Freeze-dried preservation of purified products

Freeze-dried preservation of purified products

First, the principle and advantages of freeze drying

Freeze-drying is to pre-freeze a large amount of water-containing substance into a solid, and then appropriately heat it under vacuum to sublimate the water vapor directly from the solid, while the substance itself remains in the ice shelf during freezing, so the product after drying is almost constant. The entire drying is carried out at a lower temperature.

The advantages of freeze drying:
1. Low temperature freeze drying is especially useful for many heat sensitive materials. If proteins, microorganisms, etc. do not degenerate or inactivate, they are widely used in the pharmaceutical industry.
2. At low temperature freeze-drying, the loss of volatile components of the product is small, so it is applied to food, medicine and chemical products.
3. During the low temperature freeze-drying process, the growth of microorganisms and the action of enzymes can be effectively inhibited.
4. Low-temperature freeze-drying basically guarantees the original structure of the product and does not cause concentration.
5. The dried product is porous and spongy. After adding water, it dissolves and can quickly restore its original shape.
6. Vacuum drying is basically free of oxygen, so some easily oxidizable substances are protected.
7. Low-temperature freeze-drying can eliminate more than 95% of water, and the dried product can be preserved for a long time without deterioration.

Second, freeze-drying procedures

1. Determination of the common melting point of the prepared product: The freezing point of the aqueous solution will be lower than the freezing point of the solvent (see the physical chemistry for the detailed principle). The temperature at which the cooling solvent starts to precipitate crystals is called freezing point, and the temperature of all condensation is called the freezing point of the solution. Conversely, when the solidified solvent is heated, the freezing point is the starting point of melting, so the freezing point is also called the eutectic point. The necessity of determining the eutectic point: on the one hand, since lyophilization is carried out under vacuum, it is required that the solvent liquid containing the product is completely frozen before sublimation under vacuum, otherwise the unfrozen portion of the liquid will evaporate rapidly under vacuum. The concentration of the solvent is reduced to reduce the volume of the dry product, and the gas dissolved in the water rapidly emerges under vacuum, causing the lyophilized product to bubble or even be carried out of the bottle. For this reason, the frozen solvent must be cooled to a temperature below the eutectic point at the beginning of sublimation in order to allow the dry product to actually freeze. On the other hand, the sublimation at low temperature should not exceed the temperature of the eutectic point, otherwise the product will melt, and the dried product will shrink in size, appear bubbles, deepen in color, and dissolve hard. Therefore, it can be said that the determination of the eutectic point is the key to the success of the experiment. Common methods for measuring the eutectic point in the laboratory include the Wheatstone bridge method, the critical resistance method, the differential thermal analysis method, and the direct observation method of the cryogenic microscope.

2. Pre-freezing of the product
a, the product to be lyophilized should be formulated into a certain concentration of liquid, generally 4 to 25% is appropriate.
b. Product packaging: bulk and bottled. In the bulk, metal plates, lunch boxes or glassware can be used; bottles are made of glass bottles (such as plasma bottles, vaccine bottles and penicillin vials) and ampoules (such as flat bottom ampoules, Changan bottles and garden ampoules, etc.). It is required to be cleaned and sterilized before loading. Product packaging requirements: the general thickness is not more than 15cm, the surface area is as large as possible, which is conducive to sublimation.
c. Pre-freezing method: 1. Pre-freezing method in the box: directly place the product on the multi-layer separator in the freeze-drying box of the freeze-drying machine for freezing; 2. Pre-freezing method outside the box: one method is a low-temperature refrigerator Or pre-freeze with alcohol and dry ice. Another method is a special type of refrigerating device, which can rotate the product of the big bottle to the shell structure and then into the freeze-drying box; 3. Special pre-freezing method, centrifugal pre-freezing method, using the liquid to evaporate rapidly under vacuum It absorbs its own heat and freezes, generally 800 rpm.
d. Problems to be aware of when pre-freezing: 1. Pre-freezing rate, the optimal rate should be selected according to the product; 2. The minimum temperature of pre-freezing should be lower than the temperature of the eutectic point; 3. Pre-freezing time should be just vacuuming Before (so the condenser should be operated in advance, the vacuum reaches 100μHg when the temperature reaches -40 °C). The solvent is frozen (experience value: after the general pre-freezing reaches the specified temperature, it can be vacuumed and sublimated after 1-2 hours) .

3. Drying and sublimation in the first stage of the product The heating temperature in the drying stage should be close to the temperature of the eutectic point, but not exceeding the temperature of the eutectic point.
In the first stage, most of the frozen ice in the solvent is sublimated, so the process is also referred to as the sublimation drying stage. Sublimation is an endothermic process, so the product must be heated at a low temperature, but must never exceed the eutectic temperature. If the temperature of the heating is too low than the temperature of the eutectic point, the rate of sublimation decreases and the time of the sublimation phase is prolonged.
Factors affecting the drying stage of sublimation: 

1. The product itself, the product with higher eutectic point is easy to dry, and the sublimation time is short;

2. The thickness of the product is divided, the normal sublimation rate is about 1 mm per hour; 

3. frozen The performance of the dryer itself, vacuum performance, condenser temperature and performance.

4. In the maintenance phase, the temperature of the lyophilized box heating separator is close to the temperature of the eutectic point of the product, and is maintained for about 12 hours, so that most of the moving ice in the product sublimates. (The method commonly used in the laboratory is overnight treatment, so the experimental process is expected before the experiment)

5. Drying of the second stage of the product Once most of the frozen ice in the product (about 90% of the ice has been sublimated) is sublimated, the drying of the product enters the second stage, namely the desorption drying stage.
In the desorption drying stage, the temperature of the product product can be quickly raised to the maximum allowable temperature of the product (25 to 40 ° C), and maintained at this temperature until the end of lyophilization.

6. In the final maintenance stage, that is, the end of lyophilization, how to judge the end of lyophilization? The temperature of the product reaches the maximum permissible temperature and is stored at this temperature for more than 2 hours. After lyophilization, the product should be placed in a sterile dry box and then sealed as soon as possible to prevent re-absorption of air.

Third, the production of freeze-drying curve and the formulation of timing

Freeze-dried products of biological products often require a certain physical form, such as uniform color, desirable water content, good solubility, high survival rate, and long shelf life. Therefore, to optimize the parameters of each drying step, the freeze-drying curve and timing are the basic basis for the control of the freeze-drying process.
The freeze-drying curve is the relationship between the temperature of the separator layer of the lyophilizer (the temperature of the product during drying is controlled by the temperature of the separator layer) and time. The freeze-drying sequence is the case where each device is turned on and off during the freeze-drying process.
To develop a freeze-drying curve and time, you need to determine the following data:
A. Pre-freezing rate laboratories commonly use pre-freezing temperatures and packing times to determine the pre-freezing rate. If the pre-freezing rate is required to be fast, the freeze-drying box is first lowered to a lower temperature and then repacked; if the pre-freezing rate is required to be slow, the freeze-drying box is cooled after the product is packed.
B. Minimum temperature for pre-freezing: The temperature at which the minimum temperature of pre-freezing is lower than the eutectic point of the product.
C. The pre-freezing time is generally required to be maintained for 1 to 2 hours after the sample temperature reaches the predetermined temperature. Note: It is generally not recommended to directly place the solvent directly on the separator layer of the freezer.
D. The condenser cooling time requires that the temperature of the condenser should reach -40 ° C or less at the end of the pre-freezing vacuum. The cooling of the condenser usually lasts from the beginning until the end of the freeze-drying, and the temperature should always be below -40 °C.
E. Pre-freezing end time The end of pre-freezing is to stop the cooling of the separator layer of the freeze-drying box. Usually, when the vacuum is applied (or when the degree of vacuum reaches a certain value), the temperature of the separator layer is stopped.
F. Vacuuming time, vacuuming is started at the end of pre-freezing until the end of drying
G. Vacuum Control Time Vacuum control is designed to improve the heat transfer of the freeze-drying tank. It is usually used during the second stage of drying. After the product temperature reaches the maximum allowable temperature, it is stopped and the high vacuum state is resumed.
H. During the maximum allowable temperature sublimation of the product heating, the heating temperature may slightly exceed the maximum permissible temperature of the product, but in the final stage the separator layer temperature shall be consistent with the highest permissible temperature of the product.
G. Freeze-drying time: 18~24h

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2022-11-14

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