Asparagine deamidation is a common posttranslational modification in which asparagine is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics.We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kilodalton (kDa) truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which suggests a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogendeuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide a possible explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest an approach that can be used for process control to ensure product quality and process consistency.3 Statement of SignificanceAsparagine deamidation can have a potentially significant impact on protein therapeutics.Previous studies have revealed that deamidation at a single site significantly reduces the biological activity of moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin developed for the treatment of B-cell malignancies. Surprisingly, despite the fact that deamidation introduced a negative charge, the deamidated variant eluted earlier than its unmodified form on anion exchange chromatography. In order to understand these observations, we isolated the deamidated variant using an anion exchange column and characterized it by differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry. The results revealed the conformational change caused by the deamidation, which explains the diminished biological activity of the variant and its early elution time on anion exchange chromatography.