title: Physical characterization and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting.
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.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: Srivalli
authors.initials: S
authors.lastname: Telikepalli
authors.forename: Heather E
authors.initials: HE
authors.lastname: Shinogle
authors.forename: Prem S
authors.initials: PS
authors.lastname: Thapa
authors.forename: Jae Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Meghana
authors.initials: M
authors.lastname: Deshpande
authors.forename: Vibha
authors.initials: V
authors.lastname: Jawa
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: Linda O
authors.initials: LO
authors.lastname: Narhi
authors.forename: Marisa K
authors.initials: MK
authors.lastname: Joubert
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008863:Microspheres; D053758:Nanoparticles / Q000032:analysis / Q000737:chemistry; D010316:Particle Size
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G
keywords: IgG; immune response; immunogenicity; PBMC; in vitro; monoclonal antibody; particles; protein aggregation; proteins