The Structural Fingerprint of Therapeutic Monoclonal Antibodies Determined Using a Combination of Near-UV Circular Dichroism and Statistical Approach for Comparative Analysis.

The higher-order structure (HOS) of therapeutic monoclonal antibodies is one of the important quality characteristics in terms of efficacy and safety. However, the HOS of therapeutic monoclonal antibodies has been assessed using low- to medium-resolution spectroscopy. Furthermore, because the analysis of the obtained spectra has been performed by visual evaluation in many cases, their specificity, structural similarity, and statistical significance have not been thoroughly discussed. Therefore, there is an increasing need for advanced, statistically comparable, and highly specific analytical methods for the HOS of therapeutic monoclonal antibodies. Herein, we describe an analytical method for the HOS of therapeutic monoclonal antibodies using a powerful combination of near-UV circular dichroism (CD) and statistical analysis. The obtained near-UV CD spectra of 14 therapeutic monoclonal antibodies were employed for principal component analysis. Moreover, the spectral data were converted into Euclidean distances to perform equivalence tests and significance tests. The results clearly demonstrated that each antibody had a unique near-UV CD spectrum, like a structural fingerprint. All antibodies were judged to be not equivalent to other antibodies and were also judged to be significantly different from other antibodies. Moreover, the equivalence tests were performed on several lots of antibodies, and each lot of the antibodies were judged to be equivalent. We believe that our methods are useful for identity testing and also for comparative analysis of HOS of therapeutic monoclonal antibodies.