Technical Notes

2. Incremental cycling to evaluate reversible and irreversible unfolding events in mAbs. The stability of mAbs is of major importance in the drug development process. Although the thermal stability of mAbs is routinely assessed by determination of their T m , little attention is paid to the reversibility of their folding, mainly due to the lack of high-throughput instrumentation to perform such analyses. The example shows two different mAbs, which were subjected to an incremental cycling experiment. The initial temperature was 45 °C, and each cycle increased temperature by 4 °C with respect to the previous heating cycle. This method is especially suited for finding the temperature at which the unfolding becomes irreversible. mAb1 (red) displayed a strictly irreversible unfolding, indicated by a stepwise increase in the F350/F330 ratio with each cycle without recovery to the F350/F330 ratio of the unfolded state during cooling phases. Investigation of the additionally recorded aggregation signal indicated that even the earliest cycles with little temperature increase triggered a small but significant aggregation signal, suggesting that aggregation of the partially unfolded mAb could be responsible for the observed irreversibility (lower panel). In contrast, mAb2 (blue) displayed mostly reversible unfolding up to temperatures of 69 °C, indicated by an almost complete recovery of the F350/F330 values during cooling phases. Only at temperatures >69 °C, irreversible unfolding can be observed, which again coincides with aggregation (lower panel), suggesting that these temperatures induce aggregation- prone unfolded states of the mAb which form irreversible aggregates. 3. Concentration-dependence of folding reversibility Commercial proteins are typically produced and stored at high concentrations. Highly concentrated protein solutions are however difficult to analyze by conventional methods, so that proteins are often diluted to allow for biophysical characterization. Instruments of the Prometheus series have a virtually unlimited dynamic range, allowing for the analysis of both, highly concentrated and highly diluted samples at the same time. The shown example demonstrates how protein dilution affects the reversibility of protein unfolding.

• Cover