Rivera-Trujillo, Milena; (2020) Improving therapeutic protein manufacturability in mammalian cells using lipase. Doctoral thesis (Ph.D), UCL (Univeristy College London).

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Abstract

Due to market demand for monoclonal antibodies, dramatic improvements in cell line and upstream development have made primary recovery and purification the bottle neck of bio-pharmaceutical processes. The higher concentration of impurities associated with these processes such as host cell proteins, nucleic acids, lipids and product related impurities challenge the ability of platform downstream operations to purify the product. A notable example of this is that lipid impurities change the matrix and make the purification process less reliable after repeated cycles of feed applications. To improve the purification of monoclonal antibodies (mAbs) produced by mammalian cell culture, lipid removal by lipase addition was evaluated. A workflow was developed that combined ultra-scale down centrifuge and filtration with design of experiments to screen, solids remaining, lipid concentration and depth filter capacity according to the viability of the cell culture, the enzyme addition and the equivalent flow rate to centrifuge. Results showed a statistically significant impact of lipase treatment on clarification, when CHO cell broth with low viability and high concentration of lipids was exposed to high shear forces. Addition of Candida Rugosa lipase (880 U/mL) showed a 45% improvement in depth filter capacity (L/m2) throughput. Next rational design of industrial host cells using a synthetic biology approach to remove the need for addition of exogenous lipase was used to improve manufacturability. An auto-lipolytic chassis using 293T and CHOK1SV producing IgG4 cell lines were engineered to raise the levels of robustness achieved previously in primary recovery by exogenous lipase addition. Inducible lipase was integrated by non-homologous end-joining into the host cell genome at the position of a random chromosome break, in both mammalian cell lines. Induced lipase expression was not cytotoxic and auto-lipolytic 293T cells reduced by the amount of lipids in the supernatant 40%. This shows proof of concept of the auto-lipolytic chassis as means to improve manufacturability in mAb processing.

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