Size exclusion chromatography (SEC) is a powerful tool for the separation of biotherapeutics such as monoclonal antibodies (mAb) and others such as antibody drug conjugates (ADCs), biosimilars, and bi-specific mAbs as well as other therapeutic proteins. Detection of purified protein heterogeneity is essential. Heterogenic impurities cause immunogenic response. More than 99% purity is needed for the medicinal purpose. Size exclusion chromatography (SEC) is used to monitor this purity level in the quality control (QC) process of the biopharmaceutical industry. With the increased use of ultra-high-performance liquid chromatography (UHPLC) instruments in QC laboratories today, instead of the conventional HPLC, it is important to have a size exclusion chromatography (SEC) column which is compatible with both UHPLC and conventional HPLC instruments. Orthogonal and complimentary modes such as reversed phase chromatography (RPC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography (IEC) can also be used along with SEC. SEC columns are generally modified with diol groups on the surface to prevent a secondary interaction. Surface and pore characteristics of the SEC columns are critical for the separation. Pore characteristics need to be optimized to have high resolution of mAb monomer from dimer and higher order aggregates as well as from fragments. Shallow calibration curve is necessary for the best resolution. Overall, the separation of monoclonal antibodies from the impurities by analytical size exclusion chromatography column is primarily discussed in this chapter. The evaluation of the different peak parameters such as retention time, peak asymmetry, column efficiency, peak resolution, run time, and loading capacity is also briefly discussed. Finally, the tips and tricks for the best separation and maintaining the column health are also discussed.
Part of the book: Antibody Engineering
Most clinically approved large biotherapeutics are monoclonal antibodies (mAbs), primarily belonging to immunoglobulin G subclass-1 (IgG1) and, to a lesser extent, IgG2 and IgG4. Glycosylation is the main source of post-translational heterogeneity of mAbs, impacting their drug therapeutic mechanism of action (MOA). Glycosylation is also one of the critical factors in drug product solubility, kinetics, stability and efficacy. Thus, monitoring glycan critical quality attributes (CQAs) is an essential part of any biopharmaceutical development. The binding affinity of an IgG to its cellular Fc receptor (FcR) depends on both its IgG subclass and Fc domain glycosylation pattern. Since composition of the N-glycans also correlates to the Antibody-Dependent Cellular Cytotoxicity (ADCC), the glycosylation pattern needs to be monitored for consistency in potency and efficacy. This applies for the original mAb biologics as well as biosimilars. In this chapter, we present a truly novel way to assess the variances in mAb glycoforms using FcγRIIIa-based affinity chromatography. First, a brief overview of the Fc receptor function is presented. Then, the principle of FcR-based affinity chromatography is explained including how this column’s potential to analyze a variety of mAbs according to their N-glycan content is highly selective and robust. Finally, we provide examples of the FcR column’s potential to improve analytical characterization of mAbs with practical applications such as effective cell line screening, monitoring of glycoengineering, process development and process control in manufacturing.
Part of the book: Monoclonal Antibodies