Denaturing mechanism of freeze-drying process and storage process
Many factors (eg, chemical composition, freezing rate, freezing and dehydration stress, glass transition temperature, residual moisture in dry solids, temperature and humidity of the storage environment, etc.) during lyophilization and storage of food, pharmaceuticals, and organisms It will affect the stability of the active components and even lead to deactivation.
A large number of experimental studies have shown that in addition to some foods, human plasma, milk and other materials can be directly freeze-dried; most pharmaceuticals and biological products, need to add appropriate freeze-drying protectants and additives, formulated into a mixture of liquids Perform effective freeze drying and storage.
In the freeze-drying process of biological products, there are mainly three effects that lead to the denaturation of active components in biological products: low temperature effect, freezing effect and dehydration effect.
Low temperature effect
The active component of the biological product is denatured within a certain temperature range of the temperature drop and rewarming process. For example, the study of ovalbumin found that its activity decreased significantly between -10 °C and -40 °C, and continued to cool down to -192 °C, with little change in activity.
Freezing effect
Including the increase of ion concentration, ice crystal formation and growth, pH change and phase separation, etc.
(1) During the freezing process of biological products, continuous crystallization leads to a rapid increase in the concentration of the solution. The calculated concentration of small molecular sugars in the large frozen concentrated matrix of Zui is as high as 80%. When the concentration of the solution changes, the ion concentration increases, which promotes the chemical reaction.
(2) A large amount of ice-water interface is also generated during the freezing of the biological solution. Among them, active component molecules, such as proteins, may be adsorbed to the interface, which may destroy the natural fold structure of the protein, and the Zui eventually leads to protein denaturation.
(3) During the freezing process of some biological products, the pH of the solution also changes. For example, in the freezing of a protein solution to which a phosphate buffer solution having a pH of 7 (the molar ratio of NaH2PO4 and Na2HPO4 is 0.72) is added, since the solubility of NaH2PO4 is much larger than that of Na2HPO4, when the solution reaches the three-phase eutectic point, they are The molar ratio between them is 57, and Zui eventually causes a large change in pH. Proteins undergo physical aggregation and chemical denaturation.
Dehydration effect
(1) After sufficient hydration of the protein in the aqueous solution, a single layer of water is attached to the surface of the protein molecule, which is the hydration shell. In general, the water content involved in full hydration is 0.3-0.35 g (water) / g (protein). However, the water content in the lyophilized protein product generally does not exceed 10%, and therefore, some 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 Zui eventually leads to protein denaturation. This is because the protein rich in bound water is exposed to water in the dehydration process, converting protons into charged carboxylic acid groups, destroying the charge balance in the protein, and the decrease in charge density may promote the hydrophobic interaction between protein molecules. , so that the protein is aggregated. Â
Storage process
1. Protein agglomeration: one of the main factors for the degeneration of the active components of freeze-dried biological products during storage. The resulting protein agglutination may be a physical (non-covalent) interaction or a chemical agglutination (covalent) of the protein.
2, deamidation: is also one of the main ways of protein denaturation. Asparagine (Asn) and//33, and aminoamide (Gln) are two kinds of amino acids in proteins which are prone to deamidation.
4. Non-enzymatic browning: Also known as the Maillard reaction. It causes a reducing sugar such as glucose to form a carbohydrate complex with lysine and arginine in the protein.
5. Oxidation reaction: The side chain of methionine Met, cystine Cys, histidine His, tryptophan Trp and tyrosine Tyr residues in the protein is a possible position for oxidation reaction.
6. Hydrolysis: Although the lyophilized protein contains a very small amount of water, hydrolysis still occurs during storage.
As mentioned above, the freeze-drying process of biological products is a multi-step process that produces various effects such as low temperature, freezing and dehydration; even after successful completion of the freeze-drying process, it is difficult to ensure freeze-dried biological products during long-term storage. Stability of the active ingredient. In order to prevent the denaturation of active components during freeze drying and storage of biological products, researchers have studied and explored a large number of effective protective agents.
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