title: Diffusion of Soluble Aggregates of THIOMABs and Bispecific Antibodies in Serum.
abstract: Submicrometer aggregates are frequently present at low levels in antibody-based therapeutics. Although intuition suggests that the fraction of the aggregate or the size of the aggregate present might correlate with deleterious clinical properties or formulation difficulties, it has been challenging to demonstrate which aggregate states, if any, trigger specific biological effects. One source of uncertainty about the putative linkage between aggregation and safety or efficacy lies in the likelihood that noncovalent aggregation differs in ideal buffers versus in serum and biological tissues; self-association or association with other proteins may vary widely with environment. Therefore, methods for monitoring aggregation and aggregate behavior in biologically relevant matrices could provide a tool for better predicting aggregate-dependent clinical outcomes and provide a basis for antibody engineering prior to clinical studies. Here, we generate models for soluble aggregates of THIOMABs and a bispecific antibody (bsAb) of defined size and exploit fluorescence correlation spectroscopy to monitor their diffusion properties in serum and viscosity-matched buffers. The monomers, dimers, and trimers of both THIOMABs and a bsAb reveal a modest increase in diffusion time in serum greater than expected for an increase in viscosity alone. A mixture of larger aggregates containing mostly bsAb pentamers exhibits a marked increase in diffusion time in serum and much greater intrasample variability, consistent with significant aggregation or interactions with serum components. The results indicate that small aggregates of several IgG platforms are not likely to aggregate with serum components, but nanometer-scale aggregates larger than trimers can interact with the serum in an Ab-dependent manner.
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Seattle, Washington 98195-7631, United States.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Goulet
authors.affiliation: Biologics Research, Janssen Research & Development, LLC , Spring House, Pennsylvania 19477, United States.
authors.forename: Adam
authors.initials: A
authors.lastname: Zwolak
authors.affiliation: Biologics Research, Janssen Research & Development, LLC , Spring House, Pennsylvania 19477, United States.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Chiu
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Seattle, Washington 98195-7631, United States.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Nath
authors.affiliation: Department of Medicinal Chemistry, University of Washington , Seattle, Washington 98195-7631, United States.
authors.forename: William M
authors.identifier: 0000-0002-3173-0175
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D000465:Algorithms; D018033:Antibodies, Bispecific / Q000009:adverse effects / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004058:Diffusion; D004229:Dithiothreitol / Q000494:pharmacology; D004339:Drug Compounding; D005976:Glutaral / Q000494:pharmacology; D006801:Humans; D057446:Hydrodynamics; D007074:Immunoglobulin G / Q000009:adverse effects / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D010316:Particle Size; D066329:Protein Aggregates; D011994:Recombinant Proteins / Q000009:adverse effects / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012995:Solubility; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000068878:Trastuzumab / Q000009:adverse effects / Q000032:analysis / Q000737:chemistry*; D014783:Viscosity
chemical_list: D018033:Antibodies, Bispecific; D001798:Blood Proteins; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D066329:Protein Aggregates; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D000068878:Trastuzumab; D005976:Glutaral; D004229:Dithiothreitol