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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

Gel analysis of gDNA and overview of polymerase chain reactions in this study. Primers (black triangles) detailed in Table 2 were used to amplify a mycoplasma sequence present in the plasmid pPROX2 (3010 bp) as a proxy for pathogen detection (a), target DNA within the mammalian GAPDH gene in the CHO genome (b) and the designated CAL1 locus with the lambda phage genome (c). Expected amplicon size (bp) is indicated under the bar at the bottom of each panel. (d) 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel before sonication in the lane marked ‘C’. 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel after sonication in the lane marked ‘S’. Molecular weight ladder was run in leftmost lane, uppermost band is 10 kilo-base pairs
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Fig1: Gel analysis of gDNA and overview of polymerase chain reactions in this study. Primers (black triangles) detailed in Table 2 were used to amplify a mycoplasma sequence present in the plasmid pPROX2 (3010 bp) as a proxy for pathogen detection (a), target DNA within the mammalian GAPDH gene in the CHO genome (b) and the designated CAL1 locus with the lambda phage genome (c). Expected amplicon size (bp) is indicated under the bar at the bottom of each panel. (d) 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel before sonication in the lane marked ‘C’. 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel after sonication in the lane marked ‘S’. Molecular weight ladder was run in leftmost lane, uppermost band is 10 kilo-base pairs

Mentions: Cell suspensions from the shake flask (Fig. 2a) and wave-bag bioreactor (Fig. 2b) samples were sonicated as detailed below to determine typical DNA estimations by spectrophotometry and densitometry. After this the volume of sample, ranging from 1.6 to 6.5 mL, required to provide the DNA concentration in the undiluted cell sonicate template reactions indicated in Figs. 3 and 4, was centrifuged at 10,000 RPM for 3 min and re-suspended in 400 µL dH2O. A Soniprep 150 sonicator (MSE, London, UK) was used to subject samples to three repeats of the following procedure: 10 s cycles of 100 % amplitude sonication followed by 10 s rest, for a total duration of 60 s. 60 µL of a 2.5 × 106 cell/mL cell suspension from shake flask cultivation was ran on a standard 1 % w/v agarose gel before sonication and 60 µL ran after sonication. Figure 1 shows the gel and the same pattern was observed for cells cultivated in bioreactors.Fig. 1


A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction
Gel analysis of gDNA and overview of polymerase chain reactions in this study. Primers (black triangles) detailed in Table 2 were used to amplify a mycoplasma sequence present in the plasmid pPROX2 (3010 bp) as a proxy for pathogen detection (a), target DNA within the mammalian GAPDH gene in the CHO genome (b) and the designated CAL1 locus with the lambda phage genome (c). Expected amplicon size (bp) is indicated under the bar at the bottom of each panel. (d) 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel before sonication in the lane marked ‘C’. 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel after sonication in the lane marked ‘S’. Molecular weight ladder was run in leftmost lane, uppermost band is 10 kilo-base pairs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Gel analysis of gDNA and overview of polymerase chain reactions in this study. Primers (black triangles) detailed in Table 2 were used to amplify a mycoplasma sequence present in the plasmid pPROX2 (3010 bp) as a proxy for pathogen detection (a), target DNA within the mammalian GAPDH gene in the CHO genome (b) and the designated CAL1 locus with the lambda phage genome (c). Expected amplicon size (bp) is indicated under the bar at the bottom of each panel. (d) 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel before sonication in the lane marked ‘C’. 60 µL of a 2.5 × 106 cell/mL cell suspension was ran on a gel after sonication in the lane marked ‘S’. Molecular weight ladder was run in leftmost lane, uppermost band is 10 kilo-base pairs
Mentions: Cell suspensions from the shake flask (Fig. 2a) and wave-bag bioreactor (Fig. 2b) samples were sonicated as detailed below to determine typical DNA estimations by spectrophotometry and densitometry. After this the volume of sample, ranging from 1.6 to 6.5 mL, required to provide the DNA concentration in the undiluted cell sonicate template reactions indicated in Figs. 3 and 4, was centrifuged at 10,000 RPM for 3 min and re-suspended in 400 µL dH2O. A Soniprep 150 sonicator (MSE, London, UK) was used to subject samples to three repeats of the following procedure: 10 s cycles of 100 % amplitude sonication followed by 10 s rest, for a total duration of 60 s. 60 µL of a 2.5 × 106 cell/mL cell suspension from shake flask cultivation was ran on a standard 1 % w/v agarose gel before sonication and 60 µL ran after sonication. Figure 1 shows the gel and the same pattern was observed for cells cultivated in bioreactors.Fig. 1

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