title: One single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples.
abstract: During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125mM Tris, 338mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5×1011-1.5×1011adenovirus particles per ml (~80 to 250pmol/l). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95-110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.
authors.affiliation: Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands. Electronic address: evantri@its.jnj.com.
authors.forename: Ewoud
authors.initials: E
authors.lastname: van Tricht
authors.affiliation: Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands.
authors.forename: Lars
authors.initials: L
authors.lastname: Geurink
authors.affiliation: Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands.
authors.forename: Harold
authors.initials: H
authors.lastname: Backus
authors.affiliation: Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands.
authors.forename: Marta
authors.initials: M
authors.lastname: Germano
authors.affiliation: Vrije Universiteit Amsterdam, AIMMS Divison of BioMolecular Analysis, Faculty of Sciences, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Kantisto BV, Callenburglaan 22, 3742 MV Baarn, The Netherlands; Uppsala University, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Biomedical Centre, PO Box 574, SE-751 23 Uppsala, Sweden; Australian Centre for Research on Separation Science ACROSS, School of Physical Sciences, Chemistry, University of Tasmania, Hobart, Australia.
authors.forename: Cari E
authors.initials: CE
authors.lastname: Sänger-van de Griend
mesh_terms: D000256:Adenoviridae; D019075:Electrophoresis, Capillary / Q000379:methods*; D014771:Virion / Q000302:isolation & purification*
keywords: Analytical Quality by Design; Capillary electrophoresis; Intact adenoviruses; Quantification; Upstream and downstream processing