Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment

The analytical characterization of biopharmaceuticals is a fundamental step in the early stages of development and prediction of their behavior in bioprocesses. Protein aggregation in particular is a common issue as it affects all stages of product development. In the present work, we investigate the stability and the aggregation kinetics of A33Fab, a therapeutically relevant humanized antibody fragment at a wide range of pH, ionic strength, and temperature. We show that the propensity of A33Fab to aggregate under thermally accelerated conditions is pH and ionic-strength dependent with a stronger destabilizing effect of ionic strength at low pH. In the absence of added salts, A33Fab molecules appear to be protected from aggregation due to electrostatic colloidal repulsion at low pH. Analysis by transmission electron microscopy identified significantly different aggregate species formed at low and high pH. The correlations between apparent midpoints of thermal transitions (T-m,T-app values), or unfolded mole fractions, and aggregation rates are reported here to be significant only at the elevated incubation temperature of 65 degrees C, where aggregation from the unfolded state predominates. At all other conditions, particularly at 4-45 degrees C, aggregation of A33 Fab was predominantly from a native-like state, and the kinetics obeyed Arrhenius behavior. Despite this, the rank order of aggregation rates observed at 45 degrees C, 23 and 4 degrees C still did not correlate well to each other, indicating that forced degradation at elevated temperatures was not a good screen for predicting behavior at low temperature.