(376ah) Molecular Dynamics Modeling Based Investigation of Cryoconcentration of Solutes during Freezing and Its Impact on Protein Stability

Objective: Bulk protein therapeutics are generally frozen for long term storage to prevent product degradation, and facilitate product transportation. The freezing process of bulk protein solutions leads to cryoconcentration of solutes, which may impact protein stability. However, the underlying mechanism and molecular details of proteins unfolding and aggregating at the cryoconcentration microenvironment are poorly understood. Molecular dynamics (MD) simulations have been developed to investigate how cryoconcentration of different solutes affects protein stability during the bulk freezing process. The simulations results will be further validated with previous experimental data on solutes cryoconcentration acquired by our group.

Methods: The initial conditions applied into the MD simulations were selected based on previous cryoconcentration experiments in which 1 L bottles containing 600 mL of a protein solution of 0.5 mg/mL lactate dehydrogenase (LDH) in 10 mM phosphate buffer at pH 7.0 with or without 2.5% (w/v) sucrose were frozen at -30°C. Therefore, LDH, phosphate salts and sucrose molecules were added into the simulation box. The MD simulation trajectories and their analyses were carried out using GROMACS/VMD software. We applied both MARTINI and CHARMM force-field for all-atom as well as coarse-grained simulations, respectively. The initial coordinates for LDH were taken from the protein data bank entries (pdb: 5NQB). The protein was initially modeled using the psfgen tool in VMD, and then further solvated in a simulation box and charge-neutralized with NaPO₄. The box volume was optimized in the NPT ensemble by initially applying 500 cycles of a conjugate-gradient minimization scheme followed by a short 40-ps MD run in NPT assemble. The temperature was controlled using the Langevin thermostat, and the pressure was controlled by the Nose-Hoover barostat. The simulations were carried out using periodic boundary conditions. These briefly equilibrated systems of LDH protein were further subjected to MD simulation sets in the NVT ensemble. Computations were performed on HPC, a supercomputer cluster at the University of Connecticut.

Results and Discussion: The cryoconcentration effect was evaluated applying a model protein solvated in a simulation box of aqueous solvent in which different ions and stabilizers were added for case studies. Analyses of the resulted trajectories reveal the effect of temperature, ionic strength, and solutes concentration on the protein unfolding state, biomolecular interactions, and protein aggregation.