mRNA Transfection in CHO-cells Reveals Production Bottleneck.

Abstract

Obtaining highly productive CHO-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbors potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product might prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high-qP bottlenecks. In this study we aimed to reach specific productivities approaching those of industrial production cell lines by transfection of in-vitro transcribed mRNA. The system was characterized with respect to transfection efficacy (by qPCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturated at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remained linearly correlated to the amount of mRNA taken up. Nevertheless, mass spectrometry showed a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization. This article is protected by copyright. All rights reserved.