In the solid state proteins are frequently more stable, so formulations of solid dosage forms are often used to increase the stability. However, the process of making solids is still quite challenging.
Two frequently used methods are lyophilization (freeze-drying) and spray drying; common for both processes are the formation of particles that require hydration or reconstitution before use and the removal of water (solvent) from the liquid formulation. Therefore, the effects from the removal of water and the use of excipients to substitute the hydrogen bonds are a major issue. Pharmaceutically, freeze-drying is the most commonly used process for ensuring long-term storage stability of proteins (30,73,74). Freeze-drying of proteins enhances the physical stability by inducing a rigid protein structure, due to the reduced molecular mobility in the solid state compared to protein solutions. The aim of an optimized freeze-dried process is to obtain the protein in a solidified, freeze-dried cake, which fixes the protein in a rigid structure with low moisture content and with the durability to be stored over the desired period of time.
The freeze-drying process is divided into three stages: Freezing, primary drying (where the solvent is removed by sublimation), and secondary drying (where the residual solvent is removed by desorption). When freezing, ice crystals are formed and physically separated from the proteins (74). Lowering the temperature below the freezing point makes the bulk freeze. However, some of the water remains unfrozen and is adsorbed onto the proteins. Removing water during the freezing process increases the concentration of proteins, and of excipients if added. Primary drying is the step where the bulk water is removed (74). During the primary drying step, the ice crystals are removed from the bulk material by sublimation. In the secondary drying step, the unfrozen adsorbed water is removed. The amount of residual water can be up to 20% and is adsorbed to the cake. The residual water is removed by raising the temperature (74). Hereby, the water is removed by diffusion, flowing from a region with high concentration to a region with lower concentration. The water content ought to be reduced to a level that is optimal for the stability of the protein. This is typically less than 1 to 2 w/w % (74,75). However, the level of water can also be too low causing the protein hydration layer to be disrupted. This may result in conformational changes and aggregation (44).
The freeze-drying of proteins yields a dried powder containing the protein in a glassy phase often including amorphous excipients and residual water. The amorphous state is important for maximizing the stability after freeze-drying because this state allows maximal hydrogen bonding (73). The transition between the rubbery (glass-like) and glassy (solid-like) state is called the glass transition temperature (Tg). This parameter can be used to characterize the freeze-dried product. Raising the temperature above the Tg increases the mobility. Therefore, Tg should be as high as possible so that durable storage stability, preferably at room temperature, can be obtained. Also, the storage temperature can be increased with higher T% values (73).
It is often necessary to add stabilizing excipients to stabilize proteins during the stress induced by a freeze-drying process. The stabilizing excipients can have cryoprotectant and/or lyoprotectant effects on the protein (75,76). Cryoprotection is generally believed to be accomplished by a preferential interaction. In the presence of a stabilizer, the protein thereby interacts preferentially with water, and the excipient is preferentially excluded (73). Lyoprotection effects can operate by the water-replacement theory. Stabilizing excipients can replace water by forming hydrogen bonds with the polar groups on the protein surface ensuring the protein conformation (77,78). Commonly used excipients are sugars, which have effects both during the freezing and drying processes. Furthermore, the addition of amino acids can act as cryoprotectants and/or lyoprotectants, some examples are shown in Table 6.
The addition of excipients generally lowers the Tg of the freeze-dried product when compared to the protein without added excipients. The Tg of proteins is high, above 150°C (75) and typically the Tg of excipients ranges from 50°C to 20°C. Therefore, formulation of proteins often includes stabilizing excipients, which decrease the Tg. The Tg of the freeze-dried product depends on the individual Tg and mass fraction of each compound in the solution. (75). When reducing sugars are used as protectants, they may react with the amino acid groups in the protein. This reaction is known as the Maillard reaction (described in 1912 by Maillard). The Maillard reaction occurs at high temperatures forming a browning carbohydrate complex (79). Some practical advice on how and what to take into account when formulating freeze-dried products can be found in Carpenter et al. (1997) (78) and Schwegman et al. (2005) (80).
Spray drying, is the process in which a liquid is transformed into dry particles by atomization. The process consists of a droplet formation step followed by evaporation of the liquid, typically, spraying of a liquid into hot air. The major concern when using biomacromolecules is the temperature applied and whether the protein can withstand the stress effects (81,82). Maa and Prestrelski (2000) review the different possibilities for preparing particles for pharmaceutical applications (82).
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