Effect of Sodium Tetraborate (Borax) on the Thermal Properties of Frozen Aqueous Sugar and Polyol Solutions

Abstract

Recent advances in biotechnology have focused attention on the stable storage of the biological polymers (e.g., proteins) and biological systems (e.g., cells) for medical use. Freezing and freeze-drying are popular methods to archive their long-term stability.1-4) Various co-solutes such as sugars (e.g., sucrose, trehalose) and other polyols protect proteins and cells through different mechanisms against stresses in aqueous solutions, frozen solutions, and freeze-dried solids. 5,6) Freezing an aqueous solution concentrates the solutes into a supercooled solution surrounded by ice crystals. 7,8) The sugars and polyols protect the proteins and cell membranes against dehydration-induced structural perturbation by substituting water molecules through direct molecular interactions (e.g., hydrogen bonding). Decreased molecular mobility in the polyol-based glass-state amorphous phase reduces chemical changes of the components.3,7) Controlling the physical properties of the freeze-concentrates and subsequently freeze-dried solids should provide the key to production of stable protein and other biopolymer formulations. 1,2,7) Thermal analysis of frozen aqueous carbohydrate solutions often shows several transitions of the amorphous supercooled phase including the "real" glass transition (T g ) and the glass transition of maximally freeze-concentrated solutes (T g Ј) at temperatures depending on the solute compositions. 9,10) The terming and implications of these thermal transitions are still under some debate, whereas it has been established that the changes in the solute and surrounding water mobility at the transition temperatures have significant impact on the physical and chemical stability of the components.11) The T g Ј transition, which involves ice melting around the solute molecule, is often the most apparent, and is the most important in the development of freeze-dried formulations because the increased solute mobility above the T g Ј typically induces cake collapse during the freeze-drying process.1,2) The "real" glass transition of the frozen solution (T g ) occurs without ice melting, and is often less apparent in thermal analysis.It is well known that borate forms chemical complexes with polyhydroxy compounds. [12][13][14] Addition of sodium tetraborate decahydrate (borax), which hydrolyzes to boric acid and sodium borate (Na 2 B 4 O 7 ϩ7H 2 O®2B(OH) 3 ϩ 2B(OH) 4 Na), to aqueous sugar and/or other polyol solutions results in various complexes between the borate ion and the polyols. Increase in the "effective" molecular size and changes in the molecular interactions by the complex formation (e.g., borate-trehalose) leads to reduced component mobility in the amorphous freeze-dried solids and hydrated mixtures. [15][16][17] Co-lyophilization with the borate-sugar combinations improves the protein stability at higher temperatures. 15)The large dependence of the frozen saccharide solution T g Јs on their molecular weights has suggested alternation of the T g Ј through the borate-polyol complex formation. 18,19) ...

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

“…Many other borate-saccharide complexes are expected to achieve the kinetic protein stabilization at the expense of the structure-stabilizing effect. [16][17][18] Because of this drawback, borate-saccharide combinations will be useful in only a limited number of applications, such as for high temperature storage. Possible health risks of boron-containing compounds should be also taken into consideration in pharmaceutical formulation design.…”

“…Some combination frozen solutions presented transitions at temperatures above those of the single-solute solutions, suggesting a strong interaction between the components. 18) Freeze-dried solids from solutions containing BSA (10 mg/ml) and saccharides (50 mg/ml) showed a broad glass transition in thermal analysis (Fig. 4).…”

“…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 or two saccharide molecules. [16][17][18] How the borate-saccharide complexation affects the protein structurestabilizing effect was also studied. …”

“…Co-lyophilization with sodium tetraborate raised the glass transition temperatures, indicating a reduced saccharide molecular mobility by the complexation. [16][17][18] February 2004 201 Freeze-dried solids from aqueous solutions containing BSA (10 mg/ml), dextrans (50 mg/ml), potassium phosphate buffer (20 mM), and sodium tetraborate (0-50 mM) were subjected to FT-IR analysis. Figure 5 shows the area-normalized second-derivative FT-IR spectrum of BSA freeze-dried with three molecularweight dextrans.…”

Improving Heat Transfer at the Bottom of Vials for Consistent Freeze Drying with Unidirectional Structured Ice

“…The sample is quickly cooled to a temperature of −65°C, held for 30 min, and then heated at a rate of 5°C/min back to room temperature under a continuously purged dry nitrogen atmosphere (flow rate of 50 mL/ min). The glass transition temperatures (Tg′) of the frozen solutions were obtained from the DSC thermograms (23).…”