Björn Boll1,2, Emilien Folzer1,3,2, Christof Finkler1, Jörg Huwyler2, Hanns-Christian Mahler3, Roland Schmidt3, Atanas V Koulov4. 1. Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland. 2. Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland. 3. Pharma Technical Development (Biologics) Europe, Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., Basel, Switzerland. 4. Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland. atanas.koulov@roche.com.
Abstract
PURPOSE: The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products. METHODS: Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement. RESULTS: Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 μm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time-their relative advantages and drawbacks. CONCLUSIONS: FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.
PURPOSE: The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products. METHODS: Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement. RESULTS: Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 μm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time-their relative advantages and drawbacks. CONCLUSIONS: FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.
Authors: Miranda M C Van Beers; Francesca Gilli; Huub Schellekens; Theodore W Randolph; Wim Jiskoot Journal: J Pharm Sci Date: 2011-09-14 Impact factor: 3.534
Authors: Miranda M C van Beers; Melody Sauerborn; Francesca Gilli; Vera Brinks; Huub Schellekens; Wim Jiskoot Journal: Pharm Res Date: 2011-05-05 Impact factor: 4.200
Authors: Marisa K Joubert; Martha Hokom; Catherine Eakin; Lei Zhou; Meghana Deshpande; Matthew P Baker; Theresa J Goletz; Bruce A Kerwin; Naren Chirmule; Linda O Narhi; Vibha Jawa Journal: J Biol Chem Date: 2012-05-14 Impact factor: 5.157