Statistical Validation of Reproducibility of HPLC Peptide Mapping for the Identity of an Investigational Drug Compound Based on Principal Component Analysis

Abstract

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable “fingerprint” for identity testing and process monitoring. We recently conducted a full method validation study of an optimized reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 monoclonal antibody. We have used this method routinely for over a year to test production lots for clinical trials and to support bioprocess development. One of the difficulties in the validation of the peptide mapping method is the lack of proper quantitative measures of its reproducibility. A reproducibility study may include method and system precision study, ruggedness study, and robustness study. In this paper, we discuss the use of principal component analysis (PCA) to quantitate peptide maps properly using its projected scores on the reduced dimensions. This approach allowed us not only to summarize the reproducibility study properly, but also to use the method as a diagnostic tool to investigate any troubles in the reproducibility validation process.

Key Words:

• Identity testing
• Method validation
• Monoclonal antibody
• Peptide mapping
• Principal component analysis
• Tryptic mapping