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Physical Characterization and In Vitro Biological Impact of Highly Aggregated Antibodies Separated into Size-Enriched Populations by Fluorescence-Activated Cell Sorting

Authors
  • Srivalli Telikepalli
  • Heather E. Shinogle
  • Prem S. Thapa
  • Jae Hyun Kim
  • Meghana Deshpande
  • Vibha Jawa
  • C. Russell Middaugh
  • Linda Narhi
  • Marisa K. Joubert
  • David B. Volkin
Type
Published Article
Journal
Journal of Pharmaceutical Sciences
Publisher
Elsevier
Publication Date
May 06, 2015
Volume
104
Issue
5
Pages
1575–1591
Identifiers
DOI: 10.1002/jps.24379
Source
AIMS
License
Unknown

Abstract

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 μm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed.

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