Development of an optimised icIEF method for harmonising Quality Control of Monoclonal Antibodies by using an AQbD approach

Monoclonal antibodies (mAbs) are becoming a commonly used pharmacological therapy for several oncological, inflammatory, and autoimmune diseases principally due to their high specificity in target antigen binding, reducing the need for frequent dosing. Today, more than 75 mAbs and antibody-drug conjugates (ADCs) have been approved as biotherapeutic products by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). During their production in cell culture and storage, mAbs are prone to Post-Translational Modifications (PTMs) such as deamidation, glycosylation or oxidation which produce charge heterogeneities. Since some charged-based variants can have an impact on pharmacokinetics, biological activity, and long-term storage, charge heterogeneities are considered a Critical Quality Attribute (CQA) by regulatory authorities which must be monitored and evaluated during biotherapeutics life cycle. This quality control (QC) approach is commonly achieved using ion exchange chromatography (IEC) or isoelectric focusing (IEF) techniques. The imaged-capillary IEF (icIEF) technique has gained wide applications in biopharmaceutical QC due to its improved sensitivity and robustness. Compared to conventional cIEF, icIEF allows faster separation, higher resolution, and a simpler method development procedure. These advantages offer in the regulatory context a potential analytical platform for an effective detection of several PTMsrelated charge-isoforms. To accomplish this, icIEF utilizes whole capillary imaging at 280nm - without a mobilisation step - to separate analytes and allowing the determination of the isoelectric point (pI). To strengthen their independence in QC activities, Official Medicines Control Laboratories (OMCLs) are currently involved in the effort to harmonize analytical procedures, trying to develop less productspecific method which still ensure their performance and reliability. The pI value is not typically presented in the biotherapeutics release specifications as an identity parameter because of its variability depending on the instrument employed to conduct the analytical test. Instead, the comparison of the Drug Product (DP) isoform pattern is lead with the one of the Reference Standard (RS). However, in certain situations – for example, the fight against counterfeit drugs - when a RS for comparison is unavailable, the measure of an accurate, precise, and universal pI value can be crucial in distinguishing one biotherapeutic from another. Firstly, following the ICH Q14 guideline on AQbD, we performed a DoE - tuning the icIEF fundamental parameters - using Infliximab as a mAb standard and obtaining also a good measure of the main peak pI value, according to those available on the literature. As evidenced in our preliminary results, we are confident of the achievement of a new independent, transversal, and effective icIEF analytical method with an enhanced accuracy in the measure of the pI values of mAbs.