High‐level protein expression in scalable CHO transient transfection

Abstract

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14-day fed-batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed.

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

“…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).…”

Microbial Expression Systems and Manufacturing from a Market and Economic Perspective

“…However, for early feasibility tests with a protein, transient gene expression and protein production may be sufficient (Ye et al, 2009 (Eibl et al, 2010), makes the production of several 100 g of new proteins possible in a short time.…”

Enhanced CHO Cell-Based Transient Gene Expression with the Epi-CHO Expression System

“…EBNA-1 drives the replication and maintenance of plasmid DNA containing the EBV latent origin of replication, OriP, which in turn promotes elevated and prolonged expression of the recombinant protein of interest [10][11][12][13]. Recent reports using the HEK.EBNA system have generated mAb titers in excess of 1 g/l [14], whereas, the highest reported mAb titers generated from CHO cellbased TGE in the literature are considerably lower and range from 60 to 90 mg/l [3,7,8]. Stably transfected CHO cells remain the host of choice for the large-scale commercial production of therapeutic proteins and yields in these systems now routinely exceed 1 g/l.…”

“…TGE yields have improved dramatically over the last decade due to research into a variety of parameters, including; the expression vector [1], the development of advanced chemically defined and serum-free media, their supplementation, and the feeding strategy implemented post-transfection [2,3] and the direct addition of a variety of chemical agents such as epigenetic modulators [4], cell cycle regulators [5], growth factors [6], and agents thought to directly enhance transfection efficiency and promote plasmid maintenance [3]. Culturing under mild hypothermic conditions has also resulted in significant improvements in expression levels [6][7][8].…”