Recent advances in recombinant protein production

Abstract

Designing appropriate expression vectors is one of the critical steps in the generation of stable cell lines for recombinant protein production. Conventional expression vectors are severely affected by the chromatin environment surrounding their integration site into the host genome, resulting in low expression levels and transgene silencing. In the past, a new generation of expression vectors and different strategies was developed to overcome the chromatin effects. Bacterial artificial chromosomes (BACs) are cloning vectors capable of accommodating up to 350 Kb. Thus, BACs can carry a whole eukaryotic locus with all the elements controlling the expression of a gene; therefore, BACs harbor their own chromatin environment. Expression vectors based on BACs containing open/permissive chromatin loci are not affected by the chromatin surrounding their integration site in the host cell genome. Consequently, BAC-based expression vectors containing the appropriate loci confer predictable and high levels of expression over time. These properties make BAC-based expression vectors a very attractive tool applied to the recombinant protein production field.

Advances in recombinant antibody manufacturing

“…Alternatively, bacterial artificial chromosomes (BACs) are DNA transfer vectors carrying a whole eukaryotic locus with all the elements controlling the expression of a gene. Thus, the transgene on the BAC is surrounded by an open/permissive chromatin loci and is not affected by the chromatin environment of the integration site (Kunert and Casanova 2013 ).…”

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

“…Although, the same RMCE host cell line was used and transgene copy numbers as well as levels of transcript were identical, a twofold difference in specific productivity between 2F5‐ and 3D6‐scFv‐Fc producers was observed. Following this observation, we applied two additional strategies of transgene delivery and established 2F5‐ and 3D6‐scFv‐Fc producers, once with a common plasmid strategy and secondly with the Rosa26 bacterial artificial chromosome (BAC) strategy (Blaas et al, 2009, 2012; Kunert and Casanova, 2013; Mader et al, 2013; Zboray et al, 2015), applying the same CHO DUKXB11 host cell line that was used for the generation of the RMCE cell line. Independent from the transgene delivery system, we regularly identified clones with higher specific productivities for 3D6‐scFv‐Fc compared to 2F5‐scFv‐Fc producing cell lines.…”

Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED

“…These “safe haven” loci are known to have 10-100 times higher reporter expression. 46–49 Yet even at these sites, expression of transgenes may be too low for experimental needs. For example, insertion of a single Rosa26 BAC in which a GFP reporter was placed in the first exon of transcript 1 of this locus produced higher fluorescence than was reported by insertion of a GFP reporter into the endogenous Rosa26 locus, possibly due to loss of adjacent repressive sequences 50 , and other anecdotal reports have suggested that targeting a reporter gene such that it will be under control of an endogenous promoter may produce too low expression for visualization of reporter gene activity.…”