Development of a Semi-Quantitative Multiplex PCR Method for Detecting Residual Pichia Pastoris Host Cell DNA in Biopharmaceuticals
Trends in Peptide and Protein Sciences,
Vol. 1 No. 2 (2016),
17 January 2017
,
Page 61-67
https://doi.org/10.22037/tpps.v1i2.15107
Abstract
The use of the methylotrophic yeast, Pichia pastoris, as one of the most effective and versatile systems for the expression of heterologous proteins in biopharmaceutical manufacturing has become increasingly popular in recent years. The impurity caused by residual host cell DNA is one of the major concerns in production of recombinant therapeutics. The aim of the present study was to develop a semi-quantitative, multiplex PCR method to determine the level of impurity in biopharmaceuticals produced in Pichia pastoris as the host. Primers were designed based on the rDNA repeat region and optimized to achieve the limit of detection in a multiplex PCR following by analyzing with MYImageAnalysis (Thermo Fisher Scientific, USA) software to quantify the concentration of Pichia pastoris genomic DNA in pertinent controls and drug samples. The multiplex PCR were able to detect up to 1 femtogram (fg) of genomic DNA under optimized conditions. Moreover, achieved concentration of DNA in controls and samples through relevant standard curve indicates the feasibility of this method in the presence of inhibitory effects. In comparison with other methods such as real-time PCR and Threshold assay, the assay shows acceptable sensitivity, precision and linearity along with ease of use, low equipment costs and analyte flexibility. We thus propose this method to be considered as a useful tool to estimate host cell residual DNA in biopharmaceuticals produced in Pichia pastoris.
Highlights
- The impurity of residual host cell DNA is an important concern in production of biopharmaceuticals.
- Pichia pastoris is an effective and versatile system for the expression of recombinant proteins
- Quantitative Polymerase Chain Reaction could be used for quantifying residual host-cell DNA
- We designed a sensitive and valid PCR method for detection and quantification of Pichia residual DNA
- Residual DNA
- Yeast
- Pichia pastoris
- Multiplex PCR
- Gel densitometry
How to Cite
References
Cai, H., Gu, X., Scanlan, M. S. and C. R. Lively, (2011). ″Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation.″ Journal of Pharmaceutical and Biomedical Analysis, 55: 71-77.
Cereghino, J. L. and J. M. Cregg, (2000). ″Heterologous protein expression in the methylotrophic yeast Pichia pastoris.″ FEMS Microbiology Reviews, 24: 45-66.
Durrant, I., Brunning, S., Eccleston, L., Chadwick, P. and M. Cunningham, (1995). ″Fluorescein as a label for non-radioactive in situ hybridization.″ The Histochemical Journal, 27: 94–99.
Forcic, D., Cakanic, K. B., Ivancic, J., Jug, R., Barut, M., Strancar, A. and R. Mazuran, (2005). ″Chromatographic detection of residual cellular DNA on short monolithic columns.″ Analytical Biochemistry, 336: 273-278.
Gerngross, T. U. (2004). ″Advances in the production of human therapeutic proteins in yeasts and filamentous fungi.″ Nature Biotechnology, 22: 1409-1414.
Ikeda, Y., Iwakiri, S. and T. Yoshimori, (2009). ″Development and characterization of a novel host cell DNA assay using ultra-sensitive fluorescent nucleic acid stain ″PicoGreen″.″ Journal of Pharmaceutical and Biomedical Analysis, 49: 997-1002.
Lebron, J. A., Troilol, P. J., Pacchione, S., Griffiths, T. G., Harper, L. B., Mixson, L. A., Jackson, B.E., Michna, L., Barnum, A. B., Denisova, L., Johnson, C. N., Maurer, K. L., Morgan-Hoffman, S., Niu, Z., Roden, D. F., Wang, Z., Wolf, J. J., Hamilton, T. R., Laux, K. M., Soper, K. A. and B. J. Ledwith, (2006). ″Adaptation of the WHO guideline for residual DNA in parenteral vaccines produced on continuous cell lines to a limit for oral vaccines.″ Developments in Biologicals, 123: 35-44.
Lee, D. H., Bae, J. E., Lee, J. H, Shin, J. S and I. S. Kim, (2010). ″Quantitative Detection of Residual E. coli Host Cell DNA by Real-Time PCR.″ Journal of Microbiology and Biotechnology, 20:1463-1470.
Lokteff, M., Klinguer-Hamour, C., Julien, E., Picot, D., Lannes, L., Nguyen, T., Bonnefoy, J. Y. and A. Beck, (2001). ″Residual DNA quantification in clinical batches of BBG2Na, a recombinant subunit vaccine against human respiratory syncytial virus.″ Biologicals, 29: 123–132.
Lovatt, A. (2002). ″Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products.″ Journal of Biotechnology, 82: 279-300.
Macauley-Patrick, S., Fazenda, M.L., McNeil, B. and L. M. Harvey, (2005).″ Heterologous protein production using the Pichia pastoris expression system. ″ Yeast, 22: 249-270.
Nissom, P. M. (2007). ″Specific detection of residual CHO host cell DNA by real-time PCR.″ Biologicals, 35: 211-5.
Potvin, G., Ahmad, A. and Z. Zhang, (2012). ″Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review.″ Biochemical Engineering Journal, 64: 91-105.
Schmidt, F. R. (2004). ″Recombinant expression systems in the pharmaceutical industry.″ Applied Microbiology and Biotechnology, 65: 363-372.
Shen, X., Chen, X., Tabor, D. E., Liu, Y., Albarghouthi, M., Zhang, Y. F. and M. S. Galinski, (2013). ″Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis.″ Biologicals, 41: 201-8.
Sheng-Fowler, L., Lewis, J. R. and K. Peden, (2009). ″Issues associated with residual cell-substrate DNA in viral vaccines.″ Biologicals, 37: 190-5.
Strachan, T. and A. P. Read, (1999). ″Nucleic acid hybridization assays″. In: Human Molecular Genetics. 2nd ed., Wiley-Liss, Inc., New York, pp. 95–119.
Verardo, M. L., Carvalho, J. G., Delgado, D. N. and S. T. Kuhns, (2012). ″Accuracy and sensitivity of residual DNA detection by QPCR is not predicted by target copy number.″ Biotechnology Progress, 28: 428-34.
Walsh, G. (2010). ″Biopharmaceutical benchmark.″ Nature Biotechnology, 28: 917-24.
Wang, X., Morgan, D. M., Wang, G. and N. M. Mozier, (2012). ″Residual DNA analysis in biologics development: review of measurement and quantitation technologies and future directions.″ Biotechnology and Bioengineering, 109: 307-17.
Yang, H., Zhang, L. and M. Galinski, (2010). ″A probabilistic model for risk assessment of residual host cell DNA in biological products.″ Vaccine, 28: 3308-3311.
- Abstract Viewed: 870 times
- PDF Downloaded: 476 times