Application Notes

Analysis of formulation-dependent colloidal and conformational stability of monoclonal antibodies

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3 general, pH- and salt-dependence was most pronounced for the first unfolding transition. Parallel analysis of antibody aggregation was performed by measurement of light exctinction using backreflection detection. Interestingly, the aggregation data revealed that aggregation was most pronounced under buffer conditions with higher pH values (Figure 3B and 4C). These results indicate that shifts in T agg do not correlate with the large, pH-dependent shifts in the first unfolding transition temperature T m 1 (Figure 3 and 4B), suggesting that unfolding of the corresponding mAb domain is not responsible for aggregation. In contrast, no aggregation was detected in acetate buffer pH 4 in absence of salt, showing that aggregation of the unfolded state is largely suppressed under these conditions (Figure 3B, 4B and C). Since an increase in pH resulted in an increase of the overall aggregation signal, increasing the pH-value seems to promote the formation of unfolded-state aggregation. In addition to sample pH, sodium chloride greatly affected temperature-induced mAb aggregation: The addition of 130 mM NaCl led to strong aggregation under all conditions tested, as well as to a shift of the aggregation onset temperature to lower values. Figure 3: Conformational stability and aggregation of a mAb under different buffer conditions. (A) Thermal unfolding monitored by detection of shifts in the fluorescence ratio (F350/F330) in dependence of different buffer pH values and NaCl concentrations. (B) Aggregation is detected by changes in backreflection. Since partial unfolding of proteins can be reversible under certain conditions, we next tested for reversibility of the first unfolding transition. For this, the samples were heated to temperatures either near at or ~10 °C beyond T m 1, respectively, and subsequently cooled back to 20 °C at 1 °C/min

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