Challenging applications of capillary electrophoresis in biopharmaceutical analysis
By Federica Bisceglia (Eurofins BioPharma Product Testing), Giuseppe Peddio (Eurofins BioPharma Product Testing)
1 December 2023
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After decades of development and refinement, the separation of proteins by capillary electrophoresis (CE) has moved beyond the R&D laboratory into the manufacturing quality control laboratory. Here, Giuseppe Peddio and Federica Bisceglia discuss the advancements in CE technology for development applications and batch release for biopharmaceutical products.
Since its introduction in 1980, capillary electrophoresis (CE) has been one of the most powerful techniques applicable as a choice method in the biopharmaceutical industry for the characterisation and quality control (QC) testing of biomolecules such as proteins, peptides as well as monoclonal antibodies (mAbs) and high molecular weight polysaccharides.1
In addition to the intrinsic advantages of CE, such as high separation efficiency, high resolution, low sample volume requirements, short separation time and full automation possibility, it exhibits attractive possibilities for the efficient analysis of intact large molecules, providing some information about isoforms, degradation products and impurities of biopharmaceuticals and charge heterogeneity.2
Over time, CE has advanced to become a superb complement to high-performance liquid chromatography (HPLC), and in many cases has also evolved as an automated and quantitative replacement for conventional slab gel electrophoresis methods such as SDS-PAGE and isoelectric focusing (IEF).3
What kind of CE-based methods can be applied in the QC of biopharmaceuticals?
Several approaches using CE are applicable to assess the quality of biopharmaceuticals:
- Capillary zone electrophoresis (CZE) is the simplest and most widely used method, which allows the analysis of ionised or ionisable compounds and is based on the differences in their electrophoretic mobility.4
- Capillary isoelectric focusing (cIEF) is similar to IEF–PAGE and separates proteins and peptides according to their isolectric point (pI) values with a ‘high-resolution’ response.
- Capillary gel electrophoresis (CGE) under reducing or non-reducing conditions involves the separation of molecules according to their size.
- Capillary electrokinetic chromatography (CEKC) could be a suitable method for separation of protein stereoisomers and peptides with similar charge-to-size ratios and different hydrophobicity.
Other approaches, even if less frequently applied in the QC of biopharmaceuticals, include affinity capillary electrophoresis (ACE), capillary electrochromatography (CEC) and CE‑MS.
Download the full article to learn more about the role for CE in GMP testing for biologics, including gene therapy products.
