Ultrasonic technology application

With the rapid development of biotechnology, biologically active substances have been continuously utilized.The active substances produced by the use of transgenic host cells (prokaryotic and eukaryotic) cells have been used in medicine as clinical drugs. There are hundreds of them, most of which are proteins and active peptides. The chemical structure of protein molecules determines their activity and affects many factors. There are two main aspects.One is structural factors, including molecular weight, amino acid composition, and amino acid sequence. No disulfide bond, disulfide bond position, spatial structure; second is the environmental factors around the protein molecule, protein! Peptide is subject to complex physics! Chemical factors cause condensation! Precipitation! Hydrolysis! Deamidation and other changes have been approved for domestic market There are more than 20 kinds of genetic engineering drugs and vaccines, most of which are lyophilized preparations. The reason is that lyophilized preparations can maintain protein for a long time! The activity of peptides. Therefore, in the process of research and development of new drugs, lyophilization technology is an important link.

After joining the WTO, global integration, market opening, and rapid development of the secondary drug processing market, Outsourcing has long become a practice in the European and American pharmaceutical industries. According to reports, the global drug outsourcing market reached 30 billion US dollars in 2003, 2004 The annual outsourcing of pharmaceutical preparations, estimated at US $ 34-35 billion, accounts for about 26% of the entire drug outsourcing market. The rest is outsourcing processing of APIs. After the 1990s, commissioned processing of APIs (ordered APIs) has been subject to higher technical requirements. It is replaced by increasing the processing level of APIs, that is, large companies hand over the new drugs (APIs) they develop to scientific research and development companies with strong scientific and technological strength, and the latter will transfer the new drugs (especially protein / peptide drugs and anti-drug drugs). Virus drugs! Anti-cancer drugs, etc.) are processed into nano-level preparations! Lyophilized powder injections! Oral fast-dissolving tablets! Aerosols / dry powder inhalers, and other raw materials for new administration routes. The new target freeze-dried injection for secondary processing is one of the important technical products, such as the Dutch company DSM, which is a famous European drug secondary processing company, and is good at processing freeze-dried powder injections of antibiotic raw materials in 2004. Expansion of the production capacity of freeze-dried powder injections (11 sets of large-scale freeze-drying machines, each of which covers an area of ​​more than 30 m2) with a capital of USD 62 million, and became the largest powder injection production company in Europe. Canada's Patheon is a major drug secondary processing company in North America , The main generation of processing freeze-dried powder injections The company cooperates with Italy to establish a large-scale freeze-dried powder injection production base in Italy, a large freeze-drying device covers an area of ​​271m2, it can be seen that freeze-drying technology is not only an important production of genetic engineering drugs Link, and its technological advantages can develop into an industry.

This article will combine the production of lyophilized injections of genetically engineered peptides and protein drugs, combined with the pilot work of the center, for discussion, for peer reference.

1 Principle and application of freeze-drying technology

(1) Principle

Freeze-drying refers to freezing the drug at a low temperature, then sublimating and drying under vacuum conditions, removing ice crystals, desorption and drying after the sublimation is completed, and drying methods that remove some of the bound water.This process can be divided into drug preparation! Pre-freeze! Primary drying (sublimation drying)! Secondary drying (desorption drying)! Sealing and storage steps The vertical axis shown in Figure 1 is the air pressure, the horizontal axis is the temperature, and 0 ℃ (actually 0.001 ℃) is the triple point, indicating that the water is liquid The lowest atmospheric pressure when it exists, below this point, the water can only exist as ice or steam, that is, when the temperature is raised at this time, the water can only change directly from ice to steam, and freeze-drying is far below this pressure (high vacuum Under the condition of drying moisture, usually under the vacuum of 66 ~ 133Pa and below -25 ℃, in order to ensure the smooth progress of freeze drying.
(2) Advantages and disadvantages

Compared with other drying methods, freeze-drying has the following advantages:

1) The liquid medicine is divided before lyophilization, which is convenient for dispensing! Accurate! It can be continuous;

2) Mild processing conditions, drying at low temperature and low pressure, is conducive to maintaining the activity of heat-sensitive substances, can avoid the decomposition and denaturation under high temperature and high pressure, so that the protein will not be denatured;

3) The moisture content is low, and the moisture content of freeze-dried products is generally 1% to 3%. At the same time, it can be dried and stored under vacuum, even under the protection of N2.The product is not easily oxidized, which is conducive to long-distance transportation and long-term storage;

4) The product has excellent appearance, porous and loose structure and basically unchanged color, good rehydration, and freeze-dried drugs can quickly absorb water and restore to the state before freeze-drying;

5) Closed operation of freeze-drying equipment, high cleanliness of installation environment, reduce pollution of bacteria and particles, dry and neutralize the lack of oxygen after packaging can play a role in sterilization and inhibit the vitality of certain bacteria

The disadvantages and deficiencies of freeze-drying and products:

1) High equipment requirements! Large investment! Low drying rate! Long drying time! High energy consumption;

2) The use of freeze-dried preparations for biologically active substances (such as peptides and protein drugs) is mainly to maintain activity, but the choice of ingredients (such as protective agents! Solvents! Buffers, etc.) is unreasonable! The process operation is unreasonable! The choice of freeze-drying equipment is not appropriate All may be inactivated during the preparation of lyophilized preparations, resulting in the abandonment of the product's previous efforts. This is the key to the production of lyophilized preparations, and basic research and repeated trials for specific products are required;

3) The solvent cannot be chosen at will, it is limited to water or some organic solvents with a high freezing point, so it is difficult to prepare a special crystal form. Sometimes the lyophilized product will appear cloudy when it is dissolved in rehydrated water. The formulation must be considered and experimentally studied.
(3) Application of freeze drying technology

The freeze-drying technology was invented by the British Wallaston in 1813. In 1909, the Shsckell test used this method to fight toxins! Strains! Rabies virus and other biological products were lyophilized and preserved, which achieved good results in the Second World War. Due to the large demand for blood products, freeze-drying technology has developed rapidly and entered the stage of industrial application. The large-scale development of freeze-dried food systems in the 1950s promoted the advancement of freeze-dried technology and equipment, but due to the high difficulty! High investment! High energy consumption and the backwardness of manufacturing equipment, it has experienced ups and downs for decades. In the past 20 years, as people's living standards have improved, the quality of food! Nutrition! The concept of natural pollution-free has promoted the development of freeze-drying technology. The production process has changed from intermittent to continuous, and the equipment has ranged from 0.1m2 to thousands. The m2 series is formed and has a wide range of applications: in scientific research, it is applied to, for example, analyze the measurement components in the soil; remove the solvent of the components collected by high-performance liquid chromatography; the important cultural relics found in archaeology such as cloth! leather! In industry, it is used in freeze-dried foods such as vegetables! Fruits! Seafood! Even flowers etc .; spices and condiments such as coffee! Tea and various spices! Seasonings; convenience foods that preserve nutrition and health ingredients and color and fragrance forms (Japan 50% of convenience foods are freeze-dried foods); aquatic products are the most extensive! The most stringent requirements are still the application in medicine and biological products, mainly in serum! Strains! Genetic engineering drugs! Vaccines! Natural medicines and biological products In the 2000 version of the Chinese Biological Products Regulations, 8 of the 11 recombinant therapeutic protein drugs identified were lyophilized preparations, such as recombinant human interferon α1b! Recombinant human interferon α2b! Recombinant human interferon α2b! Recombinant human interferon γ ! Recombinant Human Interleukin 22! Recombinant Human Erythropoietin! Recombinant Human Granulocyte Macrophage Colony Stimulating Factor! Recombinant Streptokinase.

Production process of lyophilized preparation

Genetically engineered peptides and protein drug freeze-dried preparations are obtained by cultivating the host (microorganism or animal cells) to express the product! After separation and purification to obtain the active substance, after preparation! Filtration and sub-packing, the sub-packed samples are sent to the freeze dryer, Pre-freeze! Sublimate! Dry, and finally seal. Therefore, the production process of the freeze-dried preparations includes pharmaceuticals. The so-called ultrasonic wave refers to sound waves with a vibration frequency ranging from 20 kHz to 1000 MHz. Since Richards and I oomis [1] reported the chemical action of ultrasound in the 1930s, various chemical effects of ultrasound have aroused widespread concern. Early research focused on the reduction of polymer viscosity (polymer degradation) under the action of ultrasound. Since 1980, with the development of polymer characterization methods, the application of ultrasound in polymer synthesis has also been carried out. Sonochemical theoretical calculations and corresponding experiments show that ultrasonication can produce extreme environments of high temperatures of thousands of K and high pressures of hundreds of atmospheres around the cavitation bubble phase interface]. Under such conditions, solvents, monomers or high The molecular chain is decomposed or broken to produce free radicals, which leads to the widespread use of ultrasound in polymer synthesis.

1 Ultrasound is used to prepare block copolymer
In 1999, Huceste et al. Dissolved 2,300,000 polyethyl methacrylate (PEMA) and 1,200,000 polystyrene (PS) in toluene, saturated them with N and irradiated them with ultrasonic waves . It was found that the polymer degraded after 2 hours of irradiation. Then they added styrene monomer to the irradiated polymer system and matched with appropriate temperature conditions to prepare a block copolymer. The non-uniformity coefficient (H) of the product was 3. The oR is as low as 1.34.
In 1998, Fujiwara H [5 et al. Studied the synthesis of polyvinyl chloride and polyvinyl alcohol block copolymers under ultrasound irradiation. They made solid polyvinyl chloride and polyvinyl alcohol into multiple aqueous solution systems and irradiated them with ultrasonic waves at 3O ° C. It was found that the average viscosity of polyvinyl chloride decreased much faster than polyvinyl alcohol. Under the action of ultrasonic waves, both polymers degraded and produced free radicals. This free radical triggered the mechanochemical reaction of the polymer, which produced a block copolymer.
In 2003, Degirmenci M et al. Synthesized block copolymers of styrene and methyl methacrylate under ultrasonic irradiation. They also studied the degradation behavior of PM-MA under ultrasonic irradiation. The theoretical molecular weight obtained was consistent with the experimentally measured value and the GPC measurement results, indicating that it was indeed the free radicals generated by ultrasonic degradation that initiated the copolymerization reaction.

2 Ultrasound is used to initiate emulsion polymerization
In 1998, Joe Chou HC et al [7.8j studied the emulsion polymerization of methyl methacrylate (MMA) when sodium lauryl sulfate was used as an emulsifier under ultrasonic irradiation, and investigated the factors affecting the polymerization rate influences. It was found that even without the addition of conventional initiators, emulsion polymerization can still be initiated by ultrasound at room temperature. In this case, the free radicals that initiate the reaction come from the degradation of the emulsifier under ultrasound irradiation. The use of ultrasound can not only initiate and accelerate emulsion polymerization, but also provide the energy required for the reaction at a lower temperature.
In 2004, Ai ZQ et al [9] studied the emulsion polymerization of styrene and butyl acrylate under ultrasonic irradiation. They dissolved PS waste in butyl acrylate, added water and initiator, and then made the graft copolymer under the action of ultrasonic irradiation and stirring. The higher the ultrasonic power, the longer the irradiation time and the higher the reaction temperature, the lower the coagulation rate of the grafted product. The types and amount of emulsifier, the total concentration of emulsifier and other factors also affect the product coagulation rate.
In 2005, Bahattab MA Ll studied the emulsion polymerization of vinyl acetate under ultrasonic irradiation. When no initiator and emulsifier are present, the ultrasonic polymerization alone can initiate the emulsion polymerization of vinyl acetate at ambient temperature. After using redox initiator system and using ultrasonic irradiation, the conversion rate and polymer yield of the polymerization reaction are improved compared to the case without ultrasonic irradiation. Ultrasound plays an important role in initiating the reaction and controlling the polymer structure. effect.

3 Ultrasound is used to modify polymers
In 1997, Santos EAGL et al [11] used ultrasound as an energy source to study the reaction of maleic anhydride modified polypropylene. It was found that an increase in the amount of maleic anhydride would reduce the grafting rate, because under the experimental conditions used, a large amount of maleic anhydride formed a homopolymer due to the presence of dibenzoyl peroxide; and with the ultrasonic power density used The increase in the grafting rate becomes more and more obvious. They also studied the effect of ultrasonic irradiation on the alum and polydispersity coefficient of the grafted product, and found that the alum of the product decreased by 13.73 and the polydispersity coefficient decreased by 1.98. These phenomena are attributed to the mechanical breakage of long chains of polymer molecules caused by ultrasound, which is consistent with the generation of free radicals and the recombination of free radicals. Filling polymer materials with ultrafine inorganic particles to improve the performance of the latter is an important direction for polymer modification. However, due to the huge surface energy, ultra-fine particles are easy to agglomerate, and it is not easy to uniformly disperse them into the polymer system. The traditional method is to modify the surface of the particles by selecting suitable surfactants, but the effect is often unsatisfactory In recent years, many people have begun to use ultrasound to disperse particles into polymer materials. Typical progress is briefly described as follows.
In 2000, Xia HS et al. Ll ¨] studied the dispersion of several inorganic nanoparticles (nano-sized siO., Al O., TiO particles) in butyl methacrylate and their effects on polymers under ultrasonic irradiation Modifications. Through the action of ultrasound, they prepared a stable emulsion containing polymer / inorganic nanoparticles. Scanning electron microscopy confirmed that the nanoparticles existed in the microcapsules formed by the polymer. The wall thickness of the microcapsules was only 5 nm to 65 nm.
In 2005, Qiu GH et al. [15] used ultrasonic waves with a power of 750 W to disperse magnetic iron oxide nanoparticles in an aqueous solution of pyrrole monomers and used an oxidation-type initiator FeC1. Polymerizing pyrrole solves the problem of easy aggregation of nanoparticles.

4 Ultrasound is used to monitor the polymer reaction process
In 2001, Kiehl C et al. C16] established an ultrasonic system to monitor the polymerization process of MMA in model batch reactors and twin-screw reactive extruders. The polymerization conversion rate of MMA in the self-made reactor was compared with the results obtained by DSC and correlated with the propagation speed of ultrasonic waves, thus establishing the relationship between the conversion rate of MMA and the propagation speed of ultrasonic waves, making it available for online MMA polymerization monitor. Mikhailyuk GM et al. [1] used ultrasound to study the polymerization process of phenolic resin. The essence is also to use the viscosity change during the polymer reaction process, and the viscosity can be more accurately reflected by the change of some properties of ultrasound.

5 Effect of ultrasound on solution polymerization
Osawa ZJ et al [1B] studied the effect of ultrasound on the stereoregularity of MMA solution polymers and oligomers. The specific method is to dissolve MMA in a mixed solvent of toluene-dioxane, comparing two cases-I. Do not use Grignard catalyst; â…¡. Using Grignard catalysts-the stereoregularity of the resulting polymers and oligomers. It was found that the stereoregularity of I was higher than that of II. However, after irradiating the reaction system with ultrasound, the result was reversed: the stereoregularity of I was lower than that of II, indicating that ultrasound changed the reaction process. In the next few years, Osawa ZJ et al. [1] conducted various researches on this issue. For example, they I. Add the catalyst directly to the mixture of reaction monomer and solvent; â…¡. The catalyst was first added to the solvent and then the monomer was added, and then the reaction results were measured. It was found that the stereoregularity of the polymer obtained by Method I was higher than that of II, and the results also occurred after the irradiation of the reaction system with ultrasound Inverted: The stereoregularity of I is lower than that of II. Generally speaking, due to the irradiation of ultrasonic waves, the properties of the polymer obtained by the solution polymerization will be changed contrary to the conventional polymerization method.

6 Ultrasonic waves are used to prepare micron / nanopolymer (or inorganic composite) particles. Ultrasound irradiation in liquid systems can produce effects that are unmatched by conventional stirring methods, making ultrasonic waves in the preparation of micron / nanopolymer (or inorganic composite) particles. It has been widely used in L2]. Wang L et al. [2] used precipitation polymerization to produce core-shell organic nanoparticles under ultrasonic irradiation. The specific method is: dissolve pyrene in acetone, add dropwise to a certain amount of water and dilute, and then irradiate with a certain power of ultrasonic for 30 rain to obtain a core of core-shell structure; then add a certain amount of six in this system Sodium metaphosphate, potassium persulfate, and acrylic acid react with 20rain under the combined action of vigorous stirring and ultrasonic waves, and the pyrene as the core is covered with polyacrylic acid to obtain nano-scale organic particles.

7 Study on the mechanism of ultrasonic polymerization
In 1999, Huceste et al. [Z73 based on the principle that ultrasound can break long polymer chains, used ultrasound technology to study the dominant chain termination mechanism of free radical polymerization of methyl methacrylate and styrene, and at the same time can obtain polymerization The ratio (d / c) of the disproportionation rate and coupling rate at the end of the chain. They dissolved the "dead" polymer that had been polymerized, and then irradiated it with ultrasound to break the chain of the polymer to obtain a long-chain free radical; then they added or did not add a chain terminator (free radical) to the system Catcher). Comparing the relative molecular masses of the resulting polymers in the two cases, it is speculated that the dominant radical polymerization chain termination mechanism in this case will be presumed.
Youn J et al. Also used ultrasound to study the formation of polyurethane foam. Nishikawa S et al. [. . Then the effect of polyvinylpyrrolidone on the proton transfer reaction in propylamine aqueous solution under ultrasound irradiation was studied.

8 Ultrasound is used to initiate bulk polymerization
Gu CB et al. [Z93 uses high energy density ultrasonic waves of hundreds of watts per square centimeter to irradiate methyl methacrylate to initiate bulk polymerization. It is found that the polymerization rate is related to the length of ultrasonic irradiation time and the size of ultrasonic energy density; for a pure MMA monomer system, there is a threshold value of ultrasonic energy density. Below this value, any extension of the irradiation time cannot initiate polymerization Reaction; adding a certain amount of polymer PMMA to the monomer system, the polymerization rate is accelerated with the increase of the amount of PMMA. ESR analysis shows that the irradiation of ultrasound does generate free radicals in the polymerization system, and the concentration of free radicals changes with the irradiation conditions.

9 Prospects Because ultrasound can produce cavitation, vigorous stirring and other effects in liquid systems, it has many unique applications in polymer synthesis. Throughout the past 10 years, the application of ultrasound, from the monitoring of polymer reaction process to the study of polymer reaction mechanism, the application of ultrasound technology has covered almost all fields of polymer science. Looking into the future, with the design and manufacture of high-power narrow-bandwidth ultrasonic generators and the continuous improvement and improvement of polymer research methods, the application and mechanism research of ultrasound will inevitably become more extensive and in-depth. In addition, compared with conventional initiation methods, ultrasonic-initiated free radical polymerization does not use an initiator, and no additional impurities are introduced into the reaction system, and high-purity polymer materials can be obtained. This feature is bound to have special characteristics for the purity of polymer materials. The required field produces a wide range of applications.