A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Abstract

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells but reported yields from Chinese hamster ovary (CHO) cells are lower at ∼300 mg/L. We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X-box binding protein (XBP-1S) and endoplasmic reticulum oxidoreductase (ERO1-Lα) and has been named CHOS-XE. CHOS-XE cells produced increased antibody (MAb) yields (5.3- 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L.

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

“…The FcRn ECD was purified by affinity chromatography on IgG (CnBr-activated Sepharose 4 Fast Flow; GE Healthcare, UK), binding at pH 5.8 and eluting at pH 8. 67,68 Rabbit, mouse and rat FcRn ECD were expressed, each with the homologous β2m, in proprietary Chinese Hamster Ovary (CHO) SXE 69 cells and protein purified.…”

“…IgG molecules were prepared from CHO SXE 69 cell culture medium by affinity chromatography on MabSelect™ SuRe™ (GE Healthcare, UK), followed by size-exclusion chromatography (SEC) on HiLoad XK 50/60 Superdex 200 PG (GE Healthcare, UK). The purity of the final product was confirmed by reducing and non-reducing SDS-PAGE, and by analytical scale SEC.…”

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

“…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).…”

“…However, the lower protein yield achieved with TGE in CHO cells has historically been a major drawback compared to the substantially higher yields obtained with stable gene expression (SGE). Thus far, extensive efforts to improve TGE yields in CHO cells have been made by optimizing the culture environment (Galbraith et al, 2006;Ye et al, 2009), transfection efficiency (Mozley et al, 2014;Rajendra et al, 2015Rajendra et al, , 2012, vector systems (Cho et al, 2001;Mariati et al, 2010) and host cell line (Cain et al, 2013;Daramola et al, 2014;Macaraeg et al, 2013). Therefore, TGE has become a robust and flexible system, applicable to multiple r-proteins, expression volumes and bioprocesses with substantially increased yields of up to 3 g/L for MAbproducing CHO cells (Liu et al, 2015).…”

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.…”