Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique

Abstract

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical techniqueFragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa () light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.

Challenging Bioanalyses with Capillary Electrophoresis

“…Other modes of capillary electrophoresis are also utilized for the evaluation of biological therapeutics. This includes the use of capillary gel electrophoresis size-based sieving of proteins to rapidly screen for antibody impurities − and to investigate protein fragmentation. , A more selective Western blot capillary electrophoresis approach was reported to identify antibody fragmentation . With this technique, antibody fragments, which had been separated with size exclusion chromatography prior to capillary electrophoresis, were visualized with fluorescent probes specific for the kappa light chain as well as the Fc and Fab regions of an IgG.…”

“…41,42 A more selective Western blot capillary electrophoresis approach was reported to identify antibody fragmentation. 43 With this technique, antibody fragments, which had been separated with size exclusion chromatography prior to capillary electrophoresis, were visualized with fluorescent probes specific for the kappa light chain as well as the Fc and Fab regions of an IgG.…”

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

“…In addition to the impurities related to the recombinant production of Fc-fusion proteins, such as host cell proteins (HCP), virus, and DNA, several other contaminants are generated ( Shukla and Gottschalk, 2013 ); for instance, one problem often encountered with Fc-fusion proteins is the formation of high molecular weight aggregates (HMWA) ( Vaniotis et al, 2018 ). Also, low molecular weight species (LMWs), which include an incomplete Fc domain, may be produced ( Zhu et al, 2019 ). To ensure safety and efficacy of the final product, it is critical to have a robust downstream process that maximizes the removal of process and product-related impurities.…”