Knowledge Bank

Familial Hypercholesterolemia is a prevalent genetic disorder characterized by elevated circulating low-density lipoprotein (LDL) cholesterol, which increases the risk of early- onset atherosclerosis and arterial blockages. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a critical role in LDL cholesterol uptake regulation by degrading LDL receptors, thereby reducing cholesterol clearance. PCSK9 inhibitors, such as monoclonal antibodies like evolocumab (Repatha), have been developed as therapeutics. As an alter- native to mAbs, adnectins—small proteins derived from the human fibronectin 10th-type III domain—have been engineered to target PCSK9. However, due to their small size (11.3 kDa), Adnectins undergo rapid renal clearance, necessitating half-life extension strategies. Previous approaches, including PEGylation (BMS-962476) and human serum albumin fusion (LIB003), have shown promise, with LIB003 demonstrating success in Phase 3 clinical trials.

This study investigates the development and characterization of adnectin fusions with small human serum albumin binding chaperone proteins, including albumin affibody and anti-albumin nanobody protein forms, as an alternative to direct human serum albumin fusion. The focus of this study was optimizing scalable bioprocessing techniques for the expression and purification of PCSK9-binding adnectin fusion proteins. Recombinant protein expression was optimized using E. coli cell lines as a cost-effective alternative to mammalian cell culture. Upstream processing was evaluated by screening soluble protein expression for extrinsic conditions using various growth media, induction temperatures, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations. Lower induction temperatures were found to significantly enhance the soluble expression of adnectin fusion proteins on a per cell basis.

Additionally, a single-step chromatographic purification strategy utilizing self-cleavingaffinity tags (split intein technology) was implemented to eliminate the need for costly polishing chromatography steps. This study characterized intein cleavage kinetics for adnectin fusion protein purification. Later, 10x scale-up purification using Npu split intein purification was demonstrated, yielding 3.9 mg of pure adnectin fusion protein.