The Role of Phase Separation and Local Mobility in the Stabilization of a Lyophilized IgG2 Formulation

Overview

Journal AAPS PharmSciTech

Publisher Springer

Specialty Pharmacology

Date 2024 Nov 19

PMID 39562383

Authors

Ashley Lay-Fortenbery

Ryan E Holcomb

Charles S Henry

Mark Cornell Manning

Eric J Munson

Affiliations

Soon will be listed here.

Abstract

The utility of employing solid-state NMR (SSNMR) to assess parameters governing the stability of a lyophilized IgG2 protein was the focus of the present work. Specifically, the interaction between the sugar stabilizer (sucrose) and protein component was measured using SSNMR and compared to physical and chemical stability data obtained from thermally stressed samples. H T and H T relaxation times were measured by SSMNR for 5 different formulation conditions, and the resultant values were used to examine local mobility and phase separation, respectively. From the SSNMR measurements, it was found local mobility decreased as the sucrose to protein weight ratio increased. The decrease in local mobility corresponded to an increase in storage stability (both chemical and physical) of the lyophilized solids up to a critical weight ratio of sucrose to protein. Additionally, H T measurements obtained on formulations having higher protein to sucrose weight ratios indicated phase separation of the protein and sucrose phases was occurring, at least on a small scale. Along with an increase in local mobility, phase separation in these specific formulations is thought to have played a role in their decreased storage stability in the solid state.

References

Hancock B, Zografi G . Characteristics and significance of the amorphous state in pharmaceutical systems. J Pharm Sci. 1997; 86(1):1-12. DOI: 10.1021/js9601896. View

Yoshioka S, Aso Y, Kojima S . Dependence of the molecular mobility and protein stability of freeze-dried gamma-globulin formulations on the molecular weight of dextran. Pharm Res. 1997; 14(6):736-41. DOI: 10.1023/a:1012194220970. View

Carpenter J, Pikal M, Chang B, Randolph T . Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res. 1997; 14(8):969-75. DOI: 10.1023/a:1012180707283. View

Chang L, Pikal M . Mechanisms of protein stabilization in the solid state. J Pharm Sci. 2009; 98(9):2886-908. DOI: 10.1002/jps.21825. View

Wang W . Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000; 203(1-2):1-60. DOI: 10.1016/s0378-5173(00)00423-3. View

Carpenter J, Crowe J . An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry. 1989; 28(9):3916-22. DOI: 10.1021/bi00435a044. View

Allison S, Chang B, Randolph T, Carpenter J . Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch Biochem Biophys. 1999; 365(2):289-98. DOI: 10.1006/abbi.1999.1175. View

Mensink M, Frijlink H, van der Voort Maarschalk K, Hinrichs W . How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm. 2017; 114:288-295. DOI: 10.1016/j.ejpb.2017.01.024. View

Tonnis W, Mensink M, de Jager A, van der Voort Maarschalk K, Frijlink H, Hinrichs W . Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins. Mol Pharm. 2015; 12(3):684-94. DOI: 10.1021/mp500423z. View

10.

Perez-Moral N, Adnet C, Noel T, Parker R . The aggregative stability of β-lactoglobulin in glassy mixtures with sucrose, trehalose and dextran. Eur J Pharm Biopharm. 2011; 78(2):264-70. DOI: 10.1016/j.ejpb.2011.02.002. View

11.

Izutsu K, Yoshioka S, Terao T . Decreased protein-stabilizing effects of cryoprotectants due to crystallization. Pharm Res. 1993; 10(8):1232-7. DOI: 10.1023/a:1018988823116. View

12.

Forney-Stevens K, Pelletier M, Shalaev E, Pikal M, Bogner R . Optimization of a Raman microscopy technique to efficiently detect amorphous-amorphous phase separation in freeze-dried protein formulations. J Pharm Sci. 2014; 103(9):2749-2758. DOI: 10.1002/jps.23882. View

13.

Izutsu K, Yomota C, Okuda H, Kawanishi T, Randolph T, Carpenter J . Impact of heat treatment on miscibility of proteins and disaccharides in frozen solutions. Eur J Pharm Biopharm. 2013; 85(2):177-83. DOI: 10.1016/j.ejpb.2013.05.004. View

14.

Hancock B, Shamblin S, Zografi G . Molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures. Pharm Res. 1995; 12(6):799-806. DOI: 10.1023/a:1016292416526. View

15.

Cicerone M, Pikal M, Qian K . Stabilization of proteins in solid form. Adv Drug Deliv Rev. 2015; 93:14-24. PMC: 4623959. DOI: 10.1016/j.addr.2015.05.006. View

16.

Bhattacharya S, Suryanarayanan R . Local mobility in amorphous pharmaceuticals--characterization and implications on stability. J Pharm Sci. 2009; 98(9):2935-53. DOI: 10.1002/jps.21728. View

17.

Chang L, Shepherd D, Sun J, Ouellette D, Grant K, Tang X . Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?. J Pharm Sci. 2005; 94(7):1427-44. DOI: 10.1002/jps.20364. View

18.

Wang B, Tchessalov S, Cicerone M, Warne N, Pikal M . Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. J Pharm Sci. 2008; 98(9):3145-66. DOI: 10.1002/jps.21622. View

19.

Giuffrida S, Cordone L, Cottone G . Bioprotection Can Be Tuned with a Proper Protein/Saccharide Ratio: The Case of Solid Amorphous Matrices. J Phys Chem B. 2018; 122(37):8642-8653. DOI: 10.1021/acs.jpcb.8b05098. View

20.

Du Y, Li J, Xu W, Cote A, Lay-Fortenbery A, Suryanarayanan R . Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions. Mol Pharm. 2023; 20(12):6380-6390. DOI: 10.1021/acs.molpharmaceut.3c00764. View