Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters

Lattenmayer, Christine; Trummer, Evelyn; Schriebl, Kornelia; Vorauer-Uhl, Karola; Mueller, Dethardt; Katinger, Hermann; Kunert, Renate

doi:10.1016/j.jbiotec.2006.12.016

Cited by 49 publications

(34 citation statements)

References 34 publications

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“…7A), indicating that higher HC and LC transgene copy numbers do not always lead to higher mAb expression. Similar observations were reported recently (Lattenmayer et al, 2007). We found that the LC to HC ratio also seems to have an effect on productivity.…”

Section: Comparison High-producer Versus Low-producer Clones At Genetsupporting

confidence: 92%

“…In general the degree of gene amplification is proportional to the level of gene expression (Guarna et al, 1995;Jiang et al, 2006;Pendse et al, 1992). However, high gene copy numbers do not always result in high productivity, probably the result of transcriptional and post-transcriptional limitations in highly amplified subclones (Kim et al, 1998b;Lattenmayer et al, 2007). Gene amplification and high-level expression of a foreign gene product can challenge the cells with different levels of toxicity and metabolic burden.…”

Section: Introductionmentioning

confidence: 99%

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A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Chusainow

Yang

Yeo

et al. 2008

Biotech & Bioengineering

279

223

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Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell À1 day À1 using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high-and lowproducer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.

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Section: Comparison High-producer Versus Low-producer Clones At Genetsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Chusainow

Yang

Yeo

et al. 2008

Biotech & Bioengineering

279

223

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“…In both, CHO as well as HEK cells, IgM-012 producing cell line showed several times lower specific productivity and antibody titer levels than IgM-617 producing cell line (sevenfold difference in CHO cells, 22-fold difference in HEK cells). Although CHO and HEK IgM-617 cell lines manifested same or higher gene copy numbers for HC, LC, and JC coding genes than IgM-012 cell lines (with dramatically higher values for CHO IgM-617 cells), this fact is not reflected at the transgene transcript and specific productivity level, similarly as shown previously (Lattenmayer et al 2007b;Reisinger et al 2008). These studies as well as our results indicated that many of coding gene copies are probably incorporated into places, which are not as favorable for transcription initiation or are not active at all.…”

Section: Discussionsupporting

confidence: 80%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulumstress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

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“…As a comparison, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin have 1.9 Â 10 4 and 5.4 Â 10 3 RPKM, respectively. The high abundance level of both heavy and light chain transcripts in hyperproducing cells is consistent with similar observations in both CHO [Lattenmayer, 2007] and mouse myeloma cells [Dorai, 2006], suggesting that abundance of heavy and light chain mRNA transcripts may be important to high productivity. Ontology terms of the 500 most abundant CHO unigenes, as determined by Illumina Solexa read mapping, were analyzed and found to be highly represented in ribosomal genes, as well as members of glycolysis and the electron transport chain, lysosomal genes, and nucleosome assembly genes.…”

Section: Mapping and Transcript Abundance Calculationsupporting

confidence: 85%

Reaching the depth of the Chinese hamster ovary cell transcriptome

Jacob

Kantardjieff

Yusufi

et al. 2010

Biotech & Bioengineering

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The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to highly variable. This observation suggests that more challenges are yet to be resolved before direct sequencing can be used as a true quantitative measure of transcript level and for differential gene expression analysis. With the depth that high-throughput sequencing methods can reach, one can expect that the entire transcriptome of this industrially important organism will be decoded in the near future.

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Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters

Cited by 49 publications

References 34 publications

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

Reaching the depth of the Chinese hamster ovary cell transcriptome

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