Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.
For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5 nm) and the size of the protein domains (∼2.5 × 2.5 × 5 nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein.
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Maarten A
MA
Mensink
Department of Pharmaceutical Sciences, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky, 40536-0596, USA.|Kansas Analytical Services, Fort Collins, Colorado, USA.
Matthew J
MJ
Nethercott
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands. W.L.J.Hinrichs@rug.nl.
Wouter L J
WLJ
Hinrichs
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.|Process Technology, Corbion Purac, Gorinchem, The Netherlands.
Kees
K
van der Voort Maarschalk
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Henderik W
HW
Frijlink
Department of Pharmaceutical Sciences, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky, 40536-0596, USA. eric.munson@uky.edu.
Eric J
EJ
Munson
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut, USA.
Michael J
MJ
Pikal
D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry*; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005612:Freeze Drying / Q000379:methods; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D055550:Protein Stability; D011506:Proteins / Q000032:analysis
D002241:Carbohydrates; D007074:Immunoglobulin G; D011506:Proteins
homogeneity; immunoglobulin G (IgG); phase separation; solid-state nuclear magnetic resonance spectroscopy (ssNMR); steric hindrance