Development of separative methods for harmonizing Quality Control of Monoclonal Antibodies by using an Analytical Quality by Design approach

Today, 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. During their production in cell culture and storage, mAbs are prone to Post-Translational Modifications (PTMs), such as deamidation, glycosylation, or oxidation. These product-related variant modifications can significantly impact the therapeutic efficacy, immunogenicity, stability, and pharmacokinetics of the final product, introducing heterogeneity in biological drugs. These heterogeneities are considered by regulatory authorities a Critical Quality Attribute (CQA) which must be monitored and evaluated throughout the biotherapeutics life cycle. Product-related variants and impurities include also components with size distributions larger or smaller than the intact mAb. High molecular weight (HMW) species are a result of aggregation and, in some instances, precipitation. Low molecular weight (LMW) species are the result of mostly non-enzymatic cleavages of the mAb, mismatching, or incomplete formations of disulfide bridges. Charge and mass variants are considered in this work. The quality control (QC) of charge variants is commonly achieved using ion exchange chromatography (IEC) or isoelectric focusing (IEF) techniques. More recently, the imagedcapillary IEF (icIEF) technique has gained wide applications in biopharmaceutical QC due to its improved sensitivity, robustness, and a simpler method development procedure. These advantages offer in the regulatory context a potential analytical platform for the effective detection of several PTMs-related charge-isoforms. To accomplish this, icIEF utilizes whole capillary imaging at 280nm to separate analytes, allowing isoelectric point (pI) determination. Size exclusion chromatography (SEC) is a widely used technique throughout the development and manufacturing of mAbs which quantifies product size variants such as aggregates and fragments. The primary advantage of SEC, as a separation technique, results from not requiring harsh elution conditions, causing minimal impact on the protein’s conformation and structure. Identification of critical parameters for the development of an analytical method is often supported by knowledge and experience. Analytical Quality by Design (AQbD) has been used to develop methods in order to ensure their quality within a defined risk of failure. AQbD is a systematic approach, described in the ICH Q14 guideline, that involves both scientific knowledge based on sound science and risk management tools. In this presentation, we will discuss the development of a new independent, transversal, and effective icIEF analytical method with enhanced accuracy in the measure of the pI values of mAbs, following the ICH Q14 guideline on AQbD. Moreover, AQbD has been used to establish a robust analytical SEC procedure by evaluating different SEC columns and mobile phase compositions. Infliximab was used as a model compound.