Protection of Protein Secondary Structure by Saccharides of Different Molecular Weights during Freeze-Drying

Abstract

Development of recombinant therapeutic proteins requires rational design of both the formulations and manufacturing processes. [1][2][3][4][5] Freeze-drying is a popular method to retain long-term stability of various proteins that are not sufficiently stable in aqueous solutions, although the stresses incurred during the freeze-drying process often induce partial unfolding and resulting protein aggregation in the rehydrated solutions. [6][7][8] Stable formulation design is required to avoid the biological activity loss and possible immunogenic effect of the structurally and/or chemically altered protein molecules.Many polyols (e.g., saccharides and sugar alcohols) protect proteins from inactivation during freeze-drying and subsequent storage. They protect native protein conformation against dehydration stresses by replacing essential water molecules through molecular interaction (e.g., hydrogen bonding) with the protein molecules.1-4,9) Some polyols also improve the physical and chemical stability of proteins during the freeze-drying process and subsequent storage by keeping the protein within a highly viscose glass-state amorphous solid with limited molecular mobility. 10,11) The choice of an appropriate saccharide and/or saccharide combination is crucial to the formulation design.1,2,12) Sucrose has been added to many freeze-drying formulations because of its potent structure-stabilizing effect and proven safety, whereas the low glass transition temperatures of the sucrose-based amorphous freeze-dried solids result in stability problems in storage at higher temperature or higher humidity. 13) Various saccharides posses different physical properties and protein-stabilizing effects.1-3) Large saccharide molecules often provide freeze-dried solids with high glass transition temperatures (T g s), which are suitable for protein storage stability. In contrast, they often show smaller effect to protect lower concentration enzymes (e.g., 1 mg/ml catalase) from inactivation during freeze-drying. 14)Protein molecules face different stresses during the freezedrying process depending on the initial concentrations, whereas the relationship between the saccharide molecular weights and the protein-stabilizing effect in freeze-drying of pharmaceutically relevant, high concentration (e.g., greater than 1 mg/ml) protein solutions has not been well elucidated.In addition to the low temperature and dehydration stresses, which are independent of the initial protein concentrations, lower concentration proteins are more likely to undergo physical stress on ice, air, and container surfaces. 3,4,15) In the present work, we used Fourier transform infrared (FT-IR) spectroscopy to study the effects of saccharides with different molecular weights on the secondary structure of proteins (BSA, ovalbumin) freeze-dried in high concentration. Addition of sodium tetraborate (Na 2 B 4 O 7 , borax) to aqueous saccharide solutions raises the "effective" saccharide molecular size through complex formation between a tetrahydroxy borate ion and one o...

Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes

“…Adding polysaccharides such as 0.5% maltose or 0.3% starch enhanced significantly the tolerance of T4 phage to the air-drying effect. This is consistent with previous reports that mentioned the usefulness of different polysaccharides in enhancing the stability of various biologically active compounds against desiccation (7,14,21). This study found that freeze-drying was an efficient method to dry phages with little decrease in phage activity, which would help in developing different paper-coating approaches for the commercial manufacture of phage-containing bioactive membranes.…”

Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations

“…Changes in the distribution of secondary structures of freeze-dried proteins are most commonly assessed by visual comparison of the second derivative of the amide I band (1700-1600 cm -1 ) in the blank corrected FTIR spectra of the sample with a native reference spectrum [10,15,25,26,27,28,29,30]. Freeze-dried formulations were designated as NL if the amide I spectrum was similar (in secondary structural elements distribution) to that of the native LDH before freeze-drying (Fig.…”

Effects of Excipients on Protein Conformation in Lyophilized Solids by Hydrogen/Deuterium Exchange Mass Spectrometry

“…Fourier transform infrared spectroscopy (FTIR) is widely used to study protein secondary structure in lyophilized solids (2,3). By comparing the FTIR spectrum of the solid protein formulation to the spectrum recorded under comparable solution conditions, an excipient_s ability to protect the protein_s native structure can be evaluated (4)(5)(6)(7). On the basis of such studies, several groups have suggested that the stabilization of the protein by the excipients is the result of protein-excipient interactions (4,6).…”

“…By comparing the FTIR spectrum of the solid protein formulation to the spectrum recorded under comparable solution conditions, an excipient_s ability to protect the protein_s native structure can be evaluated (4)(5)(6)(7). On the basis of such studies, several groups have suggested that the stabilization of the protein by the excipients is the result of protein-excipient interactions (4,6). Izutsu et al (6) reported that higher molecular weight carbohydrates had less protective effect than smaller carbohydrates such as sugars due to a reduction in the number of free hydroxyl groups available to interact with the protein.…”