(342o) Molecular Dynamics Modeling Based Investigation of Protein Instability during “Non-Isothermal” Freezing Process

Bulk protein therapeutics are generally frozen for long term storage to avoid product degradation and facilitate product transportation. As a result, freezing of water induces solute rejection, creating regions of high solute concentration, which may impact protein to be destabilized and lose its activity. However, the underlying mechanism and molecular details of cryostabilization of proteins at freezing microenvironment are still poorly understood. Molecular dynamics (MD) simulations have been developed to investigate how protein conformation and stability be impacted during the bulk freezing process. The simulation results from this current work will be further validated with previous experimental data on freezing bulk protein therapeutics.

Methods:

Lysozyme (LYZ) protein solution in 10 mM phosphate buffer at pH 7.0 was chosen as initial conditions applied into this current MD simulations. The MD simulation trajectories and their analyses were carried out using GROMACS, VMD and Pymol software. All atom MD simulations were applied using CHARMM36 force field with temperatures gradually decreased from -3ËšC to -26 ËšC with 5 – 6 ËšC temperature intervals. The initial protein crystal structure of LYZ was taken from the protein data bank entries (pdb: 1AKI). The protein was initially modeled using the psfgen tool in VMD, and then further solvated in a rectangular simulation box with the charge neutralized with NaPO₄. According to experimental work, an ice seeding approach was adopted to initiate ice formation in the MD simulation work. The simulation system was equilibrated in the NPT ensemble by initially applying 1000 cycles of a conjugate-gradient minimization scheme followed by a short 500-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. This briefly equilibrated system of LYZ protein buffer solution was further subjected to MD simulation sets in the NVT ensemble. Computations were performed on HPC, a supercomputer cluster at the University of Connecticut.

Results:

Ice formation was successfully initiated by ice seeding approach in the model protein buffer solution under freezing process. The convergence of the potential energy was monitored to reach the system equilibration along with decreased temperature, and then Root-mean-square-deviation (RMSD) analysis was applied to evaluate protein conformational changes. Significant RMSD fluctuations were captured at initial stage of freezing for the LYZ which is close to ice-water interface, whereas RMSD levels off to be stable at final freezing process. Moreover, the most flexible and unstable residues in LYZ were also identified during freezing process with the assessment of counting hydrogen bond formation within protein itself and with surrounding solvent water molecules.

Conclusions:

Therapeutic protein buffer solution under “non-isothermal” freezing process have been successfully modeled in MD simulations. Analyses of the resulted trajectories reveal the effect of temperature, ionic strength, solute concentration on the protein folding and unfolding states, biomolecular interactions, and protein aggregations.

Keywords:

Protein therapeutics, Lysozyme, Freezing, Molecular Dynamics Simulations, Non-isothermal