T_m-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions

Abstract

Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature (T) for global unfolding provide convenient surrogates for longer-term (e.g., 2-year) degradation kinetics, with which to optimize formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. T values are known to correlate with chemical degradation kinetics for wild-type granulocyte colony stimulating factor (GCSF) at pH 4-5.5. However, we found previously that the T of an antibody Fab fragment only correlated with its rate of monomer loss at temperatures close to the T. Here we evaluated T, the fraction of unfolded protein (f) at temperature T, and two additional short-term stability measures, for their ability to predict the kinetics of monomer and bioactivity loss of wild-type GCSF and four variants, at 37 °C, and in a wide range of formulations. The GCSF variants introduced one to three mutations, giving a range of conformational stabilities spanning 7.8 kcal mol. We determined the extent to which the formulation rank order differs across the variants when evaluated by each of the four short-term stability measures. All correlations decreased as the difference in average T between each pair of GCSF variants increased. The rank order of formulations determined by T was the best preserved, with R-values >0.7. T-values also provided a good predictor (R = 0.73) of the aggregation rates, extending previous findings to include GCSF variant-formulation combinations. Further analysis revealed that GCSF aggregation rates at 37 °C were dependent on the fraction unfolded at 37 °C (f), but transitioned smoothly to a constant baseline rate of aggregation at f < 10. A similar function was observed previously for A33 Fab formulated by pH, ionic strength, and temperature, without excipients. For GCSF, all combinations of variants and formulations fit onto a single curve, suggesting that even single mutations destabilized by up to 4.8 kcal mol, are insufficient to change significantly the baseline rate of aggregation under native conditions. The baseline rate of aggregation for GCSF under native conditions was 66-fold higher than that for A33 Fab, highlighting that they are a specific feature of each native protein structure, likely to be dependent on local surface properties and dynamics.

Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product

“…The filgrastim products were analyzed at low pH and the temperature of unfolding was similar in all products between 55 and 60°C. These results are in agreement with the T m previously reported ( 33 , 34 , 36 ) and consistent with those reported for other filgrastim formulations ( 24 , 35 ). PDgrastim (pink line) showed the lowest thermal stability with a drastic change in conformation at 55°C.…”

Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

“…Both T m and f 55 indicated potential correlations but with 5M/I365L as an outlier, indicating that global unfolding had a significant influence on the relative inactivation rates of the variants 55 °C. For all variants, f 55 > 0.02 (2%), which is just above the 1% threshold observed by us previously for other proteins, as the point at which the role of global unfolding in aggregation kinetics becomes relatively insignificant compared with local fluctuations in the native ensemble (23–25).…”

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

“…In conclusion, the aggregation rate was influenced by the flexibility of specific surface loops, but not for all loops. We fit the data in Figure 7B to a range of potential kinetic models as previously for GCSF 54 , assuming that all variants underwent the same aggregation mechanism, but where aggregation could occur from both a native-like species or a globally unfolded species, with relative rates that depend upon the fraction unfolded, and hence the global unfolding stability of each variant. Two numerical models gave good fits.…”