title: Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles--Differential Centrifugation and FACS.
abstract: OBJECTIVE
  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.affiliation: Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland.|Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland.
authors.forename: Björn
authors.initials: B
authors.lastname: Boll
authors.affiliation: Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland.|Pharma Technical Development (Biologics) Europe, Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.|Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland.
authors.forename: Emilien
authors.initials: E
authors.lastname: Folzer
authors.affiliation: Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland.
authors.forename: Christof
authors.initials: C
authors.lastname: Finkler
authors.affiliation: Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland.
authors.forename: Jörg
authors.initials: J
authors.lastname: Huwyler
authors.affiliation: Pharma Technical Development (Biologics) Europe, Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Hanns-Christian
authors.initials: HC
authors.lastname: Mahler
authors.affiliation: Pharma Technical Development (Biologics) Europe, Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: Roland
authors.initials: R
authors.lastname: Schmidt
authors.affiliation: Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland. atanas.koulov@roche.com.
authors.forename: Atanas V
authors.initials: AV
authors.lastname: Koulov
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification*; D010316:Particle Size; D066329:Protein Aggregates
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D066329:Protein Aggregates
keywords: particle fractionation; particle size; protein aggregation; proteinaceous particles; subvisible particles