Factors That Cause Protein Denaturation During Freeze-Drying Process

What causes denaturation of proteins during freeze-drying process? This article will list three main factors that cause protein denaturation: low temperature, freezing and dehydration.

Factors that cause protein denaturation — low temperature

The active components in biological products will be denatured within a certain temperature range during cooling and rewarming. For example, the study of ovalbumin found that between -10°C and -40°C, its activity decreased significantly, while continuing to cool to -192°C, there was almost no change in activity.Freeze-Drying-Process

Factors that cause protein denaturation — freezing

Freezing, as one of the factors that cause protein denaturation, will results in several changes during the process: increase in ion concentration and ice-water interface, and pH changes, etc.

(1) Increase in ion concentration
During the freezing process of biological products, continuous crystallization will cause the concentration of the solution to increase rapidly. The calculated concentration of small molecule sugar in the largest frozen concentrated matrix is as high as 80%. When the concentration of the solution changes, the ion concentration increases, which promotes the chemical reaction.

(2) Increase in ice-water interface
A large amount of ice-water interface will also be generated during the freezing process. Proteins may be adsorbed to the interface, which may destroy the natural fold structure of the protein and eventually lead to the denaturation of the protein.

(3) pH changes
The pH of the solution will also change. For example, during the freezing of a protein solution added with a phosphate buffer of pH 7 (the molar ratio of NaH2PO4 and Na2HPO4 is 0.72), since the solubility of NaH2PO4 is much greater than that of Na2HPO4, when the solution reaches the three-phase eutectic point, the molar ratio between them is 0.57 which ultimately results in a large change in pH. The protein undergoes physical aggregation and chemical denaturation.

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Factors that cause protein denaturation — dehydration

What causes denaturation of proteins? One of the widely-known factors is dehydration. Dehydration will result in the removal of bound water, which will eventually destroy the structure of the protein and lead to protein denaturation.

(1) After the protein in the aqueous solution has been fully hydrated, a single layer of water is attached to the surface of the protein molecule, which is called the hydration shell. Generally speaking, the water content involved in complete hydration is 0.3-0.35g (water)/g (protein). While the water content of lyophilized protein products generally does not exceed 10%, therefore, part of the bound water must be removed during the drying process.

(2) The removal of bound water is likely to destroy the natural structure of the protein and eventually lead to protein denaturation. This is because the protein rich in bound water is exposed to the water-lacking environment during the dehydration process, which converts protons to charged carboxylic acid groups, disrupts the charge balance in the protein. The decrease in charge density may promote the hydrophobic interaction between protein molecules , So that protein aggregation occurs.

The freeze-drying process of proteins is a multi-step process, which will produce various effects such as low temperature, freezing and dehydration. What causes denaturation of proteins? There is no single answer to this question. Even after successfully completing the freeze-drying process, it is difficult to ensure the stability of the protein during long-term storage. There are multiple factors that cause protein denaturation , and effective protective agents are therefore needed to keep the activity of proteins.

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