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
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