(411d) Molecular Dynamics Modeling Based Investigation of the Effect of Freezing Rate on Protein Stability

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. In addition, the stability of protein drug products frozen during fill finish operations is greatly affected by the freezing rate applied. Non-optimal freezing rates may lead to the denaturation of protein`s complex macromolecular conformation. However, limited work has been done to address the effect of different freezing rates on protein stability at nano-scale level. The stability of a model protein, lysozyme, was investigated at atomic and molecular scale under varying freezing rates and moving ice-water interface using Molecular dynamics (MD) simulations. Ice seeding approach was adopted to initiate ice formation in this present simulation. The simulation results from this current work were further validated with previous experimental data on freezing bulk protein therapeutics.

Methods:

Lysozyme 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 lysozyme 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 faster freezing rate (11-12 K/490 ns) applied resulted in overall smaller ice fraction within the simulation box with a larger freeze-concentrated liquid (FCL) region. Consequently, the faster freezing rate better maintained protein stability with less secondary structure deviations, higher hydration level and structural compactness, and less fluctuations at individual residues than observed following slow (5-6 K/490 ns) and medium (7-8 K/490 ns) freezing rates. The present study also identified the residues near and within lysozyme protein helices 3, 6, 7, and 8 dominate the structural instability of the lysozyme at 247 K freezing temperature. Moreover, after the freezing simulations, the value of lysozyme concentration increased 5.39-fold compared with the concentration at the beginning of the simulation, which is comparable with the experimental result (5.41-fold increase).

Conclusions:

The good agreement between the computational predictions and experimental results highlights the benefit of the all-atom MD approach to gain insight on ice formation and protein behavior during freezing. To the best of our knowledge, different freezing rates were studied “non-isothermally” for the first time in this current work. Thus, a good understanding of freezing rates on protein instability was revealed by applying the developed computational model, which can be utilized as an effective and powerful tool in the process of discovery, development, and optimization of new protein therapeutics.

Keywords:

freezing rate, ice-water interface, protein stability, molecular dynamics simulations