Exploration of BAC versus plasmid expression vectors in recombinant CHO cells

Mäder, Alexander; Prewein, Bernhard; Zboray, Katalin; Casanova, Emilio; Kunert, Renate

doi:10.1007/s00253-012-4498-x

Cited by 22 publications

(23 citation statements)

References 19 publications

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“…This is very similar to the herein described difference of 1.7‐fold during a shorter period of time. Mader et al (2013) described a three to fourfold different specific productivity between 2F5‐ and 3D6‐scFv‐Fc plasmid‐ and BAC‐generated producers, which is in accordance with the results presented in this study. Since both techniques are based on random integration, gene copy numbers and transcript levels can vary between 2F5‐ and 3D6‐scFv‐Fc plasmid‐ and BAC‐derived producers.…”

Section: Discussionsupporting

confidence: 92%

“…Three different strategies (RMCE, plasmid, and BAC) for transgene delivery were used to generate recombinant 2F5‐ and 3D6‐scFv‐Fc producers (Mader et al, 2013; Mayrhofer et al, 2014). The RMCE clones showed the lowest specific productivity and highest growth rate.…”

Section: Discussionmentioning

confidence: 99%

“…The two best performing clones for each strategy and product were used in this study ( n = 12). The cell line establishment of the 12 clones has been previously described (Mader et al, 2013; Mayrhofer et al, 2014). All clones were cultivated in suspension in 125‐mL spinner flasks (Techne, Thermo Fisher Scientific, Waltham, MA) at 37°C, 7% CO 2 , and 50 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…The product concentration in the cell supernatants was determined by sandwich enzyme‐linked immunosorbent assay (ELISA) as previously described (Mader et al, 2013; Mayrhofer et al, 2014). Briefly, 96‐well Maxisorp plates (Nunc, Thermo Fisher Scientific) were coated with polyclonal goat anti‐human IgG (γ‐chain specific) (I3382, Sigma–Aldrich, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Sommeregger

Mayrhofer

Steinfellner

et al. 2016

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Sommeregger

Mayrhofer

Steinfellner

et al. 2016

Biotech & Bioengineering

Self Cite

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Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis

et al. 2018

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Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.

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Synthetic promoters for CHO cell engineering

Brown

Sweeney

Mainwaring

et al. 2014

Biotech & Bioengineering

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We describe for the first time the creation of a library of 140 synthetic promoters specifically designed to regulate the expression of recombinant genes in CHO cells. Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks. The most active second generation promoters achieved approximately twice the activity of a control construct harboring the human cytomegalovirus (CMV) promoter. Lastly, we evaluated the function of a subset of synthetic promoters exhibiting a broad range of activity in different CHO cell host cell lines (CHO-S, CHO-K1, and CHO-DG44) and across extended fed-batch transient expression in CHO-S cells. In general, the different synthetic promoters both maintained their relative activity and the most active promoters consistently and significantly exceeded the activity of the CMV control promoter. For advanced cell engineering strategies our synthetic promoter libraries offer precise control of recombinant transcriptional activity in CHO cells spanning over two orders of magnitude.

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Exploration of BAC versus plasmid expression vectors in recombinant CHO cells

Cited by 22 publications

References 19 publications

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis

Synthetic promoters for CHO cell engineering

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