The pre-freezing methods of products include freeze-drying, pre-freezing inside the box, and pre-freezing outside the box.
The box pre-freezing method is to directly place the products on the multi-layer shelf in the freeze-drying box of the freeze-drying machine and freeze them by the freezer of the freeze-drying machine. When a large number of vials and ampoules are freeze-dried, in order to facilitate the entry and exit of the box, the vials or ampoules are generally sub-packed in several metal discs and then loaded into the box. In order to improve heat transfer, some metal plates are made into separable types, and the bottom is removed when entering the box so that the vial can directly contact the metal plate of the freeze-drying box; For plates that cannot be pulled low, the bottom of the plate shall be flat to obtain the uniformity of the product. The large plasma bottle with the rotary freezing method shall be frozen in advance and then added with a metal frame or block for heat conduction.
There are two methods of pre-freezing outside the box. Some small lyophilizers do not have a device for pre-freezing products. Only use a low-temperature refrigerator or alcohol and dry ice for pre-freezing. The other is a special rotary freezer, which can freeze the products of large bottles into a shell structure while rotating. Then enter the freeze-drying box.
There is also a special centrifugal pre-freezing method, which is adopted by the centrifugal lyophilizer. The liquid evaporates rapidly under vacuum, absorbs its own heat, and freezes. The centrifugal force of rotation prevents the gas in the product from overflowing so that the product can be "calmly" frozen into a certain shape.
The speed is generally about 800 rpm.
Freezing will have a certain destructive effect on cells and organisms, and its mechanism is very complex. There is no unified theory,
However, it is generally believed that it is mainly caused by mechanical effect and solute effect.
The freezing process of biological substances begins with the freezing of pure water, and the growth of ice crystals gradually leads to the concentration of electrolytes. Then the eutectic mixture solidifies. Finally, all become solid.
The mechanical effect is caused by the mechanical force generated by the growth of ice crystals inside and outside the cell. Especially for life with a cell membrane, the image is large. Generally, the larger the ice crystal is, the easier the cell membrane is to rupture, resulting in cell death; The ice crystal is small and the mechanical damage to the cell membrane is also small.
The ice crystals produced by slow freezing are larger and those produced by rapid freezing are smaller; In this regard. Rapid freezing had little effect on cells. Slow freezing is easy to cause cell death.
The solute effect is due to the gradual concentration of interstitial liquid due to the freezing of water, which increases the concentration of electrolytes. Protein is more sensitive to electrolytes. The increase of electrolyte concentration causes protein denaturation and cell death; In addition, the increase of electrolyte concentration will dehydrate the cells and die. The higher the liquid concentration in the gap. The more serious the damage caused by the above reasons. The solute effect is most obvious in a certain temperature range. This temperature range is between the freezing point of water and the full curing temperature of the liquid. If the temperature range can be crossed at a higher speed, the effect of solute effect can be greatly weakened.
In addition, the crystal size formed during freezing also affects the drying rate and the dissolution rate of dried products to a great extent. Large ice crystals are easy to sublimate, while small ice crystals are not conducive to sublimation; However, large ice crystals dissolve slowly and small ice crystals dissolve quickly. The smaller the ice crystal, the more it can reflect the original structure of the product after drying.
To sum up, it is necessary to have an optimal cooling rate. In order to obtain the highest cell survival rate, the best physical properties, and dissolution rate of the product. Of course, improving the survival rate has a lot to do with adding anti-hypothermic agents (one of the protective agents) to the products. For example, glycerol, dimethyl sulfoxide, sugars, etc. These low-temperature resistant substances can help the product expand the range of optimal cooling rates so that more cells can survive.
In order to obtain different cooling rates. Different pre-freezing methods should be adopted; For example, sometimes the temperature of the freeze-drying box needs to be lowered after packing, sometimes the machine needs to be lowered to a low temperature in advance, and then the products are loaded into the freeze-drying box.
The purpose of pre-freezing is also to fix the product for sublimation under a vacuum. If not frozen. When vacuuming, the product will come out of the bottle without a certain shape; If it is too cold, it will not only waste energy and time but also reduce the survival rate of some products.
Therefore, three data should be determined before pre-freezing. One is the rate of pre-freezing. An optimal freezing rate should be tested according to different products. The second is the minimum temperature of pre-freezing, which should be determined according to the co-melting point of the modified product. The minimum temperature of pre-freezing should be lower than the co-melting point. The third is the pre-freezing time, which is determined according to the situation of the machine to ensure that all products have been frozen before vacuum pumping. It will not come out of the bottle due to vacuum pumping. The smaller the temperature difference between each plate layer and each part of each plate layer of the freeze-drying box, the pre-freezing time can be shortened accordingly. Generally, the vacuum sublimation can be started 1-2 hours after the temperature of the product reaches the minimum pre-freezing temperature.
