Xinyi Instrument: Application of heat-resistant lyoprotectant in vaccine production Research freeze-drying protectant can affect the quality, potency and stability of biological products, change the production process of biological products, improve the drying ability of products and increase the batch of products. Stability between times. The heat-resistant lyoprotectant is immunologically active but has no pharmacological activity. It can maintain the stability of the vaccine during lyophilization and storage. The vaccine does not collapse at the appropriate temperature during the first drying, and the enterprise can obtain it at low cost.
First, classification
(1) According to the relative molecular weight
According to the relative molecular weight fraction, it can be classified into a low molecular compound and a high molecular compound.
1. Low Molecular Compounds Low molecular compounds can increase the survival rate of microorganisms and form a uniform suspension to relieve water. Such as:
Acidic substances: glutamic acid, aspartic acid, lactic acid;
Neutral substances: glucose, lactose, sucrose, trehalose, sorbitol;
Alkaline substance: arginine, histidine.
2. Polymer compounds Polymer compounds have a protective effect on microorganisms, which can promote their sublimation to form a heat-resistant skeleton to block heat conduction and heat radiation. Such as: albumin, gelatin, peptone, skim milk powder.
Hydrolyzed gelatin Hydrolyzed gelatin can remove impurity protein, no antigenicity, no allergic reaction, no heat source, and has small molecular weight, homogeneous, soluble in water, and can be filtered and sterilized. The eutectic point is -12 °C. The protection of microorganisms is higher than that of ordinary gelatin by more than 10%.
Skim milk powder promotes sublimation, heat sterilization, easy to obtain homogeneous products, and expands the distance between cells.
(2) by function and nature
1. Heat-resistant lyoprotectant The heat-resistant lyoprotectant prevents denaturation of active ingredients during freezing and drying, such as trehalose, sucrose, povidone (PVP).
2. Filler It can prevent the effective components from sublimating along with water vapor, such as mannitol, gelatin and so on.
3. The self-oxidation of the antioxidant product can consume oxygen in the lyophilized sample and in the environment; the insertion of electrons or hydrogen ions can block the oxidative chain reaction in the lyophilized sample, inhibit the oxidase activity, and prevent the sample from being freeze-dried and Oxidative deterioration during storage. Such as sodium thiosulfate, vitamin E, vitamin C and so on.
4. Acid-base conditioner During the freeze-drying and storage process, the pH of the biological product is adjusted to the most stable area of ​​the active material. Commonly used are: potassium dihydrogen phosphate, sodium hydrogen phosphate.
(3) According to the type of the substance, this report is from Xinyi Instruments.
1. Sugar/Polyols Monosaccharides (glucose, galactose): The sugar forms a hydrogen bond with the molecules of the active component of the biological product instead of the position of the original water to protect it;
Oligosaccharides (sucrose, trehalose): oligosaccharides can protect against low temperature and dehydration; trehalose has a relatively high glass transition temperature. The formation of trehalose-protein-water micro-ice crystals effectively prevents the plasticization of water in the vitrified state. And its internal hydrogen bonds are less, which is beneficial to the formation of hydrogen bonds between protein molecules.
Polyol (mannitol, sorbitol, glycerol): The polyol has the same effect as sugar. The mannitol sterile filtrate is stable, is not easily oxidized, provides a supporting structure, and does not react with the active component; sorbitol is an isomer of mannitol, but its solubility is larger than mannitol, and it is sticky at normal temperature. A thick, transparent liquid that is optically active, slightly sweet, hygroscopic, and unstable at high temperatures. Sorbitol is often used as a filler in freeze-dried formulations.
2. Surfactants Surfactants are compounds that reduce the tension of the interface and are composed of hydrophilic and lipophilic groups. Surfactants can be classified into ionic and nonionic types. Any substance that dissolves in water and ionizes into ions is called an ionic surfactant, otherwise it is called a nonionic surfactant. In the process of freezing and dehydration, the surfactant can not only reduce the freezing and dehydration deformation caused by the interfacial tension of ice and water, but also act as a wetting agent for the active component in the rehydration process. However, surfactants have no protective effect in freeze-dried biological assays and long-term storage.
3. Amino Acids Amino acids are the basic building blocks of proteins, the most important of which is a-amino acids. Commonly used amino acid protecting agents are glycine, glutamic acid, arginine and histidine, and tests have shown that amino acids are the best fillers.
Low concentration of glycine can prevent protein drug denaturation by inhibiting the change of pH caused by crystallization of 10 or 100 mmol/L phosphate buffer salt, and can increase the collapse (disintegration) temperature of the finished product and prevent the destruction of protein drugs caused by collapse. .
4. Other Additives Other additive-based protective agents include antioxidants, buffers, and freeze-drying accelerators.
Antioxidant: One is the self-oxidation of the antioxidant, consuming the oxygen inside the lyophilized sample and the environment, so that the lyophilized sample material is not oxidized; the other is to give the electron or hydrogen ion to the antioxidant, blocking the lyophilized sample An oxidative chain reaction; another way is that the antioxidant prevents oxidative deterioration of the lyophilized sample by inhibiting the oxidation activity, such as vitamin E, vitamin C, sodium thiosulfate, thiourea.
Buffer: The protein has an ampholytic electrolyte that acts both on the acid and on the base. In a neutral environment, most proteins are stable. Since the concentration of the solution in the protein solution is gradually increased during the freezing process, the pH of the solution can be changed at a high concentration, and the pH changes by 4 units to cause protein denaturation. To inactivate biological products. Therefore, in the lyoprotectant formulation, an appropriate amount of buffer should be added. For example, potassium dihydrogen phosphate, sodium dihydrogen phosphate.
Freeze-drying accelerator: The freeze-drying process takes a long time and consumes a lot of energy. It is urgent to optimize the freeze-drying cycle and reduce the production cost. Tert-butanol is a small molecule alcohol that is completely miscible with water and has low toxicity and high vapor pressure. The addition of t-butanol to an aqueous solution of the drug can serve the following effects:
It can reduce the resistance of the drying layer, thereby accelerating the drying process and shortening the drying time; for the poorly soluble water, the product can have a high specific surface area, a good appearance, and easy to rehydrate, and improve the drug solution and the lyophilized product. Stability, and has a certain antibacterial effect.
Second, the mechanism of action of lyoprotectants
(1) Mechanism of low temperature protection during freezing
The "priority effect" mechanism assumes that the protein solution preferentially interacts with water (preferential hydration) before reaching the maximum freezing concentration, while the protective agent is preferentially excluded from the protein region (priority rejection). This is because the addition of the protective agent increases the surface tension of the water molecules, prompting the protein molecules to preferentially interact with the water molecules. In this case, the outer surface of the protein molecule has relatively more water molecules and relatively less protective agent molecules than in its bulk phase, thereby protecting the native conformation of the protein.
The "priority effect" mechanism does not fully explain the phenomenon of protecting proteins at high concentrations with polymers or proteins themselves. Therefore, there must be other mechanisms of protection. The mechanism of surface tension reduction can be used to explain the protection of surfactants to the freezing process of protein solutions. The mechanism that limits the diffusion of protein molecules suggests that many protective agents can increase the viscosity of the solution and inhibit the diffusion of active molecules.
(2) Protection mechanism during drying
Glass state hypothesis In the drying process with a protective agent solution, when the concentration is sufficiently large and the protective agent does not crystallize, the mixture of the protective agent and the active component forms a glassy state. The glass state is divided into two types: strong glass and weak glass.
Cooling below the glass transition temperature, the viscosity of the weak glass increases faster than that of strong glass. Therefore, the excipients forming weak glass are much better than the strong glass. Sucrose and trehalose are very protective because they form a weak glass.
Water substitution hypothesis Because of the large number of hydrogen bonds in protein molecules, bound water is linked to protein molecules via hydrogen bonds. When the protein pushes water during the freeze-drying process, the hydroxyl group of the protective agent can replace the hydroxyl group of water on the surface of the protein, forming a "hydration layer" on the surface of the protein, so that the bonding position of the hydrogen bond can be protected from direct exposure to the surrounding environment. In order to maintain the integrity of the natural structure and function of the protein. When freeze-dried, the protective agent can form hydrogen bonds with the water-depleted parts of the biomacromolecules instead of maintaining the freeze-drying damage of the biomacromolecules.
(III) Protection mechanism during storage
The time scale that causes protein deterioration during drying is hour, while for storage, the time scale is month or year. In the correct freeze-drying process, the product temperature is required to be close to its glass transition temperature, and under the correct storage conditions, the ambient temperature should be much lower than its glass transition temperature to achieve a long relaxation time.
Third, the heat-resistant freeze-drying protective agent formula needs attention
Determining optimal pH The active ingredients (such as proteins) in biological products are stable only at very low pH values, and different pH environments can affect protein solubility. The optimal pH environment is beneficial to the stability of the protein and its solubility in solution. The pH of the freeze-dried formulation also has a great impact on the long-term storage stability of the freeze-dried biological products. In addition, pH affects the physical and chemical stability of proteins in the solid state.
Buffering Options Many buffers can be used in biologic heat protectant formulations, but not every buffer can be used in any solution; for pH sensitive protein solutions, sodium phosphate buffer should be avoided. This is because during the freezing process, Na2HPO4 tends to preferentially crystallize, causing the pH of the solution to decrease, eventually causing protein denaturation; it is also important to properly select the concentration of the buffer.
Selection of Fillers Fillers have a fairly good solubility, are compatible with the active ingredients in biological products, have little or no toxicity, and have a high eutectic temperature.
Low-temperature, dry protective agent selection Sugar is the most frequently used protective agent in the freeze-drying process of biological products. Generally, no reducing sugar is used because it may cause non-enzymatic browning reaction with protein; some salts should also be used. Used as a protective agent for biological products during freeze-drying; some polymers are often used as protective agents because they increase the glass transition temperature. However, the ability of polymers to form hydrogen bonds with protein molecules is much lower than that of sugars, so polymers and sugars are often used in combination. Of course, this method is not effective for every protein.
This report comes from Xinyi Instruments
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