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).
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).