During Erwinia chrysanthemi L-asparaginase (ErA) manufacture, minor conformational variants are formed that elute in the acidic region of "the analytical ion-exchange HPLC chromatogram. These variants retain enzymatic activity and form part of the biopharmaceutical product, but must be kept within acceptable limits through controlled operation of the manufacturing process. The high isoelectric point of the ErA native tetramer (8.6) leads to certain process steps being operated in the alkaline pH region. Previously, the formation of these species during processing was not fully understood. In this work, in process samples were analysed, and alkaline pH (8-9) and hold time were found to be the governing parameters. Formation of ErA acidic species was found to be accelerated at higher pH values and longer hold times, suggesting potential control strategies for the manufacturing process. However, the kinetics of ErA conformational variant formation were found to be slow (0.15-0.25 percent per day at pH 8.5). Changes in the ErA melt temperature (T-m) with pH as determined by both differential scanning calorimetry and differential scanning fluorimetry were found to be predictive of the tendency to form the IEX-HPLC acidic species during processing. Biopharmaceutical process developers should be aware of such changes to proteins and build relevant control strategies into process validation plans. (C) 2016 Elsevier Inc. All rights reserved.