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

Abstract

Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized process, showing that protein-specific cell/process engineering can provide a solution that exceeds the limits of genetic/functional diversity within heterogeneous host cell populations. .

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…Such a bottleneck has been reported in CHO cells upon transient expression of an Fc-fusion protein (Johari et al, 2015) and EPO (Ku et al, 2008), although only EPO titer and not q p was reported for the latter example. In addition, a post-transcriptional bottleneck has been reported in stable MAb-producing CHO cells (S. J.…”

“…However, it has been shown that both CHO-and human-derived PDI can improve q p of human MAb-related r-proteins in CHO cells (Johari et al, 2015;Mohan et al, 2007;Pybus et al, 2014) (Table 1). Moreover, XBP-1S has been shown to increase volumetric productivity and q p in…”

“…ER stress originating from r-protein expression has been observed several times. For example, q p of certain r-proteins has been shown to correlate with ER stress levels upon transient expression (Johari et al, 2015;Ku et al, 2010). Once post-transcriptional and/or secretion bottleneck conditions were established, ER stress levels increased abruptly upon a higher expression level of r-protein (Fig.…”

“…This clearly indicates that nature's physiological limit not yet has been reached and thus, intracellular rate-limiting steps in protein production remain to be resolved. Indeed, post-transcriptional rate-limiting steps in the biosynthetic pathway of r-proteins have been reported multiple times in CHO cells (Johari et al, 2015;Kallehauge et al, 2016;S. J. Kim et al, 1998;Ku et al, 2008;Schröder et al, 1999) as well as in other mammalian cells (Barnes et al, 2004;Fann et al, 1999).…”

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.…”