Protective agent in freeze-drying

Freeze-drying is a drying method in which the drug solution that needs to be dried is frozen into a solid in advance. And directly sublimated to remove water from the frozen state under low temperature and low-pressure conditions without passing through the liquid state. Since the entire operation process is in a low-temperature state, this method is especially suitable for the preparation of heat-sensitive protein pharmaceutical preparations. The freeze-dried protein drugs were loose, which was not only beneficial to the preservation but also beneficial to the refolding of protein drugs after redissolution.

Obviously, lyophilization technology provides an effective method for the preparation of protein drugs with unstable physical and chemical properties. However, the lyophilization process is a complex phase transformation process. There are many factors that induce protein denaturation in drugs during freezing, freeze-thaw, drying, and storage. Thus It is often necessary to use some protective agents to stabilize the protein in the prescription. The cryoprotectant is various, and its mechanisms are complex. In this article, the protection agents for protein lyophilized products will review.

In the whole process of the lyophilized product, exists many stresses, including low-temperature stress, freezing stress, drying stress, and so on. These stresses are often the factors that directly or indirectly lead to the instability of protein drugs. According to the difference of protective agents against stress, protein protective agents are preliminarily divided into cryoprotectants and freeze-dried protective agents. An excellent protein protectant can not only protect the protein drug during the entire freeze-drying process but also inhibit the denaturation of the protein drug during the storage period of the finished product. Since the denaturation rate of protein drugs during storage is often greater than that during freeze-drying, most types of protein cryoprotectants that are effective in solution have no protective effect on dry proteins, and even accelerate protein drugs for unstable ratio.

To expand the protective effect, it needs to use more than two types of protective agents in freeze-dried products, such as polyhydroxy compounds, sugars, proteins, polymers, amino acids, salts, amines, and surfactants.

1.Polyhydroxy compounds

Polyhydroxy compounds have long been used as antifreeze agents for proteins. Common lyophilized protective agents include glycerol, mannitol, sorbitol, inositol, mercaptan, polyethylene glycol, etc. Glycerol can promote the renaturation of lyophilized catalase. And as the glycerol concentration rises to 0.8%, catalase can completely renature. Mannitol can be a freeze-dried protective agent for protein in some prescriptions. The protective effect of mannitol on protein is related to its concentration, morphology, and structure, and its concentration is sometimes correlated with crystal morphology. It is generally believed that amorphous mannitol has the effect of stabilizing proteins, while crystalline mannitol loses its protective function. 1% or lower concentration of mannitol 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 to promote the aggregation of protein drugs.


Sugar is the most common and widely used type of lyophilized protectant. It is a non-specific stabilizer for protein, and it can protect protein drugs to a certain extent during all stages of lyophilization. Sugar protective effect related to its and type of protein and disaccharides is the most studied also recognized the most effective protective agent, in which sucrose is a disaccharide composed of a molecule of glucose and a molecule of fructose, stable chemical properties, amorphous structure. For blocking protein secondary structure changes, freeze-drying process and during extended storage period in protein’s extend and aggregates play a significant function.

Compared with sucrose, trehalose has a higher glass transition temperature, lower hygroscopicity, and no reducing properties. These advantages all indicate that trehalose may have a broader application prospect. Macromolecules sugar appears to provide less protection against proteins than micromolecule sugar. Many studies have shown that polysaccharides with high molecular weight are not the best choice of lyophilized protectant.

But some studies have shown the opposite, such as large molecular weight inulin study shown that inulin with a high degree of polymerization of 5.5-6.0 has a significantly higher protective effect on alkaline phosphatase than trehalose and inulin with a low degree of polymerization. This shows that the protective effect of polysaccharides cannot be generalized.

Especially during the storage period, dextran or polysaccharide can increase the glass transition temperature of the product and prevent the protein from being destroyed due to the collapse of the product. Glucose can partially remain amorphous forms during the freeze-drying process, which has a certain protective effect on certain protein drugs, but it should be noted that glucose-based reducing sugars should be carefully selected as protein protective agents. The protective effect of sugar on protein sometimes depends on its concentration.

Usually, the protective effect of sugar increases as the concentration increases within a certain concentration range. When it reaches a certain concentration, the protective effect reaches its maximum value, and then increasing the concentration of sugar will no longer increase the protective effect significantly, but sometimes decreases. There are different reports on the concentration of sugar when it exhibits the maximum protective effect.

For example, 100mmol/L sucrose has the best protective effect on B-galactose enzyme, while the concentration of trehalose that provides the maximum protective effect on phosphofructokinase is 300mg/ml.The lowest concentration of sugar reported for maximum stabilization is one that is sufficient to form a monomolecular layer on the protein surface.

In fact, the protective effect of sugar on protein depends not only on the overall concentration of sugar but also on the ratio of the two. Some studies have found that when the molar ratio of aD-mannopyranose lactose, trehalose, cellobiose to recombinant human growth hormone is 131:1, they can all 100% occupy the strong and weak water bind site on recombinant human growth hormone, to provide the best protection effect. When the molar ratio reaches 300:1 and 1000:1, the protective effect of sugar on recombinant human growth hormone will no longer increase and the protective effect will decrease in some prescriptions.

The reducing properties and other properties of sugar will also affect its protective effect on proteins. Glucose, lactose, maltose, cellobiose, and other reducing sugars may have mylard reaction(ammonia or brown reaction) with certain amino acid residues exposed in protein. It will make the product yellow and reduce protein activity. Maltodextrins with different optical activity showed great differences in their protective effects on lactate dehydrogenase during lyophilization.

Sugars currently used as protein protective agents include glucose, aD-mannopyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, white sugar, inulin, dextran, maltodextrin, Maiya polysaccharides, heparin, 2-hydroxypropyl-B cyclodextrin, and so on. Although sugar can provide reliable protection for most proteins, not all proteins can be protected by sugar, which still needs more detailed and in-depth research.

3.Amino acids

Amino acid is one of the common protein protective agents. During the freezing process, a low concentration of glycine can prevent the denaturation of protein drugs by inhibiting the change of pH value caused by the crystallization of 10 or 100mmol/L phosphate buffer salt. Amorphous forms of glycine can prevent the aggregation of recombinant human growth hormone during the freeze-drying process. Crystalline glycine can increase the collapse temperature of the finished product to prevent the destruction of protein drugs caused by the collapse. Commonly used amino acid protein protective agents include proline, tryptophan, sodium glutamate, alanine, glycine, and lysine Hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, etc.


Polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, polyethyleneimine, and other polymers are also commonly used as cryoprotectants for proteins. Generally, the stabilizing effect of a polymer depends on the multiple properties of the polymer. Different degrees of polymerization and concentration of polymers provide different protection. For example, PVP with a high degree of polymerization has a higher glass transition temperature. As the concentration and molecular weight of PVP increase, the viscosity of the aqueous solution will increase, and the glass transition temperature of sucrose will increase significantly, which also increase protects the protein during the freezing process.

However, when the degree of polymerization is too large, the polymer will crystallize during the freezing process and lose its protective effect on the protein. Excessive concentration and molecular weight will increase the moisture of the final product, making the protein in the product more unstable. Dextran 40 has a stronger effect on stabilizing porcine trypsin than dextran 80, suggesting that the appropriate chain length of the polymer is beneficial to the stability of trypsin.


Protein protective agents can be divided into two types: one is a protein drug, and the other is an exotic protein. The activity of some proteins after freezing and thawing is directly related to the initial concentration of the protein. An increase in the initial concentration sometimes promotes the renaturation of the protein.

A commonly used protective agent for exotic proteins is serum albumin, which is a classic and excellent protein stabilizer. For example, 1% bovine serum albumin can prevent the activity of rabbit muscle lactate dehydrogenase aqueous solution from being lost when freezing. Human serum albumin can effectively prevent the adsorption of protein surface at lower concentrations (0.05%-0.1%) and has a protective effect on the logarithmic protein during the freeze-drying process. However, due to serum albumin of potential blood borne pathogen pollution limits its application in protein products. Recombinant human albumin has been recommended as a substitute for serum albumin.


Some surfactants(Tween 80, Bridger, Pluronic, and Sodium Dodecane Sulfonate) can play a certain protective effect on the protein during the drying process of the freezer. In some freeze-dried products, adding salts and amines can obtain specific protein stabilization effects. Combining different types of protective agents can obtain freeze-dried products with better stability.

How to obtain a stable freeze-dried protein product, it is very important to choose an appropriate protective agent. So how do choose the appropriate protective agent? First of all, we must have a full understanding of the physical and chemical properties of the protein drug to be protected, and then through experiments to find out the factors that cause the protein drug to be unstable during the freeze-drying process and storage process, and then select specific protective agents for these unstable factors. Generally speaking, sugars and polyhydroxy compounds are the most commonly used freeze-dried protective agents. Among them, disaccharides such as sucrose and trehalose can show good protective effects on some proteins.

An excellent freeze-dried protective agent should have a variety of protective properties, such as high glass transition temperature, low moisture absorption, low crystallization rate, and no reducing groups. But any single protective agent cannot have all the protective properties, so sometimes 2 or more protective agents have to be considered to stabilize protein drugs.


With the rapid development of biotechnology, more and more protein drugs have been developed. Up to now, there is no more effective preparation method for preparing more stable protein products except the use of freeze-drying technology. Even if freeze-drying technology is used to prepare certain protein products, some effective auxiliary methods are still needed, such as adding freeze-drying protective agents to increase protein stability. In recent years, research on freeze-dried protective agents and their protective mechanisms has been more in-depth. In particular, the improvement of protein analysis methods provides convenience for the research of protein protective agents.

The application of computer technology in the freeze-drying process enables the establishment of corresponding mathematical models to simulate and analyze the real state of the materials during the freeze-drying process, which also enables the freeze-drying technology Can be improved and improved. However, the protein stabilization mechanism is quite complicated, and the research on protein freeze-dried products needs to be further deepened. The development of better freeze-dried protective agents and the improvement of the quality of protein drugs freeze-dried products are still the focus and direction of current biotechnology drug research.