title: Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples.
abstract: Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: wei.zhang@biogenidec.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: meng.wu@biogenidec.com.
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: emily.menesale@biogenidec.com.
authors.forename: Emily
authors.initials: E
authors.lastname: Menesale
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: tongjun.lu@biogenidec.com.
authors.forename: Tongjun
authors.initials: T
authors.lastname: Lu
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: aeona.magliola@biogenidec.com.
authors.forename: Aeona
authors.initials: A
authors.lastname: Magliola
authors.affiliation: Analytical Development, Biogen Idec, 15 Cambridge Center, Cambridge, MA 02142, United States. Electronic address: svetlana.bergelson@biogenidec.com.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Bergelson
mesh_terms: D020087:Alu Elements / Q000235:genetics*; D000818:Animals; D057205:Automation, Laboratory; D016466:CHO Cells; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers; D004340:Drug Contamination; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Alu repeats; Automation; DNA quantitation; Q-PCR; Residual host cell DNA