A new paradigm for optimized experimental design in cIEF platforms aimed at an accurate robust and reliable mAbs charge-variant assessment

The success of therapeutic monoclonal antibodies (mAbs) and their biosimilars, highlights the challenge to control their purity, identity, and stability. For this purpose, among several orthogonal methodologies, imaged capillary Iso Electric Focusing (icIEF) is one of the leading techniques. Despite the isoelectric point (pI) being a univocal parameter relying on the protein's primary sequence and its post-translational modifications (PTMs), the current Charge Variants Profile Assessment (CVPA) carried out by cIEF relies on relative comparisons with corresponding reference standards, this is because the inconsistent outputs for the same sample across different instruments preclude the uniqueness of the measured parameters. We demonstrate that refining the current calibration approach in the iciEF method allows for obtaining more reliable and objective pIs, and a deeper understanding of the pH gradients along the capillaries. We are confident our advancements will enhance CVPA, by exploring the concept of univocal charge identity. This is crucial for constructing biobanks and developing algorithms to quickly identify divergences from the originators, thus ensuring drug quality, efficacy, and safety. Moreover, our method allows an experimental design optimized to minimize bias ("Unbiased" Experimental Design-UED) to study resolution as a multivariate function of different input variables. This endeavor aims to develop optimal methods tailored to specific pH ranges.