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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 amplification efficiency for a genomic target. Real time PCR was performed using disrupted cells (grey symbols and lines) or purified DNA (black symbols and lines) from shake flask (a) and bioreactor (b) cultivation as template. Undiluted shake flask cell-sonicate and purified DNA samples were estimated to contain 115 gDNA (5.1 × 104 genome copies) by spectrophotometry and densitomery. Undiluted bioreactor cell-sonicate and purified DNA samples were estimated to contain 1 µg gDNA (3.78 × 105 genome copies) by spectrophotometry and densitomery. Cq values were plotted against tenfold dilutions of template source. Lines indicate data points for which amplification efficiency is 100 ± 10 % efficiency, at a confidence level of R2 > 0.99. Data featured is typical of n = 3 analytical repeats
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Fig4: Influence of disrupted CHO cells on amplification efficiency for a genomic target. Real time PCR was performed using disrupted cells (grey symbols and lines) or purified DNA (black symbols and lines) from shake flask (a) and bioreactor (b) cultivation as template. Undiluted shake flask cell-sonicate and purified DNA samples were estimated to contain 115 gDNA (5.1 × 104 genome copies) by spectrophotometry and densitomery. Undiluted bioreactor cell-sonicate and purified DNA samples were estimated to contain 1 µg gDNA (3.78 × 105 genome copies) by spectrophotometry and densitomery. Cq values were plotted against tenfold dilutions of template source. Lines indicate data points for which amplification efficiency is 100 ± 10 % efficiency, at a confidence level of R2 > 0.99. Data featured is typical of n = 3 analytical repeats

Mentions: DNA was purified as detailed below from the shake flask (Fig. 2a) and wave-bag bioreactor (Fig. 2b) samples to determine typical DNA measurements by spectrophotometry. After this scoping study, the volume of sample, ranging from 1.6 to 6.5 mL, required to provide the DNA concentration in the undiluted template reactions indicated in Figs. 3 and 4, was centrifuged at 10,000 RPM for 3 min, re-suspended in 400 µL of lysis buffer (2 % Triton X100, 1 % SDS, 100 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA) and freeze-thawed twice by incubating at −80 °C for 3 min and 95 °C for 1 min. Total nucleic acid was purified using standard phenol/ethanol extraction procedure and resuspended in 400 µL 10 mM Tris buffer (pH 7.5). Six aliquots of purified DNA were made and stored at −20 °C. A given aliquot was thawed once for experimentation and any unused portion of the aliquot discarded. The proxy plasmid pPROX2 was purified with a Key Prep ‘mini prep’ kit (Anachem, Luton, UK).


A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction
Influence of disrupted CHO cells on amplification efficiency for a genomic target. Real time PCR was performed using disrupted cells (grey symbols and lines) or purified DNA (black symbols and lines) from shake flask (a) and bioreactor (b) cultivation as template. Undiluted shake flask cell-sonicate and purified DNA samples were estimated to contain 115 gDNA (5.1 × 104 genome copies) by spectrophotometry and densitomery. Undiluted bioreactor cell-sonicate and purified DNA samples were estimated to contain 1 µg gDNA (3.78 × 105 genome copies) by spectrophotometry and densitomery. Cq values were plotted against tenfold dilutions of template source. Lines indicate data points for which amplification efficiency is 100 ± 10 % efficiency, at a confidence level of R2 > 0.99. Data featured is typical of n = 3 analytical repeats
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016487&req=5

Fig4: Influence of disrupted CHO cells on amplification efficiency for a genomic target. Real time PCR was performed using disrupted cells (grey symbols and lines) or purified DNA (black symbols and lines) from shake flask (a) and bioreactor (b) cultivation as template. Undiluted shake flask cell-sonicate and purified DNA samples were estimated to contain 115 gDNA (5.1 × 104 genome copies) by spectrophotometry and densitomery. Undiluted bioreactor cell-sonicate and purified DNA samples were estimated to contain 1 µg gDNA (3.78 × 105 genome copies) by spectrophotometry and densitomery. Cq values were plotted against tenfold dilutions of template source. Lines indicate data points for which amplification efficiency is 100 ± 10 % efficiency, at a confidence level of R2 > 0.99. Data featured is typical of n = 3 analytical repeats
Mentions: DNA was purified as detailed below from the shake flask (Fig. 2a) and wave-bag bioreactor (Fig. 2b) samples to determine typical DNA measurements by spectrophotometry. After this scoping study, the volume of sample, ranging from 1.6 to 6.5 mL, required to provide the DNA concentration in the undiluted template reactions indicated in Figs. 3 and 4, was centrifuged at 10,000 RPM for 3 min, re-suspended in 400 µL of lysis buffer (2 % Triton X100, 1 % SDS, 100 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA) and freeze-thawed twice by incubating at −80 °C for 3 min and 95 °C for 1 min. Total nucleic acid was purified using standard phenol/ethanol extraction procedure and resuspended in 400 µL 10 mM Tris buffer (pH 7.5). Six aliquots of purified DNA were made and stored at −20 °C. A given aliquot was thawed once for experimentation and any unused portion of the aliquot discarded. The proxy plasmid pPROX2 was purified with a Key Prep ‘mini prep’ kit (Anachem, Luton, UK).

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