   3.4.22.15 | cathepsin L exists in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaves a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Product fragmentation is caused by copurification of the cysteine protease, Cathepsin L. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography are systematically evaluated. Copurification of cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove cathepsin L but results in significant product loss, while anion exchange chromatography operates in flow-through mode does not efficiently remove cathepsin L. Mixed mode chromatography, using adsorption resin in this example, provides robust clearance over wide process parameter range (pH 7.7, 50 mM NaCl), making it an ideal technique to clear cathepsin L, method developent and evaluation, overview. Fragmentation does not occur at pH 7.0 at both 25°C and 37°C, suggesting that the protease is nearly inactive under neutral pH conditions. At pH 5.0 and pH 6.0, while the fragmentation is barely detectable at 25°C, it is pronounced at 37°C. The results suggest that the presence of the proteolytic activity under mildly acidic conditions (commonly used for formulation) still poses a significant threat to long-term product stability |
