Understanding aggregates formation and their removal from therapeutic proteins.

Recombinant DNA technology has been pivotal in transforming biologics into viable therapeutic agents by enabling the efficient and scalable production of human proteins in host expression systems. This breakthrough overcame the limitations of traditional protein sourcing and facilitated the development of safe, effective, and targeted treatments for a wide range of diseases. Aggregation in therapeutic proteins is a major challenge in biopharmaceutical development, as it can compromise product stability, efficacy, and safety. Protein aggregates may form during production, storage, or administration, often due to physical or chemical stress. These aggregates not only affect the functional integrity of the protein but also increase the risk of immunogenic responses in patients. Understanding the factors that promote aggregation, starting from the conditions at which Escherichia coli (E. coli) cells, expressing the therapeutic protein of interest, are cultivated, and developing effective processes to prevent or control it are essential steps in ensuring the quality and performance of therapeutic protein products. Towards this goal, a lysozyme-based method has been applied to extract the therapeutic protein of interest. The resulting extracts were analyzed for yield and purity using Anion Exchange chromatography with a Dimethylaminoethanol (DMAE) column. To investigate aggregate formation under different cells’ conditions, the main analytical techniques applied to the intermediates were Size Exclusion Chromatography (SEC) and Reversed-Phase Chromatography (RPC). Our study suggests that the formation of product aggregates/fragments are dependent on IPTG concentration of the induction media and potentially the temperature and duration of induction. It was proved that the level of pre-peaks by RPC decreases as the Isopropyl -D-1-thiogalactopyranoside (IPTG) concentration increases, highlighting the importance of controlling induction conditions for consistent protein production.