Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells

Self Cite

The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. Expression vector optimization is an effective strategy to increase transgene expression levels and stability, and the choice of suitable poly A element is crucial for the expression of recombinant protein. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. Five poly A elements, including bovine growth hormone (BGH), mutant BGH, herpes simplex virus type 1 thymidine kinase (HSV-TK), SV40, and a synthetic (Synt) poly A, were cloned into the expression vector and transfected into CHO cells. The results indicated the SV40 and Synt poly A sequences can significant improve eGFP transgene expression in stable transfected CHO cells and maintain long-term expression. However, qPCR results showed that the eGFP expression at protein level was not related to the gene copy number and mRNA level. Importantly, the SV40 and Synt poly A elements decreased the variation of eGFP transgene expression. Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. In conclusion, SV40 poly A and Synt poly A are stronger elements that increase stable transgene expression and decrease the variation of expression, and the choice of suitable poly A element is helpful to improve the expression of recombinant protein.

Section: Discussionmentioning

confidence: 99%

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Wang

Zhang

et al. 2022

Self Cite

“…If a two-plasmid system is utilized, the HC and LC are located on different vectors and different plasmid ratios are used for transfection. In addition, single-plasmid systems can also be used to control HC to LC expression with different ratios using IRES (internal ribosome entry site, IRES) mediated vectors up to 1.15 g/L, double or tricistronic vectors ( Li et al, 2020 ). The polycistronic vector contains IRES and Furin-2A (F2A), and the expression of multiple cistrons on a single vector is achieved by IRES and F2A ( Ho et al, 2013 ) to optimize the expression of recombinant antibodies.…”

Section: Optimization Of Expression Vectormentioning

confidence: 99%

Factors Affecting the Expression of Recombinant Protein and Improvement Strategies in Chinese Hamster Ovary Cells

Fan

Wang

et al. 2022

Self Cite

Recombinant therapeutic proteins (RTPs) are important parts of biopharmaceuticals. Chinese hamster ovary cells (CHO) have become the main cell hosts for the production of most RTPs approved for marketing because of their high-density suspension growth characteristics, and similar human post-translational modification patterns et al. In recent years, many studies have been performed on CHO cell expression systems, and the yields and quality of recombinant protein expression have been greatly improved. However, the expression levels of some proteins are still low or even difficult-to express in CHO cells. It is urgent further to increase the yields and to express successfully the “difficult-to express” protein in CHO cells. The process of recombinant protein expression of is a complex, involving multiple steps such as transcription, translation, folding processing and secretion. In addition, the inherent characteristics of molecular will also affect the production of protein. Here, we reviewed the factors affecting the expression of recombinant protein and improvement strategies in CHO cells.

“…In recent years, a prominent approach is based on self-cleavage 2A peptide-mediated multi-gene construction method ( Chng et al, 2015 ; Van der Weken et al, 2019 ). This strategy effectively avoids the disadvantage of low expression of downstream genes caused by the simultaneous expression of multiple genes and is commonly used in multi-gene expression ( Luke and Ryan, 2018 ; Li et al, 2020 ). Ho et al compared the effects of F2A and IRES on the production of a monoclonal antibody in CHO-DG44 cells.…”

Section: Optimization Of Gene Sequences Of Antibodymentioning

confidence: 99%

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

Zhang

Shan

Liang

et al. 2022

Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.