title: Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation.
abstract: Reliable and sensitive assays are required to determine whether a pharmaceutical product meets current regulatory guidelines for residual host cell DNA. In this study, the sensitivity of the qPCR assay was significantly improved by targeting the repetitive elements of mouse genome. This improved method allowed for sensitive and accurate quantitation of mouse genomic DNA in the range of 1 to 10(6)pg/mL. In addition, four sample purification methods for DNA isolation (Wako DNA extractor kit, MasterPure™ DNA purification kit, PrepSEQ™ residual DNA sample preparation kit, and phenol-chloroform extraction method with addition of glycogen), each representing a different strategy for DNA isolation from proteinaceous solutions, were evaluated by isolating DNA from a mouse monoclonal IgG antibody. Among these methods, Wako DNA extractor kit and MasterPure™ DNA purification kit demonstrated superior DNA recovery, repeatability, and sensitivity, with quantitation limits of 1pg/mL. To further evaluate these two DNA isolation methods, six replicates of an unspiked mouse polyclonal IgG antibody sample were tested by both methods, and both methods demonstrated a good degree of precision. Therefore, the residual mouse DNA quantitation methods described here represented rapid, accurate, precise, and sensitive procedures that can be used in quality control testing for regulatory compliance in the pharmaceutical industry.
authors.affiliation: Biopharmaceutical Services, PPD Inc., Middleton, WI 53562, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Cai
authors.forename: Xuelin
authors.initials: X
authors.lastname: Gu
authors.forename: Mary S
authors.initials: MS
authors.lastname: Scanlan
authors.forename: Chris R
authors.initials: CR
authors.lastname: Lively
mesh_terms: D000465:Algorithms; D000818:Animals; D000911:Antibodies, Monoclonal; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004340:Drug Contamination / Q000517:prevention & control; D007074:Immunoglobulin G / Q000627:therapeutic use; D057230:Limit of Detection; D051379:Mice; D008832:Microchemistry / Q000379:methods; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000627:therapeutic use; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015203:Reproducibility of Results; D013678:Technology, Pharmaceutical / Q000592:standards*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D004247:DNA