Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster ovary cells

Abstract

BackgroundExpression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency.ResultsAfter a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture.ConclusionsNovel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0145-9) contains supplementary material, which is available to authorized users.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

“…However, rhA1AT from these neuronal cells exhibits core-fucosylation, is not fully sialylated, and therefore differs largely from plA1AT N-glycosylation [24]. Similarly, earlier studies expressed rhA1AT in CHO with titers of up to 1.15 g/L, though differing from plA1AT by revealing core-fucosylation and alpha-2,3sialylation [25,26,43].…”

Case study on human α1‐antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

“…Since ELISA is the most widely used assay for monitoring protein titer, it seems likely that a considerable number of reported recombinant protein titers is inaccurate. Recently, a r α1AT titer of 1.15 g/L was reported , which according to the authors is the highest titer of any recombinant protein in shake flasks batch culture of stable mammalian cells. A commercially available ELISA kit was used for determining protein titer and validation of the ELISA kit by an orthogonal assay was not described.…”

“…r α1AT can be produced in transgenic sheep , but an immune response to endogenous sheep α1AT in the purified product has been observed . An attractive alternative is to produce r α1AT in CHO or human cells and efforts have been undertaken to achieve this . N ‐glycosylation patterns of r α1AT produced from CHO and human cells have been shown to be similar but not identical to pl α1AT .…”

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

“…The extracellular concentrations after feeding were computed based on the feed composition information. Specific growth rate, µ, was computed for each experimental condition separately as the slope of the linear trend line obtained by plotting ln( C XR .V R ) against time (Chin et al, ; Clarke et al, ).…”