For successful development and marketing of biosimilars with desired efficacy and safety, this industry recognizes the central importance of extensive analysis comparing innovator and biosimilar molecules. It is also recognized in the biotechnology arena that our understanding of complex biologics remains limited even though we have many analytical technologies available to us. A recent FDA guideline for biosimilar development states the following:
The three-dimensional conformation of a protein is an important factor in its biological function. Proteins generally exhibit complex three-dimensional conformations (tertiary structure and, in some cases, quaternary structure) due to their large size and the rotational characteristics of protein alpha carbons. The resulting flexibility enables dynamic, but subtle, changes in protein conformation over time, some of which may be absolutely required for functional activity. . . At the same time, a protein's three-dimensional conformation can often be difficult to define precisely using current physiochemical analytical technology. (2)
With an understanding of our current capabilities in biologics higher-order structure (HOS) characterization, we developed an antibody array enzyme-linked immunosorbent assay (ELISA) to provide a new approach for evaluation of MAb HOS.
In a previous report, we showed that antibody arrays developed specifically toward marketed MAbs could detect structural differences that correlated well with other analytical readouts, including bioassays and glycosylation analysis (8). Experiments have shown that antibody arrays can detect subtle changes that sometimes were not detected by bioassays or any other analytical technologies currently available.
The arrays use more than 30 polyclonal antibodies to cover an entire MAb molecule, thereby measuring its surface-epitope distribution systematically and sensitively, whereas other assays measure only part of the molecule or give an average status of a biologic's population. So antibody array technology should be able to provide a unique measurement of biosimilar MAb HOS comparability. We suggest that additional surface exposure from a baseline readout be termed conformational impurity(8).
Another advantage for antibody array technology is its ability to quantify small amounts of conformational impurity using an easy-to-operate ELISA format. As little as 0.1% conformational differences could be detected from all areas covered by the polyclonal antibodies, thus providing for accurate and sensitive measurement of the status of a MAb's conformation. No data yet correlate the impact of conformational impurity with efficacy and safety of a biosimilar MAb. But it is reasonable to postulate that more conformational impurities (epitope exposures) would increase the risk for potential immunogenicity if those additional epitopes were originally inside the innovator MAb molecule, which has been proven to be tolerated by patients’ immune surveillance systems. A significantly different new epitope exposure could break self-tolerance to a MAb and induce immunogenicity. Furthermore, increased exposure of new epitopes raises the possibility of a biosimilar MAb interacting with other regulatory proteins in a patient's body, causing off-target effects.
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CBER/CDER. Feb 2012. Guidance for Industry: Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product. US Food and Drug Administration, Rockville, MD www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291134.pdf
CBER/CDER. Feb 2012. Guidance for Industry: Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. US Food and Drug Administration, Rockville, MD www.basinc.com/services/gen/BiosimilarsScientific.pdf
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Schellekens, H.2009. Assessing the Bioequivalence of Biosimilars. Drug Discov. Today. 14(9):495-499
Jiskoot, W.2009. Immunological Risk of Injectable Drug Delivery Systems. Pharmaceut. Res.26(6):1303-1314
Hermeling, S. 2005. Structural Characterization and Immunogenicity in Wild-Type and Immune Tolerant Mice of Degraded Recombinant Human Interferon Alpha 2b. Pharmaceut. Res. 22(12):1997-2006
Hermeling, S. 2004. Structure–Immunogenicity Relationships of Therapeutic Proteins. Pharmaceut. Res. 21(6):897-903
Wang, X., Li, Q, and Davies, M. 2013. Development of Antibody Arrays for Monoclonal Antibody Higher Order Structure Analysis. Front. Pharmacol. 4:103 www.ncbi.nlm.nih.gov/pmc/articles/PMC3748713
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