Design and validation of a new method to detect and quantify residual host cell DNA in human recombinant erythropoietin (rEPO)

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 R 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.

Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical Products

“…Three methods(DNA hybridization assay, Threshold® assay, and quantitative PCR)have been recommended by regulatory agencies for residual host cell DNA quantitation [14, 15]. Among these methods, qPCR is considered to be the most practical for residual DNA quantitation due to their sensitivity, accuracy, precision, and time-saving features.…”

Establishment and evaluation of detection methods for process‐specific residual host cell protein and residual host cell DNA in biological preparation

“…The methods of rcDNA detection mainly include the fluorescent dye method, digoxin hybridization method, DNA-binding protein-based immunochromatography (threshold method), and qPCR method. [30][31][32][33] Among them, the qPCR method provides a reliable detection basis for the biopharmaceutical industry in process research and the quality control of finished products due to its high specificity of sequence, high speed, and high throughput. However, this method is relatively quantitative and still has defects in the accurate quantification of rcDNA.…”