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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.


Related in: MedlinePlus

Influence of disrupted CHO cells on LRE qPCR for quantification of a CHO genomic target. The same samples as used in Fig. 4 were used to assess the effect of cellular material on LRE qPCR performance. The predicted number of copies of the GAPDH target sequence in a sample, as calculated using the LRE qPCR method, were plotted as a function of sample dilution for samples derived from shake flask (a) and bioreactor (b) cultivation. Samples either underwent total DNA purification (open squares) or only mild cell disruption (open triangles) prior to LRE qPCR procedure. Genome copy number was also inferred from spectrophotometric measurement of total DNA concentration. These spectrophotometric measurements are indicated by large grey circles and are linearly extrapolated for both purified DNA (thick dashed line) or disrupted cell (fine dashed line) samples
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Fig7: Influence of disrupted CHO cells on LRE qPCR for quantification of a CHO genomic target. The same samples as used in Fig. 4 were used to assess the effect of cellular material on LRE qPCR performance. The predicted number of copies of the GAPDH target sequence in a sample, as calculated using the LRE qPCR method, were plotted as a function of sample dilution for samples derived from shake flask (a) and bioreactor (b) cultivation. Samples either underwent total DNA purification (open squares) or only mild cell disruption (open triangles) prior to LRE qPCR procedure. Genome copy number was also inferred from spectrophotometric measurement of total DNA concentration. These spectrophotometric measurements are indicated by large grey circles and are linearly extrapolated for both purified DNA (thick dashed line) or disrupted cell (fine dashed line) samples

Mentions: Reactions were carried out in a total volume of 20 µL, with each reaction containing 10 µL of 2× SsoAdvanced SYBR Green Supermix (BioRad, Hercules, CA, USA), 5 µL of material containing template DNA and 1 µL of primer at a concentration of 1 µM (to give a final concentration of 500 nM of each primer per reaction). Reactions were performed in a CFX Connect Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) with a cover heated to 105 °C. Each reaction was run at a total of 40 cycles, with the same cycling conditions as above. For qPCR experiments plotted in Figs. 4, 5, 7, 9 and 10, each sample was split into three and amplified in separate wells of a 96 well plate. The average of the resultant Cq values formed the Cq value or copy number value and the standard deviation of all three was used to plot error bars. These error balls always fell within the areas of the symbols or lines used to indicate data points.Fig. 4


A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction
Influence of disrupted CHO cells on LRE qPCR for quantification of a CHO genomic target. The same samples as used in Fig. 4 were used to assess the effect of cellular material on LRE qPCR performance. The predicted number of copies of the GAPDH target sequence in a sample, as calculated using the LRE qPCR method, were plotted as a function of sample dilution for samples derived from shake flask (a) and bioreactor (b) cultivation. Samples either underwent total DNA purification (open squares) or only mild cell disruption (open triangles) prior to LRE qPCR procedure. Genome copy number was also inferred from spectrophotometric measurement of total DNA concentration. These spectrophotometric measurements are indicated by large grey circles and are linearly extrapolated for both purified DNA (thick dashed line) or disrupted cell (fine dashed line) samples
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Influence of disrupted CHO cells on LRE qPCR for quantification of a CHO genomic target. The same samples as used in Fig. 4 were used to assess the effect of cellular material on LRE qPCR performance. The predicted number of copies of the GAPDH target sequence in a sample, as calculated using the LRE qPCR method, were plotted as a function of sample dilution for samples derived from shake flask (a) and bioreactor (b) cultivation. Samples either underwent total DNA purification (open squares) or only mild cell disruption (open triangles) prior to LRE qPCR procedure. Genome copy number was also inferred from spectrophotometric measurement of total DNA concentration. These spectrophotometric measurements are indicated by large grey circles and are linearly extrapolated for both purified DNA (thick dashed line) or disrupted cell (fine dashed line) samples
Mentions: Reactions were carried out in a total volume of 20 µL, with each reaction containing 10 µL of 2× SsoAdvanced SYBR Green Supermix (BioRad, Hercules, CA, USA), 5 µL of material containing template DNA and 1 µL of primer at a concentration of 1 µM (to give a final concentration of 500 nM of each primer per reaction). Reactions were performed in a CFX Connect Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) with a cover heated to 105 °C. Each reaction was run at a total of 40 cycles, with the same cycling conditions as above. For qPCR experiments plotted in Figs. 4, 5, 7, 9 and 10, each sample was split into three and amplified in separate wells of a 96 well plate. The average of the resultant Cq values formed the Cq value or copy number value and the standard deviation of all three was used to plot error bars. These error balls always fell within the areas of the symbols or lines used to indicate data points.Fig. 4

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.


Related in: MedlinePlus