A Process Selection Framework for Purifying Rat IgG1 Monoclonal Antibodies from Hybridoma Culture Supernatants:...

Efficient purification of rat-derived antibodies is an ongoing challenge in biologics development, particularly for isotypes that are difficult to capture using conventional affinity methods. Traditional Protein A/G affinity resins often exhibit limited efficiency and scalability for rat antibodies, particularly rat IgG1, which binds poorly to these ligands. Aside from efficiency and scalability concerns, broad application of affinity resins can be limited by sodium hydroxide ligand incompatibility. To address these limitations, we developed two alternative purification processes tailored to different production scales. For small-scale discovery screening, CaptureSelect IgG-Fc significantly outperformed Protein G across three rat IgG1 monoclonal antibodies, achieving monomer yields of 62.7-89.9% (compared to 32.1-43.1% for Protein G) and purities exceeding 90% in a straightforward bind-and-elute operation. For gram-scale production, we established a sanitizable multi-step process utilizing POROS 50 HS cation-exchange capture followed by Capto Q flow-through anion-exchange polishing. Design-of-experiments optimization of the Capto Q step (pH 7.4-7.6, 50-60 mM Tris-HCl) delivered over 90% monomer purity and overall process recoveries of 59.9-76.6%. ELISA assays verified antibody integrity and binding activity for both methods. Together, these results provide a practical process selection framework that expands production options for hybridoma-derived antibodies.