Molecular Dynamics Modeling Based Investigation of the Effect of Freezing Rate on Lysozyme Stability

Purpose 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. Methods The stability of a model protein, lysozyme, was investigated at atomic and molecular scale under varying freezing rates and moving ice-water interface. Ice seeding approach was adopted to initiate ice formation in this present simulation. Results 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 helices 3, 6, 7, and 8 dominate the structural instability of the lysozyme at 247 K freezing temperature. Conclusions For the first time, ice formation in therapeutic protein solution was studied non-isothermally at different freezing rates using molecular dynamics simulations. Thus, a good understanding of freezing rates on protein instability was revealed by applying the developed computational model.

Automated summary

This study investigates the effect of freezing rates on the stability of protein lysozyme using molecular dynamics simulations. The results show that faster freezing rates better maintain protein stability, resulting in less secondary structure deviations, higher hydration level, and structural compactness. The study also identifies the residues near and within specific helices that contribute to the structural instability of lysozyme at the freezing temperature of 247 K.