(560n) Towards Pharmaceutical Protein Stabilization: Insights from Theoretical Studies on Peptide Hydrolysis Reactions

Monoclonal antibodies (Mabs) are one of the most lucrative pharmacologics currently on the market. According to Grilo and Mantalaris, the market for Mab production has grown “between 7.2 and 18.3 percent per year”.¹ In addition to their primary use as vaccines for diseases such as the flu and HIV, Mabs are also used to treat cancer and autoimmune diseases. One such example is Avelumab, a monoclonal antibody created by Pfizer to target the rare type of skin cancer, Merkel cell carcinoma.¹ However, formulation efficiency is limited by degradation of the desired products during multiple points of the biomanufacturing process.² While the use of additives has greatly reduced degradation, determining the most effective additives is accomplished through a “guess and check” approach, instead of understanding the underlying mechanism of the degradation pathway. One such mechanism of degradation is hydrolysis. At a neutral pH, hydrolysis occurs via the fragmentation of polypeptides into individual amino acids when water molecules are introduced.³

Recently, molecular models are being developed for enhanced prescriptive stabilization of antibodies. To better understand the mechanisms, the activation barrier (E_a) and free energy (^oG) of the reaction system must be estimated. One common method of determining the activation barrier of a system is through the use of the Evans-Polanyi correlation, which requires detailed thermochemical understanding of dipeptides and their individual amino acid components. For this purpose, we conducted a computational study of a small subset of dipeptides to predict the thermodynamic properties to begin generating a library of thermodynamic and kinetic values. The optimized geometries of the dipeptides were investigated using the Gaussian n-method at the B3LYP level of theory, as well as different solvation methods to analyze the impact of solvation on thermodynamic and kinetic properties. To validate our methodology, we compared our optimized frequencies to infrared spectra and experimental rate coefficients from current literature. Our studies have established trends in thermodynamic properties (Enthalpy of reaction (^oH_R), Entropy (^oS), and Gibbs free energy of reaction (^oG_R), as a function of peptide functional groups. These learnings, which will promote more efficient additive formulation, are discussed in terms of dipeptides degraded via hydrolysis reaction, at neutral pH.

1. Grilo, A. L. and A. Mantalaris (2019). "The Increasingly Human and Profitable Monoclonal Antibody Market." Trends Biotechnol 37(1): 9-16.
2. Vazquez-Rey, M. and D. A. Lang (2011). "Aggregates in monoclonal antibody manufacturing processes." Biotechnol Bioeng 108(7): 1494-1508.
3. Pan, B., et al. (2011). "A molecular mechanism of hydrolysis of peptide bonds at neutral pH using a model compound." J Phys Chem B 115(19): 5958-5970.