied to address these challenges and ensure medicinal constituents are as they should be.
Erythropoietin (EPO) is an essential hormone that promotes the production of red blood cells from bone marrow. Synthetic human EPO is a biologic drug for the treatment of some forms of anaemia arising from various medical conditions, including kidney disease and inflammatory bowel disease (IBD). Because it has multiple potential glycosylation sites, EPO can have a large number of isoforms. In order for therapeutic recombinant EPO to be effective, it must contain a specific mix of protein isoforms. Quality control (QC) assays that are not only accurate but also precise are critical to check EPO isoform heterogeneity because if an assay is not precise, a good batch may fail the analysis or worse, a bad batch may pass.
Methods for testing biologic products to identify its constituents include chromatographic methods such as liquid chromatography (LC), which are used standalone or in combination with mass spectrometry (MS) and occasionally with alternative detection systems. EPO is not the only drug that can cause QC challenges. Gene therapies have diverse modes of action at the cellular and subcellular level. Compared with classic biologics like monoclonal antibodies (mAbs), gene therapies can have much more complex manufacturing processes, resulting in the presence of multiple types of proteins from expression systems, transgene vectors and culture media as opposed to a single protein species expressed by a single simple expression system. The QC is more complex due to the larger number of protein species that need to be detected, monitored and cleared from the final drug product.
Over the past decade, capillary electrophoresis (CE) has gained increased application and is now routinely used to check and confirm the purity, heterogeneity and glycan association of biologic drugs, as it is a simple, quick and completely effective technique, with exceptional resolving power, precision and reproducibility, specifically for large complex molecules such as biologic drugs. CE methods range from capillary zone electrophoresis (CZE) for EPO or mAbs to capillary electrophoresis sodium dodecyl sulphate (CE-SDS) for mAbs (see Figure 1) .
The power of precision
Precision speaks not only to the analytical validity of a measurement method but also, ultimately, to its utility. We understand precision as the reproducibility of a method’s quantitative and qualitative accuracy relative to data obtained using the same type of instrument and protocol. Industry guidance from regulatory authorities such as the FDA on the validation of bioanalytical methods used for the development and manufacture of biopharmaceuticals specifies that these methods must be precise, accurate, and of sufficient dynamic range . Analytical precision has been defined in various ways, often related to the approach used to measure and assess such precision. However, the critical aspect of analytical precision is in the reproducibility of the method in terms of producing the same results time and time again for a given sample. As biologics get increasingly complex, the industry and regulatory trend for ever more precise analytical methods is leading advanced analytics companies such as SCIEX to refine their CE capabilities and methods for various applications in the manufacture and QC monitoring of biotherapeutics.
Precision is one of the most important parameters for CE instrument performance, along with resolution, sensitivity and linearity. So-called ‘home brew’ methods and reagents are increasingly being replaced by specialised and standardised reagents and kits optimised for specific CE methods, such as capillary isoelectric focusing (CIEF) and CE-SDS. The aim is to develop a complete workflow solution that is simple and flexible enough for QC purposes. That way, multiple different assays can be run on the same instrument system using standardised reagents and assays. Commercially available reagents and assays that have been tested to specification and are validated and verified provide optimal precision. This is particularly important when they are used for monitoring and controlling biologic drug purity.
Precision for CE methods is evaluated using the relative standard deviation (RSD) of migration time, peak area, and proportional peak area (area percentage) across a set of samples using similar separation conditions. The lower the mean RSD value, the less deviation there is and thus the better the reproducibility. The migration time of an analyte refers to the actual time it takes to pass through a detection cell. In CE and LC, data is captured based on absorbance or relative fluorescence and is indicated as a peak. However, in CE absolute migration time (MT) does not factor in any correction for a shift caused by electromigration phenomena, it has the highest potential for variability. A better measure is the normalised or relative migration time (RMT), which is the migration time normalised against an internal reference standard. This enables the correction of the data for small shifts in the migration time, which occur to the same degree for both the standard and sample peaks.
Similarly, raw peak area is the conventional area under a peak without any corrections. Peak area % is used to express the proportion of each peak within the total. Both the raw peak area and peak area % are typically used in chromatography, but in CE both the areas must be normalised for their velocity for accurate quantitation . Otherwise, the determination of any impurities may substantially underestimate the quantity. The normalised peak area % may be referred to as the corrected peak area (CPA). The normalisation is needed for accurate quantitation and also improves precision (see Figure 2 and Table 1).
Ensuring the precision of commercially available assays is critical for the QC of drugs during clinical development and commercial manufacture. A key consideration to ensure robustness is engagement between assay manufacturers and users where critical assay specifications, including precision, of CE analyses can be assessed. These include cross-company collaborations conducted with bio/pharmaceutical companies and regulatory authorities, which have demonstrated the precision of CIEF technology for the analysis of monoclonal antibodies, CZE for charge heterogeneity testing of monoclonal antibodies, CE – laser induced fluorescence (CE-LIF) for mapping multi-site N-glycans, and CE-SDS for the analysis of biomolecules [4–7].
CE assays to bring biopharma drugs to market and clinic
Characterising molecules, large or small, using industry and regulatory accepted technology can be challenging. In order to help meet these challenges, analytical technologies such as CE, LC and MS have needed to advance in order to address new problems being identified in biopharmaceutical development.
This has become increasingly necessary as development pipelines have progressed from the conventional mAb or recombinant protein to antibody variants like bi-specific and tri-specific mAbs, antibody-drug conjugates, virus-associated moieties, and RNA therapeutics to name but a few. The quantification and characterisation of immuno-therapeutics like mAbs are critical to ensure the purity, stability and efficacy of the drug. Optimised chemistries and enhanced processing efficiency in CE methodologies have improved their utility, making them a preferred solution for both qualitative and quantitative analysis. As such, although CE technology alone is not sufficient to meet all regulatory requirements, it can be applied with other tools to achieve this.
In drug development and manufacture, CE is a simple, quick and effective characterisation technique, with exceptional resolving power and proven reproducibility, which are essential for the quantitative and qualitative assessment of drug purity and heterogeneity. Drug stability can also be assessed, including storage and transport conditions emulating conditions found around the world. Once marketing authorisation has been granted, these assessments are especially important to also ensure purity and heterogeneity of the product following scale up of manufacturing processes to characterise any modification or contamination as the result of scaling.
EPO, and its isoform composition, is routinely analysed by CZE. This is part of the European Pharmacopeia as CE provides the necessary resolution of the key isoforms required for EPO efficacy . CE has found other applications in biopharma such as routine monitoring of mAb purity during product development and QC. The ability of CE to resolve large complex molecules means it is ideally suited for analysing gene therapy products for the purity of the transgene and the overall purity of the capsid. Along with determination of the composition and purity of established medicines like EPO, the application of CE is an increasingly important tool in bringing new therapies to clinic and market, particularly new modalities in biologics like gene therapy.
Dr Mark Lies, PhD, is Senior Product Manager for new technologies in the CE & Biopharma Business Unit at SCIEX. For over 15 years, Dr Lies has worked with biopharmaceutical customers to find solutions to top analytical challenges through CE. Dr Lies was critical to the global commercialisation of the PA 800 Plus Pharmaceutical Analysis System and currently supports new biopharma-focused product development initiatives at SCIEX.
Volume 21, Issue 4 – Fall 2020
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