Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Abstract

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.

Keywords:

• protein aggregation
• antibody
• protein stability
• kinetics
• protein formulation

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

The authors thank Dr. John Miglietta, Dr. Irina Rybina, and Dr. Ertan Eryilmaz for helping design the Fc DNA construct, purify the Fc protein, and re-build the structure of the Fc from the published X-ray crystal structure of Fc with similar sequences in PDB, respectively.

Funding

Boehringer Ingelheim Pharmaceuticals Inc. is gratefully acknowledged for financial support.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.