Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system

Abstract

in Wiley Online Library (wileyonlinelibrary.com) Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems.

Integrated cell and process engineering for improved transient production of a “difficult‐to‐express“ fusion protein by CHO cells

“…Specifically, we tested genes encoding ER‐resident proteins widely reported to be involved in nascent polypeptide folding and assembly (BiP, PDI, and CypB), genes encoding activated forms of UPR transactivators (ATF6c and XBP1s) reported to generically increase the expression of ER chaperones/foldases (Dinnis and James, ), a potent small molecule inhibitor of PERK (GSK2606414, Harding et al, ) to inhibit eIF2α phosphorylation and lastly, chemical chaperones reported to alleviate deficient intracellular protein trafficking, minimize protein aggregation and relieve ER stress (e.g., Roth et al, ), specifically 4‐phenylbutyrate (PBA), dimethyl sulfoxide (DMSO), glycerol, betaine, and trimethylamine N‐oxide (TMAO). As it has been reported that the production of some DTE proteins can be improved by hypothermic culture temperatures (e.g., Estes et al, ), we also compared the effect of post‐transfection temperature shift to 32°C. For all transfections, recombinant Sp35Fc plasmid DNA load (3 μg) was kept constant.…”

“…TGE methods can be employed to fast‐track the production of multiple biologics to perform biophysical analyses and early preclinical evaluation of drug candidates. Furthermore, transient production is increasingly being employed to evaluate product manufacturability and refine process conditions (Estes et al, ).…”

“…As a result, TGE yields have improved dramatically in the last decade with Daramola et al () recently reported a transient expression productivity of 2 g/L. However, there remains little information on cellular limitations during TGE or how to improve production of DTE recombinant products other than broad generic methods such as hypothermia (Estes et al, ; Hwang et al, ). Moreover, whilst some improvements in optimized transfection protocols and transient processes have been published (Daramola et al, ; Rajendra et al, ; Thompson et al, ), there are no methods to systematically utilize TGE to determine the optimal route to engineer the CHO cell host and/or culture environment for improved product manufacturability according to set, product‐specific criteria.…”

Optimization of Protein Expression in Mammalian Cells

“…High rates of production may swamp the post-translational modification apparatus leading to heterogeneous product formation or in cellulo aggregation, like Russell bodies (Hasegawa, Woods, Kinderman, He, & Lim, 2014). Some of these problems may be ameliorated by codon optimization, by stuffer DNA, by choosing a 'weaker' promoter, or by engineering cells to have enhanced properties (B. Estes et al, 2015;Rajendra et al, 2012).…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…Limitations in the secretion of recombinant proteins can impact both protein quality and yield, which can have a negative impact on downstream processes. Published reports have focused on the characterization of such ‘difficult‐to‐express’ recombinant proteins and described the modification of culture conditions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or the design of appropriate cell/protein engineering strategies to overcome restrictions in their production 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. However, little is known regarding the mechanisms underpinning poor recombinant protein production, particularly between proteins of high sequence similarity.…”