Inverse relationship of protein concentration and aggregation

To determine the effect of protein concentration on aggregation induced through quiescent shelf-life incubation or shipping-related agitation. All aggregation was measured by size-exclusion high-performance liquid chromatography. Aggregation was induced by time-dependent incubation under stationary conditions or by agitation caused by shaking, vortexing, or vibration using simulated shipping conditions. Protein aggregation is commonly a second- or higher-order process that is expected to increase with higher protein concentration. As expected, for three proteins (PEG-GCSF, PEG-MGDF, and OPG-Fc) that were examined, the aggregation increased with higher protein concentration if incubated in a quiescent shelf-life setting. However, aggregation decreased with higher protein concentration if induced by an air/water interface as a result of agitation. This unexpected result may be explained by the rate-limiting effect on aggregation of the air/water interface and the critical nature of the air/ water interface to protein ratio that is greatest with decreased protein concentration. The non-ionic detergent polysorbate 20 enhanced the aggregation observed in the quiescently incubated sample but abrogated the aggregation induced by the air/water interface. The effect of protein concentration was opposite for aggregation that resulted from quiescent shelf-life treatment compared to induction by agitation. For motionless shelf-life incubation, increased concentration of protein resulted in more aggregation. However, exposure to agitation resulted in more aggregation with decreased protein concentration. These results highlight an unexpected complexity of protein aggregation reactions.