Monoclonal Antibody Separation: Lucentis from Genentech

Abstract

This design project focuses on the bioseparation of Lucentis (Genentech Inc.), a humanized anti-VEGF-A monoclonal antibody fragment used to treat macular degeneration. Vascular endothelial growth factor, VEGF, promotes blood vessel growth in the wet form of macular degeneration. These fragile blood vessels easily break and lead to bleeding within the macula, which causes a gradual loss of sight. Anti-VEGF-A suppresses the growth of blood vessels by binding to VEGF. The process design begins with a 1000 L batch of fermented culture. The cell culture fluid contains approximately 5 g/L of antibody and 20 to 25 % of cell material. The goal is to produce a final purified solution of 10-30 g/L antibody at 99 % purity. The bioprocesses to be investigated include: fermentation, homogenization, centrifugation, chromatography (HIC and cation exchange) and membrane utrafiltration. Alternative cases will also be explored in which the chromatography steps are replaced with G-Protein and immobilized metal chromatography. The project objective is to assemble these various separation steps into a process capable of separating and purifying the target antibody from the cell culture fluid. The final product is an aqueous solution of the pure antibody in a formulation solution. The key aspect of this study is the optimization of both purity and step yield for each process step. Experimentally, the presence and separation of Lucentis was confirmed through ELISA, SDS PAGE and mass spectroscopy. A base case of HIC and IEX was assumed. HIC was inconclusive and requires further testing to determine the parameters necessary for separation. IEX was found to successfully purify the concentrate. However, the Lucentis sample requires pretreatment and salt removal before feed into the IEX. Promising alternative cases include IMAC and Affinity G Protein which yielded the highest degree of purification experimentally with approximately 62.7% of Lucentis eluted in the protein sample. However, these base cases are limited due to the cost of the method, although preliminary economic analysis suggests that IMAC may be economically feasible. All downstream separation processes include a report on theory, experimental results and design, operational parameters, and sizing estimations and economic analysis for industrial operation. The economic analysis includes operational costs and raw material costs. An understanding of the mechanisms of separation, experimental results and economic analysis are presented in a final report and a poster.