ABSTRACT
Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for intrinsic tryptophan fluorescence emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (<5 mg/mL, equivalent to 0.8â¯mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (>40â¯mg/mL, equivalent to 6.1â¯mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223â¯mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1â¯%) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations.
