title: Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.
abstract: Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.
authors.affiliation: Novartis Pharma AG, TRD Biologics Process R&D, Basel, Switzerland.
authors.forename: Grit
authors.initials: G
authors.lastname: Peper
authors.affiliation: Novartis Pharma AG, TRD Biologics Process R&D, Basel, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fankhauser
authors.affiliation: Novartis Pharma AG, TRD Biologics QC, Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Merlin
authors.affiliation: Novartis Pharma AG, TRD Biologics Process R&D, Basel, Switzerland.
authors.forename: Ana
authors.initials: A
authors.lastname: Roscic
authors.affiliation: Novartis Pharma AG, TRD Biologics Process R&D, Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hofmann
authors.affiliation: Novartis Pharma AG, TRD Biologics Process R&D, Basel, Switzerland. Electronic address: petr.obrdlik@novartis.com.
authors.forename: Petr
authors.initials: P
authors.lastname: Obrdlik
mesh_terms: D000818:Animals; D002021:Buffers; D016466:CHO Cells; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification*; D004340:Drug Contamination; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014162:Transfection
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D019286:Endopeptidase K
keywords: Biologics; CHO; Real-time qPCR; Residual DNA; Therapeutic proteins