Ultrasonic technology application

With the rapid development of biotechnology, bioactive substances are continuously being utilized, and active substances produced by transgenic host (prokaryotic and eukaryotic) cells have been used as medicines, and more than 50 kinds of drugs approved at home and abroad have been developed. The number of hundreds of species, most of which are the chemical structure of protein and active polypeptide protein molecules determine the activity of its activities, there are many factors, mainly two aspects, one is the structural factors, including molecular weight, amino acid composition, amino acid sequence, there are No disulfide bond, disulfide bond position, spatial structure; second, environmental factors around the protein molecule, protein! Polypeptide is affected by complex physics! Chemical factors, condensation, precipitation, hydrolysis, deamidation, etc. 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, lyophilization technology is an important link in the development of new drugs.

After joining the WTO, global integration, market opening, and drug secondary processing market are developing rapidly. Outsourcing has become a practice in the European and American pharmaceutical industry. According to reports, the global drug outsourcing market reached US$30 billion in 2003. The estimated annual expenditure of 340-350 billion US dollars is about 26% of the entire drug outsourcing market. The rest is the outsourcing processing of APIs. After the 1990s, the entrusted processing of APIs (order processing APIs) has been technically more demanding. To improve the processing grade of APIs, that is, the big companies will hand over the new drugs (raw materials) they developed to research and development companies with strong scientific and technological strength, and the latter will introduce new drugs (especially protein/peptide drugs and anti-drugs). Viral drugs! Anticancer drugs, etc.) processed into nano-sized preparations! Freeze-dried powder needles! Oral fast-dissolving tablets! Aerosol/dry powder inhalers, and other novel drug delivery routes. The new target of secondary processing freeze-dried injection is one of the important technical products, such as DSM in the Netherlands, it is a famous European secondary processing enterprise, specializing in the processing of antibiotic raw materials freeze-dried In 2004, the injection of US$62 million to expand the production capacity of freeze-dried powder injections (11 sets of super-large freeze dryers, each covering more than 30 m2), became the largest powder injection production company in Europe, and the company's PATHEON company is the main North American company. Pharmaceutical secondary processing enterprise, mainly engaged in processing freeze-dried powder injection. The company cooperated with Italy to establish a large-scale freeze-dried powder injection production base in Italy. A large-scale freeze-drying device covers an area of ​​271m2. It can be seen that freeze-drying technology is not only used as genetic engineering. An important part of drug production, and its technological advantages can be developed into an industry.

This article will combine the genetic engineering of peptide and protein drug lyophilized injection production, combined with the center's pilot work, for discussion, for peer reference.

1 Principle and application of freeze drying technology

(1) Principle

Freeze-drying means freezing the drug at a low temperature, then sublimating and drying under vacuum, removing the ice crystals, and then desorbing and drying after the sublimation is finished, and removing the partially combined water drying method. The process can be mainly divided into medicine preparations! Pre-freezing! One drying (sublimation drying)! Secondary drying (desorption drying)! Sealing and other steps The ordinate on the ordinate is air pressure, the abscissa is temperature, and 0 °C (actually 0.001 °C) is a triple point, indicating that water is liquid. When there is a low atmospheric pressure of zui, below that point, the water can only exist as ice or steam, that is, when the temperature is raised at this time, the water can only be directly changed from ice to steam, and lyophilization is much lower than the pressure (high) Drying moisture under vacuum degree is usually between 66~133Pa vacuum and -25°C to ensure smooth and dry operation.
(2) Advantages and disadvantages

Freeze drying has the following advantages over other drying methods:

1) The liquid medicine is divided before the freeze-drying, and the dispensing is convenient! Accurate! Continuous can be realized;

2) mild treatment conditions, drying under low temperature and low pressure, is beneficial to the heat-sensitive substance to maintain activity, can avoid decomposition and denaturation under high temperature and high pressure, so as to achieve protein denaturation;

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

4) The product has excellent appearance, is porous and loose structure, and the color is basically unchanged. The rehydration is good, and the freeze-dried drug can quickly absorb water and return to the state before lyophilization;

5) The freeze-drying equipment is closed, the installation environment is clean, the pollution of bacteria and particles is reduced, and the lack of oxygen after drying and packaging can sterilize and inhibit the vitality of certain bacteria.

Shortcomings and deficiencies of freeze drying and products:

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

2) Biologically active substances (such as peptides and protein drugs) use lyophilized preparations mainly to maintain activity, but the choice of ingredients (such as protective agent! Solvent! Buffer, etc.) is unreasonable! The process operation is unreasonable! The selection of freeze-drying equipment is not appropriate. Both may be inactivated during the preparation of the lyophilized preparation, resulting in the product being completely discarded. This is the key to the production of lyophilized preparations, basic research and repeated trials for specific products are required;

3) Solvents cannot be selected arbitrarily. They are limited to water or some organic solvents with higher freezing point. Therefore, it is difficult to prepare a special crystal form. Sometimes the lyophilized product will appear turbid when it is dissolved in rehydration. Preparation must be considered and experimentally studied.
(3) Application of freeze drying technology

The freeze-drying technique was invented by the Englishman Wallaston in 1813. In 1909, the Shsckell test used this method to fight toxins! The strain! The rabies virus and other biological products were freeze-dried and preserved, and achieved good results in the Second World War due to The large demand for blood products, freeze-drying technology has been rapidly developed and entered the industrial application stage. The large-scale development of freeze-dried food systems in the 1950s has promoted the advancement of freeze-drying technology and equipment, but it has experienced decades of ups and downs due to high difficulty, high investment, high energy consumption and backward manufacturing equipment. In the past 20 years, with the improvement of people's living standards, the quality of food! Nutrition! The concept of natural pollution-free change has promoted the development of freeze-drying technology. The production process is from intermittent to continuous, and the equipment is from 0.1m2 to thousands. The m2 series is used in a wide range of applications: in scientific research, in the analysis of components such as soil analysis; in the removal of solvents from high-performance liquid chromatography; the important artifacts found in archaeology such as cloth, leather, bamboo, etc. In industry, it is applied to 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 nutritious health ingredients and color and flavor (Japan 50% of convenience foods are freeze-dried foods; aquatic products are wide-ranging! Zui is strictly used 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 are lyophilized preparations, such as recombinant human interferon α1b! Recombinant human interferon α2b! Recombinant human interferon α2b! Recombinant interference gamma]! Recombinant human interleukin 22! recombinant human erythropoietin! recombinant human granulocyte-macrophage colony stimulating factor! recombinant streptokinase.

Production process of cold dry preparation

The genetically engineered polypeptide and the protein drug lyophilized preparation are obtained by culturing the host (microorganism or animal cell) to obtain an expression product! The active substance is obtained by separation and purification, and is prepared, filtered, and dispensed, and the packaged sample is sent to the freeze dryer. Pre-freezing! Sublimation! Drying, sealing after zui, so the production process of lyophilized preparations includes drugs. The so-called ultrasonic refers to sound waves with vibration frequencies ranging from 20 kHz to 1000 MHz. Since the chemistry of ultrasound has been reported by Richards and Ioomis in the 1970s, the various chemical effects of ultrasound have attracted widespread attention. In the early days, people's research focused on the decrease of polymer viscosity under ultrasonic action (polymer degradation). Since 1980, with the development of polymer characterization methods, the application of ultrasonic in polymer synthesis has also been carried out. The sonochemical theoretical calculations and corresponding experiments show that the ultrasonication can generate thousands of K high temperatures and hundreds of atmospheric pressures around the interface of the cavitation bubble phase. Under such conditions, the solvent, monomer or high can be made. The molecular chain decomposes or ruptures to generate free radicals, which leads to the wide application of ultrasonic waves in polymer synthesis.

1 Ultrasonic 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, saturating with N and irradiating it with ultrasound. . It was found that the polymer was degraded 2 h after irradiation. Then they added styrene monomer to the irradiated polymer system and matched the appropriate temperature conditions to obtain a block copolymer. The non-uniform coefficient (H) of the product was 3. oR is as low as 1.34.
In 1998, Fujiwara H [5] studied the synthesis of polyvinyl chloride and polyvinyl alcohol block copolymers under ultrasonic irradiation. They made solid polyvinyl chloride and polyvinyl alcohol into a plurality of aqueous systems and irradiated with ultrasonic waves at 30 °C. It was found that the average viscosity of polyvinyl chloride was reduced much faster than polyvinyl alcohol. Under the action of ultrasonic waves, both polymers degrade and generate free radicals, which initiate the mechanochemical reaction of the polymer, thereby producing a block copolymer.
In 2003, Degirmenci M et al. synthesized a block copolymer of styrene and methyl methacrylate under ultrasonic irradiation. They also studied the degradation behavior of PM-MA under ultrasonic irradiation. The theoretical relative molecular mass was consistent with the experimental measurements and GPC measurements, indicating that the free radicals generated by ultrasonic degradation 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) with sodium lauryl sulfate as emulsifier under ultrasonic irradiation conditions, and investigated the factors of polymerization rate. influences. It was found that the emulsion polymerization reaction can be initiated by ultrasonic waves at room temperature even without the addition of a conventional initiator. In this case, the free radicals that initiate the reaction are derived from the degradation of the emulsifier under ultrasonic irradiation. The use of ultrasonic waves not only initiates and accelerates the emulsion polymerization, but also provides 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 conditions. They dissolved the PS waste in butyl acrylate, added water and an initiator, and then prepared a 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 obtained graft product; the type and amount of the emulsifier and the total concentration of the emulsifier also affect the coagulation rate of the product.
In 2005, Bahattab MA Ll studied the emulsion polymerization of vinyl acetate under ultrasonic irradiation. When no initiator and emulsifier are present, the effect of ultrasound alone can initiate emulsion polymerization of vinyl acetate at ambient temperature. When a redox initiator system is used and ultrasonic irradiation is used, the conversion rate and polymer yield of the polymerization are improved compared to the absence of ultrasonic irradiation, and the ultrasonic wave plays an important role in initiating the reaction and controlling the polymer structure. effect.

3 Ultrasound for polymer modification
In 1997, Santos EAGL et al. [11] used ultrasonic 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 reduced 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 of the grafting rate is more and more obvious. They also studied the effect of ultrasonic irradiation on the enthalpy and polydispersity coefficients of the grafted product. It was found that the enthalpy of the product was reduced by 13.73 and the polydispersity coefficient was also reduced by l1.98. These phenomena are attributed to the mechanical cleavage of long chains of polymer molecules caused by ultrasound, which is consistent with the recombination of free radicals and free radicals by chain scission. Filling the polymer material with ultrafine inorganic particles is an important direction for polymer modification to improve the performance of the latter. However, ultrafine particles are prone to agglomeration due to the large surface energy, so that it is not easy to disperse evenly into the polymer system. The traditional method is to modify the surface of the particles by selecting a suitable surfactant, but the effect is often unsatisfactory. In recent years, many people have begun to try to use ultrasonic waves to disperse particles into polymer materials. The typical progress is briefly described below.
In 2000, Xia HS et al. studied the dispersion of several inorganic nanoparticles (nano-sized siO, Al O, TiO particles) in butyl methacrylate and the polymer under ultrasonic irradiation. Modification. Through the action of ultrasonic waves, they obtained 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 into an aqueous solution of pyrrole monomer and used an oxidizing initiator FeC1. Polymerization of pyrrole solves the problem that nanoparticles are easily agglomerated.

4 Ultrasound is used for monitoring the polymer reaction process
In 2001, Kiehl C et al. (C16) established an ultrasonic system for on-line monitoring of the polymerization process of MMA in model batch reactors and twin-screw reactors. 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 MMA conversion rate and ultrasonic propagation speed, making it available for online MMA polymerization process. monitor. Mikhailyuk GM et al. [1] studied the polymerization process of phenolic resin by ultrasonic wave. The essence of the process is also to change the viscosity of the polymer during the reaction, and the viscosity can be more accurately reflected by the change of some properties of the ultrasonic wave.

5 Effect of ultrasonic wave on solution polymerization
Osawa ZJ et al. [1B] studied the effect of ultrasonic on the stereoregularity of MMA solution polymers and oligomers. The specific method is to dissolve MMA in a mixed solvent of toluene and dioxane, and compare the two cases - I. Do not use the Grignard catalyst; II. The Grignard catalyst is used - the stereoregularity of the resulting polymer and oligomer. As a result, it was found that the stereoregularity of I was higher than that of II. However, after the irradiation of the reaction system with ultrasonic waves, the result was reversed: I has a lower stereoregularity than II, indicating that the ultrasonic wave changes the reaction process. In the next few years, Osawa ZJ et al. [1] conducted a multifaceted study on this issue. For example, they I. The catalyst is directly added to the mixed solution of the reaction monomer and the solvent; II. The catalyst was first added to the solvent and then the monomer was added. Then, the reaction result was measured, and it was found that the stereoregularity of the polymer obtained by the method I was higher than that of II, and the result was also irradiated by using the ultrasonic wave to irradiate the reaction system. Upside down: I's stereoregularity is lower than II. In general, due to the irradiation of ultrasonic waves, the properties of the polymer obtained by solution polymerization are inversely changed from those of conventional polymerization.

6 Ultrasonic is used to prepare micro/nano-polymer (or inorganic composite) particles. Ultrasonic irradiation can produce unmatched effects in conventional methods of stirring in liquid systems, making ultrasonic preparation of micro/nano polymer (or inorganic composite) particles. Has obtained a wide range of applications L2]. Wang L et al [2] used a precipitation polymerization method to obtain organic nanoparticles with a core-shell structure under ultrasonic irradiation. The specific method is as follows: the hydrazine is dissolved in acetone, added dropwise to a certain amount of water, and then irradiated with ultrasonic waves of a certain power for 30 min to obtain a core of a core-shell structure; then a certain amount of six is ​​sequentially added to the system. Sodium metaphosphate, potassium persulfate, and acrylic acid were reacted for 20 hours under the action of vigorous stirring and ultrasonic waves, and the ruthenium as a core was covered with polyacrylic acid to obtain nano-sized organic particles.

7 Ultrasonics for the study of polymerization mechanism
In 1999, Huceste et al. [z73 based on the principle that ultrasonic waves can break long chains of polymers, using ultrasonic technology to study the chain termination mechanism predominating in the radical polymerization of methyl methacrylate and styrene, and at the same time can be polymerized. The ratio of disproportionation rate to coupling ratio at the end of the chain (d/c). They dissolve the "dead" polymer that has been polymerized and then use ultrasonic irradiation to break the polymer and obtain long-chain free radicals; then they add or not add chain terminators to the system (free radicals) Catching agent). The relative molecular mass of the obtained polymer in the two cases was compared, and the dominant mechanism of the radical polymerization chain termination in this case was presumed.
Youn J et al. also studied the formation of polyurethane foam using ultrasonic waves. Nishikawa S et al. . The effect of polyvinylpyrrolidone on proton transfer reaction in aqueous propylamine solution under ultrasonic irradiation was studied.

8 Ultrasound is used to induce bulk polymerization
Gu CB et al. [z93 irradiated methyl methacrylate with high energy density ultrasonic waves of several hundred watts per square centimeter to initiate bulk polymerization. It is found that the polymerization rate is related to the length of the ultrasonic irradiation time and the ultrasonic energy density. For the pure MMA monomer system, there is a threshold of ultrasonic energy density, below which the polymerization time cannot be initiated regardless of the irradiation time. Reaction; adding a certain amount of polymer PMMA to the monomer system, the polymerization rate is accelerated as the amount of PMMA increases. ESR analysis showed that the irradiation of ultrasonic waves did produce free radicals in the polymerization system, and the concentration of free radicals varied with the irradiation conditions.

9 Prospects Because ultrasonic waves can produce cavitation and vigorous agitation in liquid systems, they have many unique applications in polymer synthesis. Throughout the application of ultrasound in the past 10 years, from the monitoring of polymer reaction process to the study of polymer reaction mechanism, the application of ultrasonic technology is almost in all fields of polymer science. Looking into the future, with the continuous improvement and improvement of the design and manufacture of high-power narrow-bandwidth ultrasonic generators and polymer research methods, the application and mechanism research of ultrasonic waves will become more and more extensive and in-depth. In addition, compared with the conventional initiation method, the ultrasonic-initiated radical polymerization does not use an initiator, and the introduction of no additional impurities in the reaction system can obtain a high-purity polymer material, which is bound to be special in certain purity of the polymer material. The required fields are widely used.