title: Quantitation of the residual DNA from rice-derived recombinant human serum albumin.
abstract: 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.
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Chen
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China.
authors.forename: Huixia
authors.initials: H
authors.lastname: Dai
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China.
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China.
authors.forename: Jianlei
authors.initials: J
authors.lastname: Pang
authors.affiliation: Hubei Engineering Research Center for Molecular Pharming, Biolake, Wuhan 430076, China.
authors.forename: Jiquan
authors.initials: J
authors.lastname: Ou
authors.affiliation: College of Life Sciences, State Key Laboratory of Hybrid Rice, Wuhan University, Wuhan 430072, China; Hubei Engineering Research Center for Molecular Pharming, Biolake, Wuhan 430076, China. Electronic address: dyang@whu.edu.cn.
authors.forename: Daichang
authors.initials: D
authors.lastname: Yang
mesh_terms: D052160:Benzothiazoles; D002138:Calibration; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D003959:Diamines; D004340:Drug Contamination; D018745:Genome, Plant / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D009930:Organic Chemicals / Q000737:chemistry; D012275:Oryza / Q000235:genetics*; D011804:Quinolines; D018749:RNA, Plant / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D018744:DNA, Plant; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D018749:RNA, Plant; D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D012709:Serum Albumin; C098022:SYBR Green I; D019914:Taq Polymerase
keywords: Host cell residual DNA; Molecular pharming; OsrHSA; Rice seed bioreactor; qPCR