Small-Angle Neutron Scattering Study of Protein Crowding in Liquid and Solid Phases: Lysozyme in Aqueous Solution, Frozen Solution, and Carbohydrate Powders Joseph E. Curtis,* ,† Hirsh Nanda, † Sheila Khodadadi, † Marcus Cicerone, † Hyo Jin Lee, ‡ Arnold McAuley, ‡ and Susan Krueger* ,† † NIST Center for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 6102, Gaithersburg, Maryland 20899-6102, United States ‡ Department of Analytical and Formulation Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States * S Supporting Information ABSTRACT: The structure, interactions, and interprotein configurations of the protein lysozyme were studied in a variety of phases. These properties have been studied under a variety of solution conditions before, during, and after freezing and after freeze-drying in the presence of glucose and trehalose. Contrast variation experiments have also been performed to determine which features of the scattering in the frozen solutions are from the protein and which are from the ice structure. Data from lysozyme at concentrations ranging from 1 to 100 mg/mL in solution and water ice with NaCl concentrations ranging from 0 to 0.4 mol/L are fit to model small- angle neutron scattering (SANS) intensity functions consisting of an ellipsoidal form factor and either a screened-Coulomb or hard-sphere structure factor. Parameters such as protein volume fraction and long dimension are followed as a function of temperature and salt concentration. The SANS results are compared to real space models of concentrated lysozyme solutions at the same volume fractions obtained from Monte Carlo simulations. A cartoon representation of the frozen lysozyme solution in 0 mol/L NaCl is presented based on the SANS and Monte Carlo results, along with those obtained from other complementary methods. ■ INTRODUCTION The structure of proteins in the solid state is of interest to both the pharmaceutical and food science industries, as both industries have a need to devise ways to stabilize their products for extended periods of time without degradation. Both freezing and freeze-drying (lyophilization) are important methods used for long-term storage. However, both methods present challenges for protein stability. As therapeutic agents, proteins provide a number of treatments for human diseases and conditions. However, the development of commercial applications is challenging due to protein stability. Proteins can be degraded chemically or physically. Chemical degradation refers to modifications involving covalent bonds, such as deamidation, oxidation, and disulfide bond shuffling, while physical degradation includes protein unfolding, undesirable adsorption to surfaces, and non- native aggregation, the latter which is particularly problematic because it is encountered routinely during refolding, purification, sterilization, shipping, and storage. Factors affecting stability include temperature, solution pH, ligands and cosolutes, salt type and concentration, preservatives, and surfactants. 1,2 Lyophilized formulations are often developed to avoid protein degradation issues. 3,4 Freezing is the first step of a lyophilization process, and in many cases, especially early in the development process to manufacture a protein solution, samples are frozen to maintain biological activity. 5 Solvent additives are often introduced into protein solutions prior to lyophilization, as they have been shown to inhibit drying- induced damage, improve the activity of proteins upon rehydration, and enhance the stability of biological systems during storage. 5,6 A complete understanding of the spatial organization and interaction of proteins in heterogeneous frozen phases is lacking, although many interesting and promising studies have been recently reported. 7-10 One missing aspect from these studies is the relative interprotein distance which is an important determinant in order to understand the enhancement of deleterious chemical and conformational changes that occur at increased rates in the crowded environment. Freezing is an important preservation method used to prevent the growth of microorganisms and to slow chemical reactions, such as oxidation, to preserve the quality, nutrient Received: May 16, 2012 Revised: July 16, 2012 Published: July 23, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 9653 dx.doi.org/10.1021/jp304772d | J. Phys. Chem. B 2012, 116, 9653-9667