Research progress on freeze-drying technology of protein drugs 1 Introduction Since the lyophilized drug is porous, can be stably stored for a long time, and is easy to re-rehydrate to restore activity, the freeze-drying technique is widely used for preparing solid protein drugs, oral instant drugs, and drug embedding agent liposomes. According to the database of the State Drug Administration, there are currently recombinant human granulocyte macrophage colony-stimulating factor for injection, recombinant human interferon α2b for injection, lyophilized mouse epidermal growth factor, and lyophilized recombinant human epidermal growth factor. , recombinant streptokinase for injection, recombinant human interleukin-2 for injection, recombinant human growth hormone for injection, group A streptococci for injection, recombinant human interferon alpha 2b for injection, lyophilized human factor VIII, lyophilized human fibrinogen Lyophilized drugs such as pyrogallol oral freeze-dried tablets are approved for marketing. As of February 2000, the United States FDA has approved a total of 76 biotech drugs. The freeze-drying technology Zui was invented by the English Wollaston as early as 1813. In 1909, the Shsckell test used this method to freeze and preserve toxins, strains, rabies virus and other biological products, and achieved good results. In the Second World War, the large demand for blood products greatly stimulated the development of freeze-drying technology, and the technology entered the industrial application stage. Since then, the rapid development of refrigeration and vacuum equipment has provided strong material conditions for the rapid development of freeze-drying technology. In the 1980s and 1990s of the last century, the rapid development of science and technology and the people's demand for health protection provided a powerful driving force for the rapid development of drug freeze-drying technology. In the freeze-drying damage and protection mechanism of drugs, the freeze-drying process of drugs , pharmaceutical freeze dryers and other aspects have achieved great results. However, drug freeze-drying technology is a marginal discipline that requires the intersection and integration of knowledge in biology, pharmacy, refrigeration, vacuum and control. Therefore, there are still problems to be solved. 2 Principle and characteristics of drug freeze drying The lyophilization of the drug refers to a method in which the drug solution is frozen at a low temperature, then sublimed and dried under vacuum to remove ice crystals, and then desorbed and dried after the sublimation is completed to remove part of the bound water. The process can be divided into five steps: drug preparation, pre-freezing, primary drying (sublimation drying) and secondary drying (desorption drying), sealed storage. After the drug is lyophilized as described above, it can be stored in the dark for a long time at room temperature. When it is needed, it can be reconstituted to the state before lyophilization by adding distilled water or physiological saline to make a suspension. Compared with other drying methods, the drug freeze-drying method has outstanding advantages and features: a) The liquid is dispensed before freezing, and the dosage is accurate; b) drying at a low temperature to retain the heat sensitive substance in the dried drug; c) drying under low pressure, the dried drug is not easy to oxidize and deteriorate, and at the same time can sterilize or inhibit the vitality of certain bacteria due to lack of oxygen; d) The dried drug can form a "skeleton" when frozen, and can maintain its original shape after drying, forming a porous structure and the color is basically unchanged; e) Rehydration is good, and the lyophilized drug can be quickly hydrated and reduced to the state before lyophilization; f) Dehydrated thoroughly, suitable for long-distance transportation and long-term storage. Although the freeze drying of the drug has the above advantages, the low drying rate, long drying time, high energy consumption in the drying process, and large investment in drying equipment are still outstanding shortcomings of the technology. 3 drug freeze-drying damage and protection mechanism Freeze drying of drugs is a multi-step process that produces a variety of stresses that degrade the drug, such as low temperature stress, frozen stress, and dry stress. The freezing stress can be divided into the formation of dendritic ice crystals, the increase of ion concentration, the change of PH value and phase separation. Therefore, in order to protect the activity of the drug, a protective agent for the active substance is usually added to the drug formulation. It requires four characteristics: high glass transition temperature, poor water absorption, low crystallization rate, and no reduction groups. Commonly used protective agents are the following types of substances: a) sugars/polyols: sucrose, trehalose, mannitol, lactose, glucose, maltose, etc.; b) polymer: HES, PVP, PEG, dextran, albumin, etc.; c) anhydrous solvent: ethylene glycol, glycerin, DMSO, DMF, etc.; d) Surfactant: Tween 80, etc.; e) amino acid: L-serine, sodium glutamate, alanine, glycine, sarcosine, etc.; f) salts and amines: phosphates, acetates, citrates, etc.; Since there are many kinds of stress damage in the freeze-drying process, the mechanism of the protective agent to protect the activity of the drug is also different, and can be divided into low temperature protection and freeze-drying protection. For cryoprotection, one of the mechanisms currently accepted for protein stabilization in liquid state is the principle of preferential action. Preferential action means that the protein preferentially interacts with a protective agent in water or an aqueous solution. In the presence of a stabilizing protective agent, the protein preferentially interacts with water (preferential hydration), while the protective agent is preferentially excluded from the protein region (prioritized rejection). In this case, the protein surface has more water molecules and fewer protectant molecules than its interior. The principle of priority action also applies to the freeze-thaw process. A protein protectant that is repelled from the surface of the protein in solution and stabilizes the protein during freezing. However, the mechanism of preferential action does not fully explain the phenomenon of protecting proteins at high concentrations with polymers or proteins themselves. During the lyophilization process, the preferential mechanism of action is no longer applicable as the hydration layer of the protein is removed. For the freeze-drying protection mechanism, it is still under study and there are two main types: a) Water substitution hypothesis. Many researchers believe that due to the presence of a large number of hydrogen bonds in protein molecules, bound water is linked to protein molecules through hydrogen bonds. When the protein loses moisture 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, so that the surface of the protein forms a hypothetical hydrated film, so that the bonding position of the hydrogen bond can be protected from direct exposure to the surrounding environment. It stabilizes the high-grade structure of the protein and prevents the protein from denaturation due to lyophilization, so that it maintains the structural and functional integrity of the protein even in the case of low temperature freezing and drying and water loss. b) Glass state hypothesis. The researchers believe that the protective agent-water mixture will vitrify during the drying process with the protective agent solution when the concentration is large enough and the crystallization of the protective agent does not occur. It has been found that in the glass state, the substance has both solid and fluid behavior, the viscosity is extremely high, crystals are not easily formed, and the molecular diffusion coefficient is very low, so that a viscous protective agent surrounds the protein molecules to form a structure. The carbohydrate glass body similar to the glassy ice hinders the chain forging movement of the macromolecular substance, prevents the protein from stretching and precipitating, and maintains the stability of the three-dimensional structure of the protein molecule, thereby protecting. At present, most scholars agree with the "water substitution hypothesis" because the hydrogen bond between the protein and the protective agent can be detected experimentally to provide evidence for the theory. In fact, whether it is the "water substitution hypothesis" or the "glass state hypothesis", their basis is based on the partial or complete vitrification freezing of the liquid. 4 freeze-drying process and optimization Since the freeze-drying process of the drug will produce a variety of stresses, which have a great influence on the medicinal properties of the freeze-dried drug, rational design of the freeze-drying process of the drug is of great significance for reducing the freeze-drying damage and improving the quality of the freeze-dried drug. 4.1 Freezing research The freezing process during lyophilization is very important because the morphology and size of the ice crystals formed during freezing and the degree of vitrification not only affect the subsequent drying rate, but also affect the quality of the lyophilized drug. Therefore, the formulation, freezing rate, freezing method, and annealing should be considered during the freezing process. 4.1.1 Effect of the formula The solids content of the formulation affects the freezing and drying process. If the solids content is less than 2%, the mechanical properties of the lyophilized drug structure will be unstable. Especially during the drying process, the drug particles cannot stick to the substrate, and the escaped water vapor will bring the particles to the stopper of the vial, and sometimes even to the vacuum chamber. In addition, in order to obtain a uniform, smooth and stable protein drug, the formulation must contain a protective agent such as a filler, an excipient, a stabilizer, etc., which have a significant influence on the vitrification freezing of the drug. Many sugars or polyols are often used as stabilizers for non-specific proteins during solution freeze-thaw and lyophilization. They are both effective cryoprotectants and good lyoprotectants. Their effect on freezing depends on the type. And concentration. In the literature [16 ~ 23], different protective agents were studied in detail, and their freezing characteristics were discussed. The freezing properties of other protective agents have also been studied in [4]. However, there are many kinds of proteins and different physicochemical properties. Therefore, different proteins require different protective agent formulations, so their freezing characteristics are different, and experiments are generally required. 4.1.2 Freeze mode Different freezing methods result in different forms and sizes of ice crystals, which will affect the subsequent drying rate and the quality of the freeze-dried drugs. According to the freezing mechanism, the freezing can be divided into two types: global supercooled crystallization and oriented crystallization. Global supercooled crystallization refers to the way in which all liquids are frozen under the same or similar subcooling. In global supercooled crystallization, freezing rate and ice crystal nucleation temperature are important parameters. The global supercooled crystallization can be divided into slow freezing and fast freezing according to the freezing rate. The fast frozen ice crystals are small and there is no freezing and concentration, but there is incomplete freezing. In contrast, slow cooling produces larger ice crystals and freezes and concentrates. Thomas W Patapoff et al. found that if the drug was directly immersed in a liquid nitrogen or dry ice-ethanol solution tank (rapid freezing), the crystal nucleus was first produced on the wall of the bottle, and then the ice crystals spread toward the center and then spread vertically upward. Due to the small size of the ice crystals and the horizontal structure, the mass transfer resistance in the drying stage is large, and the sublimation rate is lowered. Experiments show that rapid freezing results in low sublimation rate, fast desorption rate, slow freezing, fast sublimation rate and slow desorption rate. James A Searles et al. believe that ice crystal nucleation temperature is an important factor in the global supercooling crystallization because it is the main determinant of sublimation rate. They found in the study that the ice crystal nucleation temperature is inherently random and unstable, and is not easy to control, but is affected by the content of particles in the solution and the presence or absence of ice crystal nucleation. It is the randomness of the ice crystal nucleation temperature that results in sublimation drying rate non-uniformity and morphology-related parameters such as lyophilized drug surface area and desorption drying rate. Directional crystallization refers to the way in which a small portion of the drug solution is frozen under too cold conditions. Thomas W Patapoff introduced a vertical freeze method. The solution was cooled with wet ice, cooled at the bottom of the bottle with dry ice to form crystal nuclei, which were then frozen on a shelf at -50 °C. The ice crystals of the sample frozen in this way are chimney-like in the vertical direction, there is no frozen layer on the surface of the drug, and the structure of the whole drug is uniform, so the mass transfer resistance during drying is small, and the freeze-drying rate is accelerated. . Martin Kramer et al. used another way to achieve directed freezing. Under the condition that the pressure in the vacuum chamber is 0.1 kPa and the shelf temperature is +10 ° C, the solution is allowed to freeze on the surface to form a thin layer of ice crystals of about 1 to 3 mm. The vacuum is then released and the shelf temperature is lowered below the crystallization temperature for freezing. Under these conditions, the ice crystals grown are thick and chimney-like. At the same time, it was found in the drying stage that the sublimation drying time was 20% less than the general freezing time. When analyzing lyophilized drugs, it was also found that for mannitol, the lyophilized product frozen in this way had more residual water content than that of normal freezing; however, for sucrose and glycine, the difference was small. H Schoof et al. also used directional crystallization when lyophilizing collagen. Different freezing methods result in different ice crystal forms and sizes, and subsequent drying rates are also different. Experiments have shown that the rate of drying of frozen drugs using directed crystallization is faster than that of global supercooled crystallization. However, no matter which freezing method is adopted, the drug solution must be partially or completely vitrified to protect the drug properties. 4.1.3 Annealing Annealing refers to the process of raising the temperature of a frozen drug below the eutectic temperature, holding it for a period of time, and then lowering the temperature to a freezing temperature. There are at least three reasons for adding an annealing step before sublimation drying: a) Strengthening crystallization. During the freezing process, especially during the rapid freezing process, the crystalline components in the formulation are often too late to crystallize completely. However, if the ingredient provides the necessary support for the structure of the lyophilized drug or if the protein is more stable after the ingredient has completely crystallized, then complete crystallization is necessary. In addition, some of the frozen concentrate will not be able to precipitate, so that it does not reach the large concentration of zui. Experiments have shown that when the annealing temperature is higher than the glass transition temperature Tg' of the large concentration of the zui of the formulation, the formation of recrystallization is promoted to complete the crystallization of the crystalline component and the unfrozen water. b) increasing the glass transition temperature Tg' of the zoi large concentrate of the amorphous phase. By removing the crystal component having a lower Tg' from the amorphous phase, the Tg' of the amorphous phase can be increased. Barry J Aldous studied the crystallization of amorphous carbohydrates and found that the glass transition temperature of the trehalose drying solution after annealing increased from 31 ° C to 79 ° C, greatly improving the stability. c) Change the ice crystal morphology and size distribution to improve drying efficiency. James A Searles et al. have suggested that different nucleation temperatures produce different ice crystal forms and particle sizes, which in turn leads to uneven rates of sublimation drying. However, the drying rate in one process is determined by the slow drying of the drug, so uneven drying rate will affect the quality of the drug and the economics of production. Studies have shown that phase behavior and recrystallization during annealing can reduce ice crystal size differences and drying rate non-uniformity due to nucleation temperature differences, improve drying efficiency and drug uniformity. In order to achieve the annealing purpose, parameters such as heating rate, annealing temperature, annealing time, etc. must be considered in the annealing operation. However, due to the lack of advanced experimental methods and lack of theoretical knowledge, the annealing mechanism is still in doubt, and the selection of annealing parameters is still unfounded. 4.2 drying The drying process of the drug freeze-drying can be divided into two stages, one drying and two drying. Free water is removed in one drying stage and a portion of the bound water is removed in the secondary drying stage. The drying process occupies most of the energy consumption of the freeze-drying process of the drug, so it is very meaningful to take effective measures to increase the drying rate. At present, most of the measures to control the temperature of the shelf and the temperature of the drug, the temperature of the cold trap and the degree of vacuum are used to achieve an increase in the drying rate. Control of drug temperature. Temperature control including frozen and dried layers. The principle of controlling the temperature of the frozen layer is to ensure that the frozen layer does not melt (below the eutectic point), the higher the temperature, the better. The principle of controlling the dry layer temperature is to use a higher drying temperature as much as possible without denaturation of the material or collapse of the dried layer structure. The shelf temperature control is to meet the drug temperature control standard. Cold trap temperature. The driving force for water sublimation during lyophilization is the temperature difference between the drug and the cold trap. Since the temperature of the drug is limited by the heating mode and cannot be higher than the eutectic temperature, the lower the temperature of the cold trap, the better. In order to improve economic efficiency, it should be at least 20 °C lower than the drug temperature during the sublimation drying process; in the desorption drying process, the cold trap temperature requirements are lower for those formulations requiring very low residual moisture. Vacuum degree. It is generally believed that pressure has both positive and negative effects on the freeze-drying process: a) Below the eutectic temperature and collapse temperature of the drug, the higher the sublimation interface temperature, the more sublimated water vapor, the greater the heat required. The higher the pressure, the higher the thermal conductivity of the dried layer, and the greater the surface convection. Therefore, the faster the sublimation water vapor, that is, the higher the lyophilization rate. b) The sublimation interface is related to the difference between the water vapor escaping velocity from the dry layer to the outside and the pressure difference between the interface and the surface, that is, the saturation pressure corresponding to the interface temperature and the vacuum degree of the drying chamber. This pressure difference is large, which helps the water vapor to escape. The smaller the pressure difference, the slower the escape and the smaller the drying rate. If freeze drying is a heat transfer control process, the drying rate increases as the drying chamber pressure increases; if freeze drying is a mass transfer control process, the drying rate decreases as the drying chamber pressure increases. Experience has shown that the vacuum in the sublimation stage is between 10 and 30 Pa, which is beneficial to the transfer of heat and to the sublimation. If the pressure is too low, the heat transfer is unfavorable, the medicine is not easy to obtain heat, the sublimation rate is lowered, and the requirements for the equipment are higher, which increases the cost. When the pressure is too high, the sublimation speed of the internal ice of the medicine is slowed down, and the heat absorbed by the medicine is reduced, so that the temperature of the medicine itself rises. When the temperature is higher than the eutectic temperature, the medicine will melt and cause the freeze-drying failure. According to the influence of vacuum on the freeze-drying rate, the literature [40] adopted the cyclic pressure method and obtained good results. The freeze-drying process of the drug is a continuous operation. Different drug formulations have different freezing characteristics and the freeze-drying curve is different. Therefore, individual research should be carried out on the basis of basic research to optimize the freeze-drying curve and increase the drying rate. Reduce energy consumption. 5 drug freeze dryer There are many classification methods for drug freeze dryers. According to their shelf area, they can be divided into three types: large, medium and small. Generally, the freeze-dry area is less than 1.5m2, which is small, between 1.5m2 and 50m2, medium size, and larger than 50m2. According to its purpose and use, it can be divided into experimental freeze dryer, pilot freeze dryer and industrial production freeze dryer. The drug freeze dryer is mainly composed of a drying box, a vacuum system, a refrigeration system, a cold trap system, a heating system, a capping system, and an automatic control system. In addition, large and medium-sized freeze dryers often have a steam sterilization system (SIP) and a in-situ cleaning system (CIP). The refrigeration system provides a cold source for the freeze drying oven and the cold trap system, respectively. The laminar cooling temperature of the single-stage refrigeration compression cycle currently used is between -35 ° C and -40 ° C, the cold trap temperature is about -50 ° C; the laminar cooling temperature of the two - stage refrigeration compression cycle is -45 ° C ~- Between 50 ° C, the cold trap temperature is around -65 ° C; the laminar cooling temperature of the cascade refrigeration cycle is between -55 ° C and -60 ° C, and the cold trap temperature is around -75 ° C. The control of the freeze dryer is mainly for the refrigeration machine, the vacuum unit, the start and stop of the heating power and the temperature control, the determination of the vacuum degree, the temperature, the monitoring, and the automatic protection and alarm devices. The lyophilizer with fully automatic control or microcomputer control can display the working status of each main component, and display the temperature, vacuum degree and water trap temperature of the shelf and medicine in the drying cabinet, and can be parameterized, modified and real-time. display. The drug freeze dryer must implement GMP standards to achieve high degree of sterility and dust-free, achieving high reliability, safety and easy maintenance. For this purpose, the drug freeze dryer often uses a steam sterilization system (SIP) to ensure complete sterilization and no dead angle. At the same time, supplemented by the in-place cleaning system (CIP), the drying chamber, condenser, main valve and pipeline are cleaned in advance to preset the drainage gradient to ensure no liquid retention. At the same time, it has protection measures against power outage, water stoppage and misoperation. Once a fault occurs, it can protect the medicine; realize computer control of the operation and operation of the freeze dryer, and have three countermeasure systems for power failure and water stop, and can automatically alarm by multiple interlocks. Since the drug freeze dryer must implement the GMP standard, the research of the drug freeze dryer will continue to be carried out in the direction of sterilizing and high reliability, such as automatic feeding mode and vacuum control mode. With the rapid development of biotechnology, peptide protein drugs continue to emerge, and can be applied to clinical polypeptides, proteases, hormones, vaccines, cell growth factors and monoclonal antibodies. In order to prevent drug denaturation, a solid-state drug is currently widely prepared by freeze-drying. After decades of development, although the pharmaceutical freeze-drying technology has made great progress, there are still many problems that need to be solved. During the freeze-drying process, a variety of freezing and drying stresses are generated to cause different degrees of denaturation of the drug, and the freeze-drying method itself has the disadvantages of low drying rate, long drying time, high energy consumption in the drying process, and large investment in drying equipment. Therefore, in order to improve the stability and economy of the drug, it is necessary to further study the damage and protection mechanism of the drug during lyophilization, and at the same time develop advanced and low-cost freeze dryer with advanced refrigeration and vacuum equipment and control means. Continue to improve the theory of heat and mass transfer under low temperature and low pressure, and optimize the freeze-drying process. Ningbo DOKEE Medical Technology Co., Ltd. , https://www.dokeemedical.com