Quantitation of the residual DNA from rice-derived recombinant human serum albumin
| Autor: | Huixia Dai, Daichang Yang, Zhenwei Liu, Liping Zhang, Zhen Chen, Jiquan Ou, Jianlei Pang |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
DNA
Plant Biophysics Diamines Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Biochemistry law.invention chemistry.chemical_compound Limit of Detection law medicine TaqMan Humans Taq Polymerase Benzothiazoles Organic Chemicals Molecular Biology Gene Escherichia coli Serum Albumin Polymerase chain reaction DNA Primers Chinese hamster ovary cell RNA Ribosomal 5S Reproducibility of Results food and beverages Oryza Cell Biology Molecular biology Recombinant Proteins chemistry RNA Plant Calibration Quinolines Recombinant DNA SYBR Green I Drug Contamination Genome Plant DNA |
| Zdroj: | Analytical Biochemistry. 450:4-10 |
| ISSN: | 0003-2697 |
| Popis: | Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect