Types Of Cryoprotectants
There are various stresses throughout the freeze-drying process, usually including low-temperature stress, freezing stress, drying stress, etc. The protective agents used in the freezing process are called cryoprotectants, while in the drying process are lyoprotectants. Today’s topic is cryoprotectants, and types of cryoprotectants include polyhydroxy compounds, sugars, proteins, polymers, amino acids, salts, amines, surfactants, and the like.
1.Types of cryoprotectants — polyhydroxy compounds
The polyhydroxy compound is one of the typical types of cryoprotectants. Common cryoprotectant examples of this type include glycerin, mannitol, sorbitol, inositol, thiol, and polyethylene glycol. Glycerin can promote the renaturation of lyophilized catalase, and as the concentration of glycerin rises to 0.8%, the catalase can completely renature. Mannitol can not only be used as an excellent matrix agent, but also a cryoprotectant for proteins in some prescriptions. The protective effect of mannitol on protein is related to its concentration and morphological structure, and its concentration is sometimes related to the crystal form. It is generally believed that amorphous mannitol has a protein-stabilizing effect and crystalline mannitol loses its protective function; mannitol at a concentration of 1% or lower prevents the aggregation of protein drugs through the formation of an amorphous structure, but a high concentration of mannitol is easy to form a crystalline state and promote the aggregation of protein drugs.
2. Types of cryoprotectants — sugar
Among various types of cryoprotectants, sugar is the most common and widely used one. It is a non-specific stabilizer for protein. It can play a certain role in the protection of protein drugs at various stages of lyophilization (such as freezing, freeze-thaw, and sublimation drying). Disaccharide is the most studied and effective protective agent. And sucrose as a a disaccharide, is composed of a molecule of glucose and a molecule of fructose, which is chemically stable and mostly amorphous. playing a significant role in preventing protein secondary structure changes, protein extension, and aggregation during freeze-drying and storage. Sucrose and trehalose are all cryoprotectant examples. Compared with sucrose, trehalose has a higher glass transition temperature, lower moisture absorption, and less reducibility. These advantages all indicate that trehalose may have a broader application prospect.
3. Types of cryoprotectants — amino acids
Amino acids are one of the common types of cryoprotectants. During freezing, a low concentration of glycine can prevent the denaturation of proteins by inhibiting the change of PH value caused by the crystallization of 10 or 100 mmol/L phosphate buffer salt. Amorphous glycine can prevent the aggregation of recombinant human growth hormone during lyophilization; crystalline glycine can increase the collapse temperature of the finished product to prevent the destruction of protein drugs caused by the collapse. Common amino-acid cryoprotectant examples are proline and tryptophan, sodium glutamate, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, etc.
4. Types of cryoprotectants — polymer
Polymers such as polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, and polyethyleneimine are also cryoprotectant examples for proteins. Generally, the stabilizing effect of the polymer depends on multiple properties. Polymers with different degrees of polymerization and concentrations provide different protections. For example, PVP with a large degree of polymerization has a higher glass transition temperature. As the concentration and molecular weight of PVP increase, several factors will consequently increase one after another: the viscosity of the aqueous solution, the glass transition temperature of sucrose, and the protective effect on the protein in the freezing process. But when the degree of polymerization is too large, the polymer will crystallize during the freezing process, thereby losing the protective effect on the protein. And when the concentration and molecular weight are too large, the moisture of the final product will be increased, making the protein in the product more unstable.
5. Types of cryoprotectants — protein
Protein is also one of the types of cryoprotectants. It can be classified into two groups: one is the protein drug itself, and the other is the foreign protein. The activity of some proteins after freezing and thawing is directly related to the initial concentration of the protein, and an increase in the initial concentration sometimes promotes the renaturation. Serum albumin is one of the common foreign protein cryoprotectant examples. It is a classic and excellent protein stabilizer. This serum albumin has a protective effect on most proteins during the lyophilization process, so it is often used in the lyophilization prescription of hydrophobic cytokines such as interleukins and macrophage colony-stimulating factor (M-CSF). However, due to the potential bloodborne pathogen contamination of serum albumin, its application in protein products is limited. Recombinant human albumin has been recommended as a substitute for serum albumin.
6. Types of cryoprotectants — others
Some surfactants such as Tween 80, Bridger, Planic, and sodium dodecyl sulfonate sometimes play a certain role in protecting proteins during freezing and drying. In some freeze-dried products, the addition of salts and amines can achieve specific protein stabilization effects.
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