Model‐directed engineering of “difficult‐to‐express” monoclonal antibody production by Chinese hamster ovary cells

Abstract

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions.

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

“…In fact, PDI overexpression only increased q p for one of four monoclonal antibody (MAb) variants in a parallel experimental setup (Pybus et al, 2014). However, many cellular and experimental factors are at play when examining how an effector gene affects volumetric productivity and q p (Fig.…”

“…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…”

“…CHO cells -both the human (Becker et al, 2008;Cain et al, 2013;Pybus et al, 2014) and murine (Hansen et al, 2015;Ku et al, 2008) orthologues. In contrast, only the CHO-derived and not the human orthologue of YY1 was able to increase volumetric productivity in CHO cells and vice versa in human cells (Tastanova et al, 2015).…”

“…Naturally, the occurrence of a secretion bottleneck is protein-specific, and it is likely that, for some difficult-to-express proteins, a secretion bottleneck can be readily obtained. A post-transcriptional bottleneck in CHO cells has been reported upon expression from replicating episomes (Pybus et al, 2014); however, this was without antibioticbased selection and is therefore not regarded as SEE (Table 2). A relatively high average q p (10 pcd for MAbs) can be obtained using the SEE platform (personal communication with Dr Meelis Kadaja, Icosagen Cell Factory Ltd, Estonia), implying that post-transcriptional and/or secretion bottleneck conditions can be established using the SEE platform.…”

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.…”