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 to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to 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 is discussed.

Effect of growth hormone and IgG aggregates on dendritic cells activation and T‐cells polarization

“…Again this latter result suggested differences in the mechanism of DC stimulation by Rituximab vs purified IgG1. Interleukin‐6, CCL2, CCL3 and CCL4 are part of the identified cytokine signature identified by Joubert et al, 25 using PBMCs and aggregated antibodies 37 . In this latter work, several cell subsets can contribute to cytokine and chemokine production that can explain some discrepancies with our results.…”

“…In contrast, circular dichroïsm analysis was comparable for Rituximab and IgG1, showing a dramatic perturbation of the signal in relation with the tremendous increase in the light scattering after stirring. Previous structural studies on mAbs aggregates showed that the stirring stress indeed induced intramolecular loss of β‐sheet structures in IgG1 aggregates, whereas some intermolecular β‐sheet structures were detected 37 …”

“…Among others, it has been shown that stirring stress applied to IgG1‐antibody induced high amounts of homogenous small protein aggregates, between 100 and 1000 nm, detected by dynamic light scattering 36 . Another work showed that an IgG2 antibody undergoing a stir stress showed nanometer‐sized particles (0.2–1.5 μm) as well as micron‐sized particles 37 …”

Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges

“…Furthermore, current available technology used to fractionate, purify, stabilize and deliver aggregates and particles in narrow, well-defined size ranges is limited. 67 Even when aggregates are fractionated, they may be unstable, and any changes in aggregate distributions that may occur upon administration are unknown. The reversibility/dissociability of aggregates under in vivo conditions could be important in this regard.…”

Novel chemical degradation pathways of proteins mediated by tryptophan oxidation: tryptophan side chain fragmentation

“…Protein pharmaceuticals can degrade via multiple chemical and physical processes, and detailed knowledge of these processes is critical to prevent degradation and to maintain stability of formulations . The last years have witnessed significant progress in our understanding of physical degradation processes, for example leading to aggregation and particle formation, in part due to the development of new analytical methodology and the critical evaluation of its potential and limitations . In contrast, the characterization of chemical degradation processes is far from complete, for the most part due to the continuous discovery of new reaction pathways and products, which can be unique to specific protein sequences.…”