Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR−/− cells in fed-batch processes – Implications for designing novel CHO-based expression platforms

Florin, Lore; Lipske, Carolin; Becker, Eric; Kaufmann, Hitto

doi:10.1016/j.jbiotec.2011.02.002

Cited by 6 publications

(9 citation statements)

References 23 publications

(23 reference statements)

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“…These results indicate that cell growth may be affected more by the DHFR expression level than by the production of monoclonal antibody. CHO DG44 cells with reintroduced DHFR show different DHFR expression levels, and the growth of these cells is linearly correlated with the level of DHFR expression . In this study, cell growth differed between the two rCHO DG44 cell lines.…”

Section: Discussionmentioning

confidence: 62%

“…CHO DG44 cells with reintroduced DHFR show different DHFR expression levels, and the growth of these cells is linearly correlated with the level of DHFR expression. 12 In this study, cell growth differed between the two rCHO DG44 cell lines. This difference may be because of clonal variability between the recombinant cell lines caused by variability in the gene amplification process following integration of the DHFR-containing plasmid to different random sites of the host cell genome.…”

Section: Discussionmentioning

confidence: 73%

“…rCHO DG44 cell lines derived from DHFR‐deficient CHO DG44 cells achieve maximum viable cell densities more than 2‐fold greater than those achieved by the DHFR‐deficient CHO DG44 cells under the same culture conditions . Reintroduction of the DHFR gene to DHFR‐deficient CHO DG44 cells yields subclones with different integrals of viable cell density, and some of these subclones exhibit growth comparable to that of the rCHO DG44 cell line . These results indicate that exogenous H/T is not sufficient to completely compensate for DHFR deficiency when cells are grown in CDM.…”

Section: Introductionmentioning

confidence: 93%

“…DHFR‐deficient CHO DG44 cells are auxotrophs for hypoxanthine and thymidine (H/T), which are believed to compensate for DHFR deficiency . However, DHFR‐deficient CHO DG44 cells grow poorly in suspension culture in CDM even if the medium is supplemented with H/T . rCHO DG44 cell lines derived from DHFR‐deficient CHO DG44 cells achieve maximum viable cell densities more than 2‐fold greater than those achieved by the DHFR‐deficient CHO DG44 cells under the same culture conditions .…”

Section: Introductionmentioning

confidence: 99%

“…12 Reintroduction of the DHFR gene to DHFR-deficient CHO DG44 cells yields subclones with different integrals of viable cell density, and some of these subclones exhibit growth comparable to that of the rCHO DG44 cell line. 12 These results indicate that exogenous H/T is not sufficient to Correspondence concerning this article should be addressed to H. W. Park at hhwwpp75@gmail.com completely compensate for DHFR deficiency when cells are grown in CDM.…”

Section: Introductionmentioning

confidence: 99%

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Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Kim

Park

2016

Biotechnology Progress

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Adaptation of dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted DHFR-deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted DHFR-deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2-359 μM enhanced cell growth in in-house CDM. Addition of 3.6 μM THF to in-house CDM resulted in a more than 2.5-fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9-fold enhancement of maximum viable cell density. An anchorage- and serum-dependent DHFR-deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in-house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension-adapted DHFR-deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR-deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539-1546, 2016.

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Section: Discussionmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Kim

Park

2016

Biotechnology Progress

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Multi‐omics profiling of CHO parental hosts reveals cell line‐specific variations in bioprocessing traits

Lakshmanan

Kok

Lee

et al. 2019

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines has been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11, and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N-and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics and subsequent genome-scale metabolic network model-based enrichment analyses indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell-cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N-and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced. K E Y W O R D S CHO parental cell lines, critical quality attributes, mammalian systems biotechnology, multi-omics analysis, N-glycosylation

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Accelerating and de‐risking CMC development with transposon‐derived manufacturing cell lines

Rajendran

Balasubramanian

Webster

et al. 2021

Biotech & Bioengineering

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The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein-based biologic drugs. Here, we describe the Leap-In Transposase® platform, a novel transposon-based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high-titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources.

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Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR−/− cells in fed-batch processes – Implications for designing novel CHO-based expression platforms

Cited by 6 publications

References 23 publications

Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Multi‐omics profiling of CHO parental hosts reveals cell line‐specific variations in bioprocessing traits

Accelerating and de‐risking CMC development with transposon‐derived manufacturing cell lines

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