Tibo Duran1, Bruna Minatovicz2, Ryan Bellucci3, Jun Bai4, Bodhisattwa Chaudhuri5,6,7. 1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, 06269, USA. 2. Drug Product Development, BioTherapeutics Development, Janssen Research and Development, Malvern, PA, 19355, USA. 3. Department of Chemical & Biomolecular Engineering, University of Connecticut, Storrs, CT, 06269, USA. 4. Department of Computer Sciences and Engineering, University of Connecticut, Storrs, CT, 06269, USA. 5. Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, 06269, USA. bodhi.chaudhuri@uconn.edu. 6. Department of Chemical & Biomolecular Engineering, University of Connecticut, Storrs, CT, 06269, USA. bodhi.chaudhuri@uconn.edu. 7. Institute of Material Sciences (IMS), University of Connecticut, 69 N. Eagleville Road, Storrs, CT, 06269, USA. bodhi.chaudhuri@uconn.edu.
Abstract
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.
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.
Authors: Yemin Xu; Pawel Grobelny; Alexander Von Allmen; Korben Knudson; Michael Pikal; John F Carpenter; Theodore W Randolph Journal: J Pharm Sci Date: 2014-03-12 Impact factor: 3.534