New biological entities, protein-based pharmaceuticals, are now routinely obtained by genetic engineering with host cells that are mostly mammalian and microbial. It is essential that robust analytical methods are developed to identify and monitor aggregation and accurately quantify the aggregate content of a biopharmaceutical preparation. Size-exclusion chromatography is an important technique in biopharmaceutical characterization, and this article discusses its use for soluble aggregation analysis and quantitation.
New biological entities, protein-based pharmaceuticals, are now routinely obtained by genetic engineering with host cells that are mostly mammalian and microbial. The cellular processes are complex. Often the resultant recombinant proteins are unstable and aggregate or do not adopt the native conformation that imparts the required biological activity. The subsequent multistep purification procedure subjects the target protein to numerous changes in its environment with an associated risk of further conformational changes and increased levels of aggregation, visible precipitation, and invisible soluble aggregates. The impact of aggregation on the process economics, efficacy, and immunogenicity of a biopharmaceutical are considerable, and so reliable and accurate methods of analysis and quantitation that can be applied to the various scenarios encountered in development and production are required.
It is therefore essential that robust analytical methods are developed to identify and monitor aggregation and accurately quantify the aggregate content of a biopharmaceutical preparation. One technique that is used for soluble aggregation analysis and quantitation is size-exclusion chromatography (SEC).Aggregation
Protein aggregation can impact both the economic viability of a biopharmaceutical product and its efficacy. The reduction in the economic viability of the process is seen through a reduction in product yield or decreased bioactivity of the product. An increase in the level of aggregation can increase the immunogenicity of the final product because the recipient's immune system may recognize the protein complex as nonself and trigger an antigenic response.
At the molecular level, the formation of protein aggregates is complex, but it is accepted that as part of the mechanism of formation the protein must at some point lose its three-dimensional structure to interact with other protein molecules. The mechanism of interaction of unfolded proteins can result in the formation of irreversible aggregation, but if there was minimal disruption to the three-dimensional structure then aggregation may be reversible. In the worst case, the proteins can irreversibly denature and the three-dimensional structure and, hence, bioactivity, is lost. In this case the protein no longer functions as a biopharmaceutical, efficacy is reduced, and the process yield is decreased (1).