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A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction

View Article: PubMed Central - PubMed

ABSTRACT

Background: Chinese Hamster Ovary (CHO) cells are the current industry standard for production of therapeutic monoclonal antibodies at commercial scales. Production optimisation in CHO cells hinges on analytical technologies such as the use of the polymerase chain reaction (PCR) to quantify genetic factors within the CHO genome and to detect the presence of contaminant organisms. PCR-based assays, whilst sensitive and accurate, are limited by (i) requiring lengthy sample preparation and (ii) a lack of standardisation.

Results: In this study we directly assess for the first time the effect of CHO cellular material on quantitative PCR (qPCR) and end-point PCR (e-pPCR) when used to measure and detect copies of a CHO genomic locus and a mycoplasma sequence. We also perform the first head-to-head comparison of the performance of a conventional qPCR method to that of the novel linear regression of efficiency (LRE) method when used to perform absolute qPCR on CHO-derived material. LRE qPCR features the putatively universal ‘CAL1’ standard.

Conclusions: We find that sample preparation is required for accurate quantitation of a genomic target locus, but mycoplasma DNA sequences can be detected in the presence of high concentrations of CHO cellular material. The LRE qPCR method matches performance of a conventional qPCR approach and as such we invite the synthetic biology community to adopt CAL1 as a synthetic biology calibration standard for qPCR.

No MeSH data available.


Cultivation of CHO cells. Growth profile of CHO cells in 1L Erlenmeyer flasks (a) and in an Applikon 3L rocked bag bioreactor fermentation (b). Samples for PCR experiments were taken at the time points indicated (closed symbols)
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Fig2: Cultivation of CHO cells. Growth profile of CHO cells in 1L Erlenmeyer flasks (a) and in an Applikon 3L rocked bag bioreactor fermentation (b). Samples for PCR experiments were taken at the time points indicated (closed symbols)

Mentions: A clonally derived glutamine synthase (GS) CHO cell line stably expressing an antibody-based therapeutic was cultivated in 1L Erlenmeyer shake flasks (SF) according to the protocol described by Velez-Suberbie et al. (2013) until a viable cell count of 2.5 × 106 cell/mL was reached, as determined by ViCell-XR cell viability analyser (Beckman Coulter, USA). At this point a sample was taken for further PCR experiments (Fig. 2a). This stage represents a critical point of industrial scale cultivation where the seed train is used to inoculate the larger scale growth vessel. The shake flask culture was added to CD-CHO media in a rocked bag bioreactor to an initial concentration of 2.5 × 105 cells/mL. Bioreactor cultivation was performed in a 3L Applikon Appliflex (Applikon, Holland) flexible rocked bag bioreactor, controlled by an Applikon EZ controller system. Temperature was kept at 37 °C with the dissolved oxygen (DO) set-point at 30 % and the pH set-point at 7.1 ± 0.05. Glucose concentration was maintained at 150 g/L, as determined by NOVA Bioanalyser 400 (NOVA Biomedical, Waltham, USA), by supplementing with 10× concentrated CD-CHO media (Life Technologies, Paisley, UK). Cells grew to achieve 1 × 107 cells/mL with ~99 % viability (Fig. 2b) in the bioreactor and a sample was removed for PCR.


A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction
Cultivation of CHO cells. Growth profile of CHO cells in 1L Erlenmeyer flasks (a) and in an Applikon 3L rocked bag bioreactor fermentation (b). Samples for PCR experiments were taken at the time points indicated (closed symbols)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016487&req=5

Fig2: Cultivation of CHO cells. Growth profile of CHO cells in 1L Erlenmeyer flasks (a) and in an Applikon 3L rocked bag bioreactor fermentation (b). Samples for PCR experiments were taken at the time points indicated (closed symbols)
Mentions: A clonally derived glutamine synthase (GS) CHO cell line stably expressing an antibody-based therapeutic was cultivated in 1L Erlenmeyer shake flasks (SF) according to the protocol described by Velez-Suberbie et al. (2013) until a viable cell count of 2.5 × 106 cell/mL was reached, as determined by ViCell-XR cell viability analyser (Beckman Coulter, USA). At this point a sample was taken for further PCR experiments (Fig. 2a). This stage represents a critical point of industrial scale cultivation where the seed train is used to inoculate the larger scale growth vessel. The shake flask culture was added to CD-CHO media in a rocked bag bioreactor to an initial concentration of 2.5 × 105 cells/mL. Bioreactor cultivation was performed in a 3L Applikon Appliflex (Applikon, Holland) flexible rocked bag bioreactor, controlled by an Applikon EZ controller system. Temperature was kept at 37 °C with the dissolved oxygen (DO) set-point at 30 % and the pH set-point at 7.1 ± 0.05. Glucose concentration was maintained at 150 g/L, as determined by NOVA Bioanalyser 400 (NOVA Biomedical, Waltham, USA), by supplementing with 10× concentrated CD-CHO media (Life Technologies, Paisley, UK). Cells grew to achieve 1 × 107 cells/mL with ~99 % viability (Fig. 2b) in the bioreactor and a sample was removed for PCR.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Chinese Hamster Ovary (CHO) cells are the current industry standard for production of therapeutic monoclonal antibodies at commercial scales. Production optimisation in CHO cells hinges on analytical technologies such as the use of the polymerase chain reaction (PCR) to quantify genetic factors within the CHO genome and to detect the presence of contaminant organisms. PCR-based assays, whilst sensitive and accurate, are limited by (i) requiring lengthy sample preparation and (ii) a lack of standardisation.

Results: In this study we directly assess for the first time the effect of CHO cellular material on quantitative PCR (qPCR) and end-point PCR (e-pPCR) when used to measure and detect copies of a CHO genomic locus and a mycoplasma sequence. We also perform the first head-to-head comparison of the performance of a conventional qPCR method to that of the novel linear regression of efficiency (LRE) method when used to perform absolute qPCR on CHO-derived material. LRE qPCR features the putatively universal ‘CAL1’ standard.

Conclusions: We find that sample preparation is required for accurate quantitation of a genomic target locus, but mycoplasma DNA sequences can be detected in the presence of high concentrations of CHO cellular material. The LRE qPCR method matches performance of a conventional qPCR approach and as such we invite the synthetic biology community to adopt CAL1 as a synthetic biology calibration standard for qPCR.

No MeSH data available.