When the product is freeze-dried, it needs to be put into a suitable container and then pre-frozen before sublimation drying. The pre-freezing process is not only to keep the main properties of the material unchanged; but also to obtain a reasonable structure of the frozen product to facilitate the sublimation of water; and to have an appropriate amount for future application.
There are usually two ways to pack the product: bulk and bottled. Bulk can be in metal trays, lunch boxes or glassware; bottled in glass vials and ampoules. Glass bottle and plasma bottle. Vaccine bottles and penicillin vials, etc., ampoules also include flat-bottom ampoules, long ampoules and round ampoules, etc. These need to be determined according to the future use of the product, and the bottle needs to be equipped with a suitable rubber stopper.
Table 22 The eutectic point of some substances (℃)
substance eutectic point
0.85% sodium chloride solution -22
10% sucrose solution -26
40% sucrose solution -33
10% glucose solution -27
2% gelatin, 10% glucose solution -32
2% gelatin, 10% sucrose solution -19
10% sucrose solution, 10% glucose solution, 0.85% sodium chloride solution -36
Skim milk -26
Horse Serum -35
Various containers are required to be cleaned and sterilized before being subpackaged.
The product that needs to be freeze-dried needs to be formulated into a liquid of a certain concentration. In order to ensure a certain shape after drying, the content of the substance is optimal between 10 and 15%.
The product is dispensed into the container with a certain surface area to thickness ratio. The surface area is larger and the thickness is smaller. Large surface area is conducive to sublimation, and large product thickness is not conducive to sublimation. Generally, the thickness of the packaging is not more than 10mm. Some products require large bottles. When lyophilizing a large amount of product, it can be frozen into a shell by the method of spin freezing, or frozen into a sloped surface by tilting the container, so as to increase the surface area and reduce the thickness.
The pre-freezing method of the product includes the pre-freezing method in the freeze-drying box and the pre-freezing method outside the box.
The pre-freezing method in the box is to directly place the product on the multi-layer shelf in the freeze-drying box of the freeze-drying machine, and freeze it by the freezer of the freeze-drying machine. When a large number of vials and ampoules are lyophilized, in order to facilitate the loading and unloading of the vials or ampoules, the vials or ampoules are generally divided into several metal trays and then packed into the boxes. In order to improve heat transfer, some metal pans are made detachable, and the bottom is removed when entering the box, so that the vial is directly in contact with the metal plate of the freeze-drying box; for pans that cannot be drawn low, the bottom of the pan is required to be flat to obtain a uniform product. sex. The large plasma bottle using the spin-freezing method should be frozen in advance, and then a metal rack or block for heat conduction should be added before freezing.
There are two ways to prefreeze outside the box. Some small freeze dryers do not have a device for pre-freezing product. Pre-freezing can only be done using a low temperature freezer or alcohol and dry ice. The other is a dedicated cyclone, which freezes large bottles of product into a shell-like structure while rotating. And then into the freeze-drying box.
There is also a special centrifugal pre-freezing method, which is used in centrifugal freeze dryers. Using the rapid evaporation of liquid under vacuum, it absorbs its own heat and freezes. The centrifugal force of the rotation prevents gas from escaping the product, allowing the product to freeze "quietly" into a certain shape.
The speed is generally around 800 rpm.
Freezing will have a certain destructive effect on cells and living organisms, and the mechanism is very complicated. There is no unified theory,
But it is generally believed that it is mainly caused by mechanical effects and solute effects.
The freezing process of biological substances starts with the freezing of pure water, and the growth of ice crystals gradually causes the concentration of electrolytes. This is followed by solidification of the eutectic mixture. In the end it all turned solid.
Mechanical effects are caused by mechanical forces generated by the growth of ice crystals inside and outside the cell. Especially for living organisms with cell membranes, the images are larger. Generally, the larger the ice crystal, the easier the cell membrane is to rupture, resulting in cell death; the smaller the ice crystal, the less mechanical damage to the cell membrane.
Slow freezing produces larger ice crystals, fast freezing produces smaller ice crystals; for that matter. Rapid freezing has less effect on cells. Slow freezing can easily cause cell death.
The solute effect is that the interstitial fluid is gradually concentrated due to the freezing of water, thereby increasing the concentration of electrolytes, and proteins are more sensitive to electrolytes. The increase in electrolyte concentration causes protein denaturation and cell death; in addition, the increase in electrolyte concentration causes cell dehydration and death. The higher the interstitial fluid concentration. The damage caused by the above reasons is also more severe. The solute effect is most pronounced in a certain temperature range. This temperature range is between the freezing point of water and the full solidification temperature of the liquid. If this temperature range can be crossed at a higher speed, the effect of the solute effect can be greatly weakened.
In addition, the size of the crystals formed during freezing also greatly affects the drying rate and the dissolution rate of the dried product. Large ice crystals are easy to sublime, and small ice crystals are not conducive to sublimation; but large ice crystals dissolve slowly, and small ice crystals dissolve quickly. The smaller the ice crystal, the more it reflects the original structure of the product after drying.
To sum up, there needs to be an optimal cooling rate. In order to obtain the highest cell viability, the best product physical properties and dissolution rate. Of course, improving the survival rate has a lot to do with adding an anti-low temperature agent (one of the protective agents) to the product. For example, glycerol, dimethyl sulfoxide, sugars, etc. These low temperature resistant substances 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 are required; for example, sometimes the cooling of the freeze-drying box is started after packing, and sometimes the machine needs to be lowered to a low temperature in advance, and then the product is put into the freeze-drying box.
The purpose of pre-freezing is also to fix the product for sublimation under 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 for some products.
Therefore, three data should be determined before prefreezing. One is the rate of pre-freezing, and 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 eutectic point of the product. The minimum temperature of pre-freezing should be lower than the temperature of the eutectic point. The third is the pre-freezing time, which is determined according to the condition of the machine to ensure that all products are frozen before vacuuming. It will not come out of the bottle due to vacuuming. The smaller the temperature difference between each layer of the freeze-drying box and the various parts of each layer, the shorter the pre-freezing time. Generally, the temperature of the product reaches the pre-freezing time. Vacuum sublimation can be started 1-2 hours after the lowest temperature.
