The latest vacuum freeze drying technology

First quarter    Principle of freeze drying technology

Drying is one of the ways to keep the material from spoilage. There are many methods of drying, such as drying, boiling, drying, spray drying, and vacuum drying. However, these drying methods are carried out at temperatures above 0 ° C or higher. The product obtained by drying generally shrinks in size and hardens in texture. Some substances are oxidized, and most of the volatile components are lost. Some heat-sensitive substances such as proteins and vitamins are denatured. Microorganisms lose their vitality, and the dried substances are not easily dissolved in water. Therefore, the dried product has a large difference in properties compared with that before drying. The freeze-drying method is different from the above drying method, and the drying of the product is basically carried out at a temperature below 0 ° C, that is, in a state where the product is frozen, until the later stage, in order to further reduce the residual moisture content of the dried product, the product is allowed to be Raise to a temperature above 0 °C, but generally does not exceed 40 °C.

Freeze-drying involves freezing a large amount of water, freezing it to a solid beforehand, and then directly sublimating the water vapor under vacuum. The substance itself remains in the ice shelf when it is frozen, so it has a constant volume after drying and is loose and porous. The ice or other solvent that freezes the product during sublimation absorbs heat. It causes the temperature of the product itself to decrease and slows down the sublimation speed. In order to increase the sublimation speed and shorten the drying time, the product must be properly heated. The entire drying is carried out at a lower temperature.

Freeze drying has the following advantages:

(1) Freeze-drying is carried out at a low temperature, and thus is particularly suitable for many heat-sensitive substances. Such as protein, microorganisms and the like will not degenerate or lose biological vitality. Therefore, it is widely used in medicine.

(2) When drying at low temperature, some volatile components in the material are lost little, which is suitable for drying some chemical products, medicines and foods.

(3) During the freeze-drying process, the growth of microorganisms and the action of the enzyme cannot be performed, so that the original trait can be maintained.

(4) Since drying is carried out in a frozen state, the volume is almost constant, the original structure is maintained, and concentration does not occur.

(5) The material after drying is porous and spongy. After adding water, the solution dissolves quickly and completely, and the original trait is almost immediately restored.

(6) Since drying is carried out under vacuum, there is little oxygen, so some easily oxidizable substances are protected.

(7) Drying can exclude more than 95-99% of water, so that the dried product can be preserved for a long time without deterioration.

Therefore, freeze drying is currently widely used in the pharmaceutical industry, food industry, scientific research and other sectors.

Second quarter   Freeze dryer composition and lyophilization procedures

The freeze-drying of the product needs to be carried out in a certain device. This device is called a vacuum freeze dryer or a freeze-drying device, referred to as a freeze dryer.

The lyophilizer is divided into four parts: the refrigeration system, the vacuum system, the heating system, and the control system. According to the structure, it consists of a freeze-drying box or a drying box, a condenser or a water vapor condenser, a refrigerator, a vacuum pump and a valve, and electrical control components. Figure 13 is a schematic diagram of the composition of the freeze dryer.

The freeze-drying box is a high-temperature box that can be cooled to about -55 ° C and can be heated to about +80 ° C. It is also a closed container that can be vacuumed. It is the main part of the lyophilizer. The lyophilized product is placed on the layered metal plate in the box, the product is frozen, and heated under vacuum to make the moisture in the product sublimate and dry.

The condenser is also a vacuum-tight container with a large surface area of ​​metal adsorption surface inside, and the temperature of the adsorption surface can be lowered to below -40 ° C to -70 ° C, and this low temperature range can be maintained. The function of the condenser is to freeze and adsorb the water vapor sublimated from the product in the freeze-drying box on its metal surface.

The freeze-drying box, the condenser, the vacuum pipe, the valve, the vacuum pump and the like constitute a vacuum system of the freeze-drying machine. The vacuum system requires no air leakage, and the vacuum pump is an important part of the vacuum system to establish vacuum. The vacuum system is essential for the rapid sublimation of the product.

The refrigeration system consists of a chiller and a lyophilizer, a pipe inside the condenser, and the like. The refrigerator can be two or more independent of each other, or a set can be used in combination. The function of the chiller is to cool the lyophilizer and condenser to generate and maintain the low temperatures required for their operation. It has two modes: direct cooling and indirect cooling.

The heating system has different heating methods for different lyophilizers. Some use direct electric heating; others use intermediate media for heating, and a pump (or a spare pump) keeps the intermediate medium circulated. The function of the heating system is to heat the product in the lyophilization box so that the moisture in the product is continuously sublimated and the required residual moisture content is met.

The control system consists of various control switches, indicating adjustment instruments and some automatic devices. It can be simple or complicated. The control system is more complicated in general lyophilizers with a higher degree of automation. The function of the control system is to manually or automatically control the lyophilizer to operate the machine in order to make the lyophilizer produce the desired product.

Freeze-drying procedure:

(1) Before lyophilization, the products that need to be lyophilized are placed in suitable containers, usually glass mold bottles, glass tube bottles or ampoules, the volume should be uniform, the evaporation surface should be as large as possible and the thickness should be as thin as possible;

(2) Then placed in a metal pan adapted to the size of the lyophilized box. It is generally used for the bottle to remove the chassis, which is beneficial to the effective transfer of heat.

(3) Before packing, freeze the freeze-drying box to empty the box, then put the product into the freeze-drying box for pre-freezing; or put the product into the inner layer of the freeze-drying box and pre-freeze;

(4) Before the vacuum is applied, the condenser should be operated according to the cooling rate of the condenser refrigerator, and the condenser should reach a temperature of at least -40 °C when vacuuming;

(5) After the vacuum degree reaches a certain value (usually it should reach the vacuum degree within 13Pa~26Pa), or some freeze-drying process requires to reach the required vacuum degree and continue to vacuum for more than 1~2h; heating. Generally, the heating is carried out in two steps. The first step of heating does not cause the temperature of the product to exceed the temperature of the eutectic or the eutectic point; after the moisture in the product is substantially dried, the second step is performed, and the product can be quickly made. The maximum permissible temperature for the rise. After the maximum allowable temperature is maintained for more than 2 hours, the lyophilization can be ended.

The entire sublimation drying time is about 12~24h or even longer, which is related to the quantity of the product in each bottle, the total capacity, the shape and specifications of the glass container, the type of the product, the freeze-drying curve and the performance of the machine, etc. .

After lyophilization, it should be filled with dry and sterile air into the drying oven, and then sealed as soon as possible to prevent re-absorption of moisture in the air.

In the lyophilization process, the temperature of the product and the layer, the condenser temperature and the vacuum control time are plotted as a curve, which is called a freeze-drying curve. Generally, the temperature is the ordinate and the time is the abscissa. Different freeze-dried curves were used for lyophilization of different products. When the same product uses different freeze-drying curves, the quality of the product is also different, and the freeze-drying curve is also related to the performance of the freeze dryer. Therefore different products, different freeze dryers apply different freeze-drying curves. Figure 14 is a schematic diagram of the freeze-drying curve (in which there is no temperature curve and vacuum curve of the condenser).

Third quarter   Co-melting point and its measuring method

The product that needs to be lyophilized is usually a solution or suspension pre-formed into water, so its freezing point is different from water. The water freezes at 0 °C, but the sea water is below 0 °C. Icing, because seawater is also an aqueous solution of many substances. The experiment indicated that the freezing point of the solution will be lower than the freezing point of the solvent.

In addition, the icing process of the solution is not the same as the pure liquid. The pure liquid such as water freezes at 0 ° C, the temperature of the water does not decrease, and the temperature does not decrease until all the water freezes, indicating that the pure liquid has a fixed knot. freezing point. The solution is different. It does not completely condense into a solid at a certain fixed temperature. At a certain temperature, the crystal begins to precipitate. As the temperature decreases, the number of crystals increases continuously until the solution is completely condensed. Thus, the solution does not condense at a certain fixed temperature. Instead, it condenses in a certain temperature range. The temperature at which crystals begin to precipitate when cooled is referred to as the freezing point of the solution. The temperature at which the solution is completely condensed is called the freezing point of the solution. The freezing point is the starting point of melting (ie, the melting point), which is the point at which the solution and the solvent melt together. So it is also called the eutectic or eutectic point. It can be seen that the freezing point and the eutectic point of the solution are different. The eutectic point is the temperature at which the solution actually solidifies completely into a solid.

It is clear that the concept of eutectic is important for freeze drying. Because lyophilized products may have substances such as salts, sugars, gelatin, proteins, blood cells, tissues, viruses, bacteria, and the like. Therefore, it is a complex liquid, and its freezing process is certainly a complicated process. Similar to the solution, there is also a temperature at which all of it actually condenses into a solid, that is, the eutectic point. Since freeze drying is carried out under vacuum. Sublimation drying can only be carried out under vacuum after the product is completely frozen. Otherwise, in the presence of some liquid, not only will it evaporate rapidly under vacuum, but the concentration of the liquid will reduce the volume of the lyophilized product; and the gas dissolved in the water will be under vacuum. It quickly pops up, causing the liquid to boil, causing the freeze-dried product to bubble or even emerge out of the bottle. This is what we don't want. To this end, the lyophilized product must be cooled to a temperature below the eutectic point at the beginning of sublimation, so that the lyophilized product is completely frozen.

During the freezing process, it is impossible to determine whether the product is completely frozen into a solid from the observation of the appearance; it is impossible to determine the structural state inside the product by measuring the temperature. The change in electrical properties is extremely useful as the product structure changes. Especially when the temperature is measured during freezing, we can know whether the freezing is in progress or has been completed. The resistivity will be very large after all freezing, so the solution It is ion conductive. When frozen, the ions will be fixed and cannot move, so the resistivity is significantly increased. The resistivity will decrease significantly when a small amount of liquid is present. Therefore measuring the resistivity of the product will determine the eutectic point of the product.

The normal eutectic measurement method is to immerse a pair of platinum electrodes in a liquid product, insert a thermometer into the product, cool them to a low temperature below -40 ° C, and then slowly heat the frozen product. The Wheaton bridge is used to measure its resistance. When a sudden drop in resistance occurs, the temperature at this time is the eutectic point of the product. The bridge should be powered by alternating current, because the direct current will be electrolyzed and the whole process will be recorded by the instrument (Figure 16).

It can also be measured in a simple way, as shown in Figure 15. Two suitable thick and mutually insulated copper wires are inserted into the container holding the product as an electrode. Insert a thermometer near the copper electrode, the insertion depth is similar to that of the electrode, put them together near the observation window in the freeze-drying box, fix them with appropriate methods, and then pre-freeze them with other products. The multimeter constantly measures the resistance value during the cooling process and determines the eutectic point based on the change in the resistance value.

The electrode lead is connected to the multimeter through a switch, and the positive and negative electrodes can be separated. If the freeze-drying box does not have a wire lead-out joint, you can use two thin wires to pull out from the door joint and apply some vacuum sealing wax near the wire so as not to affect the vacuum. The measurement is started and recorded after the thermometer has dropped to 0 °C. Place the multimeter's transfer switch at the highest level of the measuring resistor (×1K or ×10K). Since the DC is used in the multimeter, in order to prevent electrolysis, the switch should be turned off immediately after each measurement, and the temperature and resistance values ​​of each measurement are recorded one by one. The resistance value is small at the beginning and gradually increases gradually. At a certain temperature, the resistance suddenly increases, almost infinite, and the temperature value at this time is the value of the eutectic point.

The eutectic point measured by this method has a certain error because there is some electrolysis at the copper electrode. The multimeter has no bridge sensitivity for high resistance; in addition, the change of the resistance of the freezing process and the melting process is not exactly the same, but the measured value still has practical reference value.

The values ​​of the eutectic point range from 0 ° C to -50 ° C, depending on the product variety, the type and concentration of the protective agent. A list of eutectic points for some substances is provided for reference, as the actual lyophilized product has other ingredients. So it is different from this.

Table 22 The eutectic of some substances (°C)

Section 4 Disintegration temperature of freeze-dried products

For the eutectic of freeze-dried products, we are already familiar with it, it is the real cure point of the product. That is, the temperature at which the product must be cooled before vacuuming, otherwise the product will foam when it is pumped out, and the product will not exceed this temperature when sublimation is heated, otherwise the product will melt. Therefore, the eutectic point is a temperature value that needs to be controlled during the pre-freezing phase and the sublimation phase.

A concept of disintegration temperature is now introduced, which is another temperature different from the eutectic.

A normal sublimation product, when the sublimation is carried out to a certain extent, the upper layer of the dry layer and the lower layer of the frozen layer will appear. The interface between the two layers is the sublimation surface, and the sublimation surface is declining as the sublimation progresses. of.

The dried product should be loose and porous and kept in this stable state so that the water vapor which is sublimated from the lower frozen product can pass smoothly, so that all products are well dried.

However, some products that have been dried will lose their rigidity and become sticky when the temperature rises to a certain value. A collapse-like phenomenon occurs, which causes the dried product to lose its loose and porous state, and closes the water vapor of the lower frozen product. The escape path hinders the continuation of sublimation.

As a result, the sublimation rate is slowed down, and the sublimation heat absorbed from the frozen product is also reduced, and the heat supplied by the ply layer will be excessive, which causes the temperature of the frozen product to rise, and when the temperature rises to a temperature above the eutectic point, the product Melting or foaming will occur, causing lyophilization to fail.

The temperature at which disintegration occurs is called the disintegration temperature of the product. For such products to obtain good drying, only the temperature of the dried product in the sublimation is below the disintegration point until the frozen product is fully sublimated, so that the product temperature continues to rise. At this time, due to the absence of frozen ice in the product, even if the dried product disintegrates, it will not affect the drying of the product, because the product has been transferred from the sublimation stage to the desorption drying stage.

The dry product which does not disintegrate and the dry product which disintegrates are not visually different from the naked eye, and the structural change can only be seen under the microscope. When the freeze-drying process of the product is observed under a microscope, if the disintegration is observed, the temperature at this time is the disintegration temperature of the product.

Some products have a disintegration temperature higher than the eutectic temperature, so it is only necessary to control the product temperature below the eutectic temperature during sublimation; however, some products have a disintegration temperature lower than the eutectic temperature, then the sublimation is controlled according to the general method. Disintegration may occur, and such products are only sublimed at a lower temperature, so the freeze-drying time must be extended.

The eutectic point of the product can be known by electrical resistance, differential thermal analysis and direct observation by cryogenic microscopy, but the disintegration temperature of the product can only be known by direct observation under a freeze-drying microscope.

The disintegration temperature of the product depends on the variety of the product itself and the type of protective agent; the disintegration temperature of the mixed substance depends on the disintegration temperature of each component. Therefore, when selecting the lyoprotectant of the product, the material with higher disintegration temperature should be selected so that the sublimation drying can be carried out at a very low temperature to save the energy consumption and time of lyophilization and increase the productivity.

Protective agents such as glycine, mannitol, dextran, xylitol, polyhexyrolidone and protein mixtures can increase the disintegration temperature of the product. The disintegration temperature of some substances °C is shown in Table 23

Table 23 The disintegration temperature of some substances °C

Section 5 Freeze-drying protectant

In freeze-dried liquid preparations, in addition to those active, toxic or therapeutically effective ingredients, they are collectively referred to as lyoprotectants. It is different from the adjuvant, the adjuvant has a therapeutic effect, and the protective agent has no therapeutic effect.

Some liquid products can be lyophilized separately, but some liquid products are often not easily lyophilized. In order to make certain products successfully freeze-dried, improve the solubility and stability of the freeze-dried products, or make the freeze-dried products have an aesthetic appearance, it is necessary to add some additional substances to the products, which are protective agents, sometimes called The protective agent is a suspending medium, a filler, an excipient, a buffer, a base, and the like. The protectant must be chemically inert to the lyophilized product.

The role of protective agents:

1. Bacteria and viruses need to grow under specific culture media, but some culture media are often difficult to separate from bacteria and viruses, and they are generally successfully lyophilized in these culture media. For example, broth, skim, protein, etc.

2. Some active substances have a very small concentration and very little dry matter. Substances that have dried during lyophilization are carried away by the sublimated gas stream. In order to improve the concentration and increase the dry matter content, the lyophilized product can form a better agglomerate. Therefore, it is necessary to add a filler such that the concentration of the solid matter is between 4 and 25%. These fillers or excipients are: sucrose, lactose defatting, protein and hydrolysate, polyvinylpyrrolidone, dextran, sorbitol, and the like.

3. Some active substances are particularly fragile and may be harmed by physical or chemical reasons during freeze-drying. Therefore, some protective agents or antifreeze agents are added to reduce damage in freeze-drying. For example, dimethyl hydrazine, glycerin, dextran (dextran), saccharides, polyvinylpyrrolidone, and the like are added.

4. Adding certain substances can increase the disintegration temperature of the product to obtain a good product and easy to freeze. They are mannitol, glycine, dextran, xylitol, polyvinylpyrrolidone and the like.

5. In order to change the pH of the lyophilized liquid preparation, thereby changing the eutectic point to facilitate lyophilization, they are sodium hydrogencarbonate, sodium hydroxide and the like.

6. In order to change the stability of product storage, increase storage temperature and increase storage time, they are: antioxidants such as vitamin C, vitamin E, amino acids, sodium thiosulfate, thiourea and the like.

The range of protective agents is quite wide and varied, but no ideal protective agent can be found. There is also no universal formulation of a protective agent for different lyophilized products. Suitable protective agents for each product are subject to repeated testing.

The types of protective agents can be classified into the following categories according to their chemical composition:

Section VI   Factors affecting the drying process

The freeze-drying process is actually a physical state change of water and its transfer process. The biological product containing a large amount of water is first frozen into a solid, and then directly sublimed into water vapor by solid ice under vacuum, and the water vapor is condensed into frost in the condenser, and the frost melts and is discharged after drying. The desired freeze-dried product was obtained in a lyophilized box, and the drying process is shown in Figure 17.

The lyophilization process has two exothermic processes and two absorption processes: the liquid biological product emits heat to solidify into a solid biological product as an exothermic process; the solid biological product absorbs heat under vacuum to sublimate into water vapor as an endothermic process; The heat released from the condenser is condensed into frost for the exothermic process; after the lyophilization, the frost absorbs heat in the condenser and melts into water as an endothermic process.

Heat and mass transfer occurs throughout the lyophilization process. The transfer of heat throughout the entire process of freeze drying. The pre-freezing stage, the first stage and the second stage of drying, and the defrosting stage all carry out heat transfer; the mass transfer is only carried out in the drying stage, and the water vapor generated in the freeze-dried box product is condensed into frost in the condenser. The process is actually a process of mass transfer. Only when quality is passed can the product be dried. In the drying phase, heat is transferred to promote mass transfer, and improved heat transfer also improves mass transfer.

If the heat is not supplied during the sublimation of the product, the product is lowered by its sublimation to absorb its own heat, and the sublimation rate is gradually decreased until the product temperature is equal to the surface temperature of the condenser, and the drying is stopped. At this time, the number of water vapor molecules from the frozen product to the surface of the condenser is equal to the number of water vapor molecules returning from the surface of the condenser to the frozen product, and the water vapor pressure between the lyophilization tank and the condenser is equal to zero, reaching a state of dynamic equilibrium.

If an external heat is added to the frozen product, the dynamic equilibrium state is destroyed, the temperature of the frozen product is higher than the temperature of the condenser surface, and a water vapor pressure difference is generated between the lyophilization tank and the condenser, forming a The water vapor flow from the lyophilization tank to the condenser. Since the surface of the condenser is dehydrated and the water vapor is frost, the water vapor from the lyophilization tank in the condenser is continuously adsorbed, and the vapor pressure is kept in the condenser; and the lyophilization tank flows away. The water vapor is continuously replenished by the sublimated water vapor in the product, maintaining a high water vapor pressure in the freeze-drying tank. This process continues, and the product is gradually dried.

Sublimation begins with the surface of the product. After drying for a period of time, a layer of dried product is formed on the frozen product, creating an interface between the dried product and the frozen product (also known as the sublimation interface). The interface continues to decrease as the drying progresses until the interface disappears after sublimation. When the interface is created, the water molecules must pass through the dried product to enter the space; after the water molecules run out of the interface, they enter a certain gap of the dried product. In the future, it may be necessary to pass through many such gaps before entering the space from the gap of the product. It can also go back to the frozen product after some turning, and the gap in the dry product is sometimes like a labyrinth.

When the water molecules run out of the surface of the product, its path of motion is still very tortuous. It may collide with the glass bottle wall, may collide with the rubber stopper on the glass bottle, may collide with the metal plate wall of the freeze-drying box, and often collide with water molecules, and then enter the condenser. When the water molecules collide with the refrigerating surface of the condenser, because the temperature of the surface is very low, the low temperature surface absorbs the energy of the water molecules, so that the water molecules lose their kinetic energy, leaving no energy to leave the cooling surface of the condenser. The water molecules are then "captured". A large amount of water molecules are trapped to form a layer of frost on the surface of the condenser, so that the surface temperature of the condenser rises slightly, but as the water vapor load from the freeze-drying box gradually decreases, the temperature of the condenser frost surface gradually decreases. Thereby, the water vapor pressure in the system is gradually reduced, so that the water vapor in the freeze-drying tank continuously flows to the condenser. As time goes on, the product is continuously heated in the freeze-drying box and the condenser is continuously worked, and the product is gradually dried.

The rate of drying is proportional to the difference in water vapor pressure between the lyophilizer and the condenser and inversely proportional to the resistance of the water vapor flow. The greater the pressure difference of water vapor, the smaller the resistance of the flow, and the faster the rate of drying. The pressure difference of the water vapor depends on the temperature difference between the effective temperature of the condenser and the temperature of the product. Therefore, it is necessary to reduce the effective temperature of the condenser as much as possible and to maximize the temperature of the product.

The flow resistance of water vapor comes from the following aspects:

(1) Internal resistance of the product: the resistance of the water molecules through the layer of the already dried product. The magnitude of this resistance is related to the structure of the dry material layer and the type, composition, concentration, and protective agent of the product.

(2) Resistance of the container: The resistance of the container mainly comes from the mouth of the bottle. Because the cross section of the bottle mouth is small, there may be some items at the mouth of the bottle. For example: a rubber stopper with a groove, gauze, etc., the cross section of the bottle mouth is large, and the resistance is small.

3 The resistance of the machine itself: mainly the pipe resistance between the freeze-drying box and the condenser. The pipe is thick, short and straight and the resistance is small. The resistance is also related to the structure and geometry of the freeze-drying box.

The method for accelerating the sublimation rate of freeze-dried products is as follows:

1 Increase the temperature of the product in the freeze-drying tank: increase the water vapor pressure in the freeze-drying tank, accelerate the flow of water vapor to the condenser, accelerate the mass transfer, and increase the drying rate. However, there is a limit to increasing the temperature of the product, and the temperature of the product cannot exceed the temperature of the eutectic point.

2 lowering the temperature of the condenser: it also reduces the pressure of water vapor in the condenser, and also accelerates the flow rate of water vapor from the freeze-drying tank to the condenser. It also speeds up the transfer of mass and increases the drying rate. However, more cooling of the condenser temperature requires increased investment and operating costs.

Reducing the flow resistance of water vapor also accelerates mass transfer and increases drying rate. The methods for reducing the resistance to water vapor flow are as follows: 1 reducing the thickness of the product and increasing the sublimation area of ​​the frozen product;

2 Reasonable design of bottles, stoppers, and reduction of bottle mouth resistance;

3 Reasonable design of the freeze dryer to reduce the pipe resistance of the machine;

4 select the appropriate concentration and protective agent to make the structure of the dried product loose and porous, reducing the resistance of the dry layer;

5 Experiment with the optimal pre-freezing method to create an ice crystal structure that is favorable for sublimation. These methods can promote the transfer of mass,

Increase the drying rate.