RESUMO
An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.
Assuntos
Células CHO , Engenharia Celular/métodos , Engenharia Genética/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO/metabolismo , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Via Secretória/genética , Via Secretória/fisiologiaRESUMO
A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.