Engineering Enhanced Alkaline Stability of Recombinant Protein A for Improved Monoclonal Antibody Affinity Purification in Industrial Applications

Native Staphylococcus aureus protein A exhibits strong affinity to the Fc and VH regions of human IgG1, IgG2, and IgG4, making it a valuable tool for monoclonal antibody (mAb) purification. However, its low stability under conditions such as increased alkaline concentrations during cleaning-in-place (CIP), protease exposure, thermal stress, and shear forces limits its usability for large-scale industrial applications. Recombinant Protein A (rProtein A) can be modified to improve key properties, including alkaline stability. In this study, we present targeted modifications to the C domain of native Protein A, evaluating multimeric variants for structural and functional improvements. The selected variant demonstrated extremely high stability after 60 h incubation at 0.5 M NaOH by maintaining more than >90% initial dynamic binding capacity (DBC) and up to 80% DBC after 40 h in 1.0 M NaOH. However, the most impressive result obtained was the stability of the ligand in 1.5 M NaOH, retaining 80% DBC after 22 h and 60% DBC after 40 h. To the best of our knowledge, this is the first time that such high alkaline stability is reported for a rProtein A. To assess its application in monoclonal antibody purification, the optimized rProtein A ligand was immobilized on agarose resin and tested in chromatography processes. The resulting chromatography resin functionalized with the CmZmb ligand (now commercialized by Sunresin, China under the name of rProtein A Seplife Suno) exhibited a high dynamic binding capacity of 70 mg/mL, minimal ligand leaching under operational conditions (~15 ppm), and extended lifecycle performance (88% DBC retained after 120 purification cycles with 0.5 M NaOH CIP), making it well-suited for industrial-scale applications.