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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 e-pPCR detection of a mycoplasmal target sequence. 5 ng of plasmid encoding a mycoplasmal DNA sequence (1.54 × 109 copies) was used as e-pPCR template either as purified DNA or purified DNA plus disrupted cells derived from a sample containing a total of 2 × 106 cells from shake flask (a) and 2.5 × 105 cells from bioreactor (b) cultivation. For both cultivation methods the following data are depicted. The mass of amplicon produced in a reaction is plotted as a function of sample dilution (i). Inlaid graphs (ii) plot the area (arbitrary units) under each curve as a bar chart. Agarose gel images show the 184 bp amplicons generated from the purified plasmid DNA (iii) and plasmid DNA plus disrupted CHO cells (iv). The number of copies of the plasmid molecule in a given sample is indicated in the row labelled (v)
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Fig8: Influence of disrupted CHO cells on e-pPCR detection of a mycoplasmal target sequence. 5 ng of plasmid encoding a mycoplasmal DNA sequence (1.54 × 109 copies) was used as e-pPCR template either as purified DNA or purified DNA plus disrupted cells derived from a sample containing a total of 2 × 106 cells from shake flask (a) and 2.5 × 105 cells from bioreactor (b) cultivation. For both cultivation methods the following data are depicted. The mass of amplicon produced in a reaction is plotted as a function of sample dilution (i). Inlaid graphs (ii) plot the area (arbitrary units) under each curve as a bar chart. Agarose gel images show the 184 bp amplicons generated from the purified plasmid DNA (iii) and plasmid DNA plus disrupted CHO cells (iv). The number of copies of the plasmid molecule in a given sample is indicated in the row labelled (v)

Mentions: A common application of ePCR is the binary detection of organisms known to contaminate cultures of mammalian cells at industrial scale. As such we designed the 3010 bp pPROX2 plasmid containing a 300 bp sequence conserved across five species of mycoplasma (Kong et al. 2001) and used this as a safe proxy test of the sensitivity of e-pPCR for mycoplasma detection. We used a mycoplasma genomic locus sequence present in pPROX2 as this afforded us (i) exquisite control over the copy number of the gene achievable by serial dilution into our samples and (ii) a safer option than using material directly derived from mycoplasma which could possibly be contaminated with live mycoplasma cells. Serial dilutions of a solution containing 5 ng (1.54 × 109 copies) of the pPROX2 plasmid were made and to each dilution a constant volume of either water or disrupted cells was added (Fig. 8). Disrupted cells were generated from a sample containing 2 × 106 cells/mL from shake flask cultivation (Fig. 8a) or from a sample of 2.5 × 105 cells/mL from bioreactor cultivation (Fig. 8b). The LOD for naked DNA template was 154 copies (0.5 fg pDNA). This was increased tenfold to 1540 copies (5 fg pDNA) by the presence of disrupted cells, from either shake flask (Fig. 8a) or bioreactor (Fig. 8b) cultivation. Total amplicon production, with either pure DNA or disrupted cells as template, was similar for shake flask (Fig. 8a-ii) and bioreactor (Fig. 8b-ii) cultivation.


A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction
Influence of disrupted CHO cells on e-pPCR detection of a mycoplasmal target sequence. 5 ng of plasmid encoding a mycoplasmal DNA sequence (1.54 × 109 copies) was used as e-pPCR template either as purified DNA or purified DNA plus disrupted cells derived from a sample containing a total of 2 × 106 cells from shake flask (a) and 2.5 × 105 cells from bioreactor (b) cultivation. For both cultivation methods the following data are depicted. The mass of amplicon produced in a reaction is plotted as a function of sample dilution (i). Inlaid graphs (ii) plot the area (arbitrary units) under each curve as a bar chart. Agarose gel images show the 184 bp amplicons generated from the purified plasmid DNA (iii) and plasmid DNA plus disrupted CHO cells (iv). The number of copies of the plasmid molecule in a given sample is indicated in the row labelled (v)
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Related In: Results  -  Collection

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Fig8: Influence of disrupted CHO cells on e-pPCR detection of a mycoplasmal target sequence. 5 ng of plasmid encoding a mycoplasmal DNA sequence (1.54 × 109 copies) was used as e-pPCR template either as purified DNA or purified DNA plus disrupted cells derived from a sample containing a total of 2 × 106 cells from shake flask (a) and 2.5 × 105 cells from bioreactor (b) cultivation. For both cultivation methods the following data are depicted. The mass of amplicon produced in a reaction is plotted as a function of sample dilution (i). Inlaid graphs (ii) plot the area (arbitrary units) under each curve as a bar chart. Agarose gel images show the 184 bp amplicons generated from the purified plasmid DNA (iii) and plasmid DNA plus disrupted CHO cells (iv). The number of copies of the plasmid molecule in a given sample is indicated in the row labelled (v)
Mentions: A common application of ePCR is the binary detection of organisms known to contaminate cultures of mammalian cells at industrial scale. As such we designed the 3010 bp pPROX2 plasmid containing a 300 bp sequence conserved across five species of mycoplasma (Kong et al. 2001) and used this as a safe proxy test of the sensitivity of e-pPCR for mycoplasma detection. We used a mycoplasma genomic locus sequence present in pPROX2 as this afforded us (i) exquisite control over the copy number of the gene achievable by serial dilution into our samples and (ii) a safer option than using material directly derived from mycoplasma which could possibly be contaminated with live mycoplasma cells. Serial dilutions of a solution containing 5 ng (1.54 × 109 copies) of the pPROX2 plasmid were made and to each dilution a constant volume of either water or disrupted cells was added (Fig. 8). Disrupted cells were generated from a sample containing 2 × 106 cells/mL from shake flask cultivation (Fig. 8a) or from a sample of 2.5 × 105 cells/mL from bioreactor cultivation (Fig. 8b). The LOD for naked DNA template was 154 copies (0.5 fg pDNA). This was increased tenfold to 1540 copies (5 fg pDNA) by the presence of disrupted cells, from either shake flask (Fig. 8a) or bioreactor (Fig. 8b) cultivation. Total amplicon production, with either pure DNA or disrupted cells as template, was similar for shake flask (Fig. 8a-ii) and bioreactor (Fig. 8b-ii) cultivation.

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