Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo)

Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo) Publisher Pubmed

Zamanian S¹ ; Mohammadiyeganeh S^{2, 3} ; Kia V⁴ ; Kaghazian H⁵ ; Paryan M⁵

Show Affiliations

1. Faculty of Science, University of Guilan, Department of Biology, Rasht, Iran
2. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3. Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4. Faculty of Medicine, Department of Medical Biotechnology, Zanjan University of Medical Sciences, Zanjan, Iran
5. Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran

Source: Preparative Biochemistry and Biotechnology Published:2017

Abstract

During the purification of human recombinant erythropoietin (rEPO) from host cells, residual DNA may remain in final products. This contamination is a risk factor for patients and may result in the inactivation of some tumor suppressor genes or activation of oncogenes if its concentration is more than the standard defined by WHO. Based on WHO’s criteria, acceptable level of residual DNA in biopharmaceuticals is less than 10–100 pg/dose. In this study, we have designed a sensitive and specific quantitative real-time polymerase chain reaction (PCR) assay for the detection of residual DNA in human rEPO products. All reported sequences of CHO’s GAPDH gene were retrieved from GenBank, and a multiple alignment was performed using Mega 6 software to find conserved regions of the gene. Primers and probe were designed by AlleleID7 software for the highly conserved region. Quantitative real-time PCR showed an R2 value more than 0.99 and the efficiency equal to 101% indicating a highly accurate and efficiency of the reaction, respectively. Based on the standard curve, the limit of detection of the assay was determined to be 10 copies/µL (0.00967 fg/µL). In addition, the inter- and intra-assay of the test were determined to be 1.14% and 0.65%, respectively, which are in acceptable range according to the WHO’s guidelines. © 2017 Taylor & Francis.

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Style	Citing Format
MLA	Zamanian S, et al.. "Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo)." Preparative Biochemistry and Biotechnology, vol. 47, no. 9, 2017, pp. 847-851.
APA	Zamanian S, Mohammadiyeganeh S, Kia V, Kaghazian H, Paryan M (2017). Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo). Preparative Biochemistry and Biotechnology, 47(9), 847-851.
Chicago	Zamanian S, Mohammadiyeganeh S, Kia V, Kaghazian H, Paryan M. "Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo)." Preparative Biochemistry and Biotechnology 47, no. 9 (2017): 847-851.
Harvard	Zamanian S et al. (2017) 'Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo)', Preparative Biochemistry and Biotechnology, 47(9), pp. 847-851.
Vancouver	Zamanian S, Mohammadiyeganeh S, Kia V, Kaghazian H, Paryan M. Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo). Preparative Biochemistry and Biotechnology. 2017;47(9):847-851.
BibTex	@article{ author = {Zamanian S and Mohammadiyeganeh S and Kia V and Kaghazian H and Paryan M}, title = {Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo)}, journal = {Preparative Biochemistry and Biotechnology}, volume = {47}, number = {9}, pages = {847-851}, year = {2017} }
RIS	TY - JOUR AU - Zamanian S AU - Mohammadiyeganeh S AU - Kia V AU - Kaghazian H AU - Paryan M TI - Design and Validation of a New Method to Detect and Quantify Residual Host Cell Dna in Human Recombinant Erythropoietin (Repo) JO - Preparative Biochemistry and Biotechnology VL - 47 IS - 9 SP - 847 EP - 851 PY - 2017 ER -

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