RESUMO
Monoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune system, anti-drug antibodies are often formed with continuous use. Particles formed during stress conditions, which can be used to simulate storage and handling conditions of commercial antibodies, have previously been associated with the formation of anti-drug antibodies. This study investigates the relationship between particles, oligomerization, folding and chemical degradation on the in vitro cytokine response toward the TNFα inhibitor adalimumab. Adalimumab aggregates generated using stir and heat stress were fractionated into distinct sub-populations, and their structure and immunogenic potential were evaluated. A chemically degraded sample of adalimumab was included to compare particle composition with the milder accelerated heat and stir stressed conditions. Particles from stressed adalimumab samples induced elevated cytokine levels and CD4+ T cell proliferation in vitro compared to non-stressed samples. Samples enriched with both submicron and subvisible particles of adalimumab induced the strongest cytokine release and the strongest CD4+ T cell proliferation despite maintaining some TNFα inhibitory functionality. Samples that were stressed and subsequently purified of subvisible and submicron particles did not elicit a significantly higher cytokine response or show increased CD4+ T cell proliferation compared to a non-stressed sample. Oxidation-induced chemical modifications in adalimumab, mainly in Met, His, Trp, and Tyr, were not found to be sufficient in absence of particle formation to induce increased CD4+ T cell proliferation or cytokine release despite less decreased TNFα inhibitory activity of adalimumab. These observations provide further evidence that particles do indeed potentiate the immunogenic potential of adalimumab.