Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates

Abstract

Aggregates of therapeutic proteins have been associated with increased immunogenicity in pre-clinical models as well as in human patients. Recent studies to understand aggregates and their immunogenicity risks use artificial stress methods to induce high levels of aggregation. These methods may be less biologically relevant in terms of their quantity than those that occur spontaneously during processing and storage. Here we describe the immunogenicity risk due to spontaneously occurring therapeutic antibody aggregates using peripheral blood mononuclear cells (PBMC) and a cell line with a reporter gene for immune activation: THP-1 BLUE NFκB. The spontaneously occurring therapeutic protein aggregates were obtained from process intermediates and final formulated drug substance from stability retains. Spontaneously occurring aggregates elicited innate immune responses for several donors in a PBMC assay with cytokine and chemokine production as a readout for immune activation. Meanwhile, no significant adaptive phase responses to spontaneously occurring aggregate samples were detected. While the THP-1 BLUE NFκB cell line and PBMC assays both responded to high stress induced aggregates, only the PBMC from a limited subset of donors responded to processing-induced aggregates. In this case study, levels of antibody aggregation occurring at process relevant levels are lower than those induced by stirring and may pose lower risk in vivo. Our methodologies can further inform additional immunogenicity risk assessments using a pre-clinical in vitro risk assessment approach utilizing human derived immune cells.

A Retrospective Analysis of the Potential Impact of Differences in Aggregates on Clinical Immunogenicity of Biosimilars and their Reference Products

“…13 However, artificially induced aggregates may differ from aggregates occurring spontaneously in terms of aggregate shapes and chemical modifications. 18,19 Swanson et al 20 used process-induced aggregates to investigate their immunogenic potential in vitro using peripheral blood mononuclear cells and a cell line with a reporter gene for immune activation. They found that whereas artificially induced aggregates induced both adaptive and innate immune responses, spontaneous aggregates only induced innate responses in a limited subset of donor peripheral blood mononuclear cells, suggesting that data obtained in stressed samples may not be representative for what is happening in marketed products that are used in daily clinical practice.…”

“…However, artificially induced aggregates may differ from aggregates occurring spontaneously in terms of aggregate shapes and chemical modifications 18,19 . Swanson et al 20 . used process‐induced aggregates to investigate their immunogenic potential in vitro using peripheral blood mononuclear cells and a cell line with a reporter gene for immune activation.…”

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2

“…Unlike other antibody-derived fragments, knob peptides do not rely on the β-barrel core scaffold of Ig domains but instead use disulfide bonds and a short internal β-sheet to maintain their compact structure ( 19 , 21 ). This structure may also be an advantage given the propensity of some Ig domains to aggregate and produce amyloid ( 62 – 66 ), which can be especially problematic in lung tissue where delivery regimes like inhalers or nebulizers can be harsh on protein molecules ( 67 ) where denatured or aggregated forms can cause immunogenicity ( 68 – 70 ). The compact, disulfide-bonded knob may enhance stability to proteases or heat, which would be advantageous in extreme environments in vivo, like the GI tract, or ex vivo, to enable long shelf lives or eliminate the need for cold-chain distribution of therapeutic products ( SI Appendix , Fig.…”

Stabilization challenges and aggregation in protein-based therapeutics in the pharmaceutical industry

“…Furthermore, the impact of additional clinical factors on immunogenicity is a complex area requiring further investigation. 67,68 Immunogenicity refers to the development of serum anti-drug antibodies (ADAs) that specically target and bind to the proteins of interest, such as a therapeutic proteins or drugs. This immune response can have signicant implications for the efficacy and safety of the protein products and can lead to reduced their effectiveness.…”