CN118064419A - Method for preparing pancreatin by removing viruses - Google Patents
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- CN118064419A CN118064419A CN202410325498.2A CN202410325498A CN118064419A CN 118064419 A CN118064419 A CN 118064419A CN 202410325498 A CN202410325498 A CN 202410325498A CN 118064419 A CN118064419 A CN 118064419A
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- 210000000496 pancreas Anatomy 0.000 claims abstract description 42
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- 244000005700 microbiome Species 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000000415 inactivating effect Effects 0.000 abstract description 3
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- 102000038379 digestive enzymes Human genes 0.000 description 4
- 108091007734 digestive enzymes Proteins 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- 238000001042 affinity chromatography Methods 0.000 description 2
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- 206010033649 Pancreatitis chronic Diseases 0.000 description 1
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- 238000001493 electron microscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002641 enzyme replacement therapy Methods 0.000 description 1
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- 235000019421 lipase Nutrition 0.000 description 1
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- 230000001717 pathogenic effect Effects 0.000 description 1
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- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/94—Pancreatin
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
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Abstract
The invention relates to the technical field of pancreatin preparation, and in particular discloses a method for preparing pancreatin by removing viruses, which comprises the following steps: step one: soaking the pig pancreas in a peroxyacetic acid solution and then washing; step two: mincing and pulping the washed pig pancreas, and adding a calcium chloride solution for autolysis to obtain slurry; step three: sequentially activating and quenching the slurry obtained in the step two; step four: carrying out solid-liquid separation on the quenched slurry to obtain filtrate; step five: adding an organic solvent effective for the lipid viruses into the filtrate obtained in the step four to precipitate pancreatin; step six: washing and separating wet precipitate obtained in the fifth step; step seven: the precipitate is obtained by dehydration and dried at high temperature. The present invention is capable of reducing or inactivating viruses and microorganisms during the production of pancreatin without compromising purity, composition or potency as measured by enzyme activity or ratio of enzyme activities.
Description
Technical Field
The invention relates to the technical field of pancreatin preparation, in particular to a method for preparing pancreatin by removing viruses.
Background
Pancreatin is produced from porcine pancreatic tissue of animals found to be suitable for human consumption after veterinary examination. Pancreatin is a mixture of digestive enzymes, mainly amylase, protease and lipase extracted from porcine pancreas. Pancreatin has long been used as a pharmaceutical formulation for enzyme replacement therapy because of its important therapeutic properties and high safety. Various pancreatin is commercially available as a digestive enzyme supplement to aid digestion and enhance nutrient absorption. Clinical pancreatin replacement therapy is the primary means for treating pancreatic exocrine insufficiency associated with cystic fibrosis, chronic pancreatitis, post-gastrointestinal bypass surgery (post-gastrointestinal bypass surgery), post-pancreatectomy, and the like. A common feature of all biological products of any animal or human raw material is the risk of viral contamination. Biological contamination may come from raw materials or foreign matter introduced during production.
Viruses are small infectious agents that replicate only within the living cells of other organisms. Viruses consist of nucleic acids (RNA or DNA) surrounded by a protein shell and, in some cases, a lipid layer. Viruses that produce only a protein shell are often referred to as non-enveloped viruses, and viruses that contain both protein and lipid components in the shell are often referred to as enveloped viruses. They range in size from about 15nm to about 450nm and cannot be seen with an optical microscope. The shape and structure of viruses have been studied by electron microscopy, NMR spectroscopy and X-ray spectroscopy.
Viruses can infect all types of life forms, from animals and plants to bacteria and archaea. Viruses depend on the host because they cannot replicate independently. Thus, they are found in almost all organisms in the world. Known viruses are rarely pathogenic to humans because of their high host specificity. Improved attempts have met with limited success because most conditions that remove or inactivate viruses also result in an inactivated product (disrupted enzymatic activity). Regulatory and safety issues require viral clearance (viral removal or inactivation) in biopharmaceuticals such as pancreatin Active Pharmaceutical Ingredients (APIs).
Current measures of biological virus inactivation/removal processes include inactivation techniques including dry heat treatment inactivation, pasteurization, organic solvent/surfactant (S/D) methods, and low pH incubation, and removal techniques. Wherein, the dry heat treatment inactivation method is used as a non-specific virus inactivation method, can inactivate lipid-coated and partially non-lipid-coated viruses, and can treat coagulation factors and the like. It is desirable that the pasteurization process effectively inactivate both lipid-enveloped and non-enveloped virus-treated blood products. The organic solvent/surfactant (S/D) method can destroy lipid envelope, is effective for lipid-enveloped viruses, and is ineffective for non-lipid-enveloped viruses. Low pH incubation is effective against lipid-enveloped viruses and weak against non-lipid-enveloped viruses. The removal technique includes membrane filtration techniques such as chromatography (chromatographic) and affinity chromatography. Wherein the chromatography (chromatograph) includes ion exchange chromatography, hydrophobic chromatography and reverse chromatography. The membrane filtration technology such as affinity chromatography is to remove virus by nano membrane physical filtration.
However, to date, no reliable method has been developed to remove or inactivate all viral contaminants in pancreatin samples. This is due to the fact that the active enzymes in pancreatin are incompatible with many known inactivation conditions including heating, low pH, oxidation and ionizing radiation.
Disclosure of Invention
The object of the present invention is to provide a method for removing viruses for preparing pancreatin, which can reduce or inactivate viruses and microorganisms during the production of pancreatin without impairing the purity, composition or potency as measured by the enzyme activity or the ratio of the enzyme activities.
The invention is realized by the following technical scheme:
a method for removing viruses to produce pancreatin, comprising the steps of:
step one: soaking the pig pancreas in a peroxyacetic acid solution and then washing;
Step two: mincing and pulping the washed pig pancreas, and adding a calcium chloride solution for autolysis to obtain slurry;
Step three: sequentially activating and quenching the slurry obtained in the step two;
step four: carrying out solid-liquid separation on the quenched slurry to obtain filtrate;
step five: adding an organic solvent effective for the lipid viruses into the filtrate obtained in the step four to precipitate pancreatin;
Step six: washing and separating wet precipitate obtained in the fifth step;
step seven: the precipitate is obtained by dehydration and dried at high temperature.
Wherein, the peroxyacetic acid is the peroxide of acetic acid, is a stronger disinfection than hydrogen peroxide, and has good rapid sterilization activity on mycobacteria, fungi, viruses, bacterial spores and the like.
The organic solvent effective for the lipid virus may be isopropanol, which is not active for the non-lipid enterovirus but is effective for the lipid virus.
The high temperature drying is aimed at high temperature disinfection, and the principle is that virus proteins are denatured at high temperature for a long time so as to inhibit replication of virus genetic material, and finally the virus loses infectivity.
The invention firstly soaks pig pancreas with oxyacetic acid solution, can realize the quick sterilization and disinfection of pig pancreas, so as to reduce viruses and microorganisms, then adds organic solvent which is effective for lipid viruses into slurry, carries out inactivation treatment for lipid viruses, has no activity for non-lipid enteroviruses, finally carries out high-temperature sterilization on pancreatin precipitation to ensure that viruses lose infectivity, namely the combination of peracetic acid and the organic solvent which is effective for lipid viruses and high-temperature sterilization is used for preparing pancreatin, which not only can reduce or inactivate viruses and microorganisms in the process of producing pancreatin, but also does not damage the purity, the composition or the titer which are measured by enzyme activity or the enzyme activity proportion, namely does not influence the activity of enzymes contained in biological extracts and does not change the composition of enzymes contained in the biological extracts.
Further, in the first step, the concentration of the peracetic acid solution is 10000-40000ppm.
Further, in the first step, the soaking time is more than or equal to 30 minutes.
Further, in the second step, the mass fraction of calcium chloride in the calcium chloride solution is 1-2%.
Further, in the second step, the autolysis time is 10 hours or more.
Further, in the third step, the specific process of activation is as follows:
activating in water bath at 20-25deg.C for 2-3 hr.
Further, in the third step, quenching is performed by adding an organic solvent to the activated ground pulp.
Further, in the fifth step, the organic solvent is isopropyl alcohol.
Further, the temperature of the high-temperature drying in the fifth step is 80-100 ℃.
Further, the method further comprises the following steps:
Step eight: and D, crushing the precipitate obtained in the step seven to obtain pancreatin after virus removal.
Compared with the prior art, the invention has the following advantages and beneficial effects:
The present invention provides methods for reducing or inactivating microbial and viral contamination of a biological extract (peracetic acid + isopropyl alcohol + high temperature sterilization) without affecting the activity of enzymes contained in the biological extract and without altering the composition of enzymes contained in the biological extract. Furthermore, the process does not produce any toxic compounds or residues in the final product.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be described in further detail with reference to the following examples, which are illustrative embodiments of the present invention and the description thereof are intended to be illustrative of the present invention and not limiting of the present invention, and the examples described below are some, but not all, examples of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: no such specific details are necessary to practice the invention. In other instances, well-known structures, materials, or methods have not been described in detail in order to avoid obscuring the present invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
Inactivation of pancreatin has been studied more, for example:
Tijsensen et al (US 2014/0017223 A1) disclose a method of preparing a pancreatin (PEP) comprising reacting beta-propiolactone (BPL) with a formulation comprising one or more pancreatins for a time sufficient to reduce viral infectivity in the formulation.
Et al (U.S. patent 2011/0268844A 1) disclose treating pancreatin with high pressure and/or screening filtration followed by treatment with 4000, 5000 or 6000 bar high pressure for 5 minutes at 15 ℃. According to/>This applies to all viral forms, such as DNA and RNA viruses, enveloped and non-enveloped viruses and bacteria and fungi, and comprises at least 50% biological activity. He further discloses the unpredictability of whether inactivation of certain viruses using high pressure treatment was successful. Because of the compressibility of the samples, different process conditions must be selected depending on whether the sample is a liquid or a solid.
Kurfurst et al (US 2009/023344 A1) disclose a method for reducing viral and microbial contamination of a sample by treating a sample having 0.5% or less by weight residual moisture, heat treating a pancreatin at a temperature of 84 ℃ for 48 hours or 30 hours (preferably 80 ℃ and below), wherein the activity of the resulting pancreatin is at least 50% biological activity. The disclosed pancreatin viral infectivity reduced the log10 reduction factor by greater than 1log 10.
Mann (US 2009/6749861) discloses a method for sterilizing digestive enzyme preparations to reduce the level of active biological contaminants such as viruses, bacteria, yeasts, moulds and fungi. Treatment of the composition comprising digestive enzymes comprises stabilizing the composition by (a) reducing the temperature, (b) reducing the solvent, or (c) adding a stabilizer to the composition, followed by irradiation of the composition.
Becher et al (US 2009/013063 A1) disclose a method of isolating infectious viral load from a pancreatin sample, quantitatively determining viral load in a pancreatin sample by using centrifugation and ultracentrifugation. The method is limited to liquid samples suitable for centrifugation.
Braeuniger et al (int.J.hyg.environ.health (2000) 203:71-75) disclose the use of heat inactivated Niu Xixiao virus (BPV). BPV has been demonstrated to be deactivated by exposure to heat and residual moisture. Braeuniger et al, however, do not disclose any effect of heating on changes in enzyme activity and composition.
Lewis (U.S. 1971/3,956,483) discloses methods for pancreatin processing and bacterial reduction while maintaining amylolytic, proteolytic and lipolytic activity. The method comprises heating the pancreatin to a sufficiently high temperature between 49-82 ℃. However, lewis does not provide a method of inactivating or reducing the number of viruses.
However, since the active enzymes in pancreatin are incompatible with many known inactivation conditions (including heat, low pH, oxidation and ionizing radiation), no reliable method has been developed to date to remove or inactivate all viral contaminants in pancreatin samples. A particular challenge is to inactivate or reduce viruses from a biological extract matrix in which the active substance is a mixture of enzymes without disrupting or altering the enzymatic activity or proportion of the protein in the process. There is a need for a method in which the viral and bacterial content of a solid-containing biological extract is reduced or minimized to the greatest extent possible and consistent with maintaining the purity, composition and potency of the desired pharmaceutical active ingredient. The process must be equally applicable to solids and suspensions.
In order to solve the above problems, the present invention provides a method for preparing pancreatin by removing viruses, comprising the steps of:
Step one: soaking the pig pancreas in a peroxyacetic acid solution and then washing; the concentration of the peracetic acid solution is 10000-40000ppm; the soaking time is more than or equal to 30min; the pig pancreas can be frozen pig pancreas;
Step two: mincing and pulping the washed pig pancreas, and adding a calcium chloride solution for autolysis to obtain slurry; the mass fraction of calcium chloride in the calcium chloride solution is 1-2%; the autolysis time is more than or equal to 10 hours;
step three: sequentially activating and quenching the slurry obtained in the step two; the specific process of activation is as follows: activating in water bath at 20-25deg.C for 2-3 hr; quenching is to add an organic solvent into the activated ground pulp for activation quenching, wherein the organic solvent is isopropanol;
Step four: carrying out solid-liquid separation on the quenched slurry to obtain filtrate; the solid-liquid separation mode is filtration and the like;
Step five: adding an organic solvent effective for the lipid viruses into the filtrate obtained in the step four to precipitate pancreatin; the organic solvent is isopropanol;
Step six: washing and separating wet precipitate obtained in the fifth step;
step seven: dehydrating to obtain a precipitate and drying the precipitate at a high temperature; the high-temperature drying temperature is 80-100 ℃;
Step eight: and D, crushing the precipitate obtained in the step seven to obtain pancreatin after virus removal.
The combination of peracetic acid + an organic solvent effective for lipid viruses + high temperature sterilization according to the present invention for preparing pancreatin not only reduces or inactivates viruses and microorganisms during the production of pancreatin without compromising the purity, composition or potency as measured by the enzyme activity or the ratio of enzyme activities, i.e. without affecting the activity of the enzymes contained in the biological extract and without changing the composition of the enzymes contained in the biological extract.
Example 1:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: 1kg of frozen porcine pancreas was immersed in 10000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 80 ℃ for 16 hours;
step eight: pulverizing to obtain pancreatin.
Example 2: the concentration of peracetic acid in step one of example 1 was set to 20000ppm, the other steps being unchanged.
Example 3: the peracetic acid concentration in step one of example 1 was set to 30000ppm, the other steps being unchanged.
Example 4: the peracetic acid concentration in step one of example 1 was set to 40000ppm, the other steps being unchanged.
Comparative example 1:
this comparative example is based on example 1, and differs from example 1 in that: in step seven, the filter cake is dried for 16 hours at 45 ℃, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: 1kg of frozen porcine pancreas was immersed in 10000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 45 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 2:
this comparative example is based on example 1, and differs from example 1 in that: in step seven, the filter cake is dried for 16 hours at 65 ℃, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: 1kg of frozen porcine pancreas was immersed in 10000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 65 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 3:
This comparative example is based on example 1, and differs from example 1 in that: frozen porcine pancreas was not soaked with peracetic acid solution, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
step two: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step three: filtering insoluble substances by using a screen to obtain filtrate;
Step four: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step five: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step six: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 80 ℃ for 16 hours;
Step seven: crushing to obtain the pancreatin with virus removed.
Comparative example 4:
this comparative example is based on example 2, and differs from example 2 in that: in step seven, the filter cake is dried for 16 hours at 45 ℃, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: 1kg of frozen porcine pancreas was immersed in 20000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 45 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 5:
this comparative example is based on example 2, and differs from example 2 in that: in step seven, the filter cake is dried for 16 hours at 65 ℃, specifically:
Step one: 1kg of frozen porcine pancreas was immersed in 20000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 65 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 6:
This comparative example is based on example 3, and differs from example 3 in that: in step seven, the filter cake is dried for 16 hours at 45 ℃, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
step one: 1kg of frozen porcine pancreas was immersed in 30000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 45 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 7:
this comparative example is based on example 3, and differs from example 3 in that: in step seven, the filter cake is dried for 16 hours at 65 ℃, specifically:
step one: 1kg of frozen porcine pancreas was immersed in 30000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 65 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 8:
this comparative example is based on example 4, and differs from example 4 in that: in step seven, the filter cake is dried for 16 hours at 45 ℃, specifically:
a method for removing viruses to produce pancreatin, comprising the steps of:
Step one: 1kg of frozen porcine pancreas was immersed in 40000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 45 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Comparative example 9:
This comparative example is based on example 4, and differs from example 4 in that: in step seven, the filter cake is dried for 16 hours at 65 ℃, specifically:
Step one: 1kg of frozen porcine pancreas was immersed in 40000ppm peracetic acid solution. Washing the pig pancreas with purified water after soaking for 30 min;
step two: mincing pancreas, pulping, adding 1% calcium chloride solution, stirring, and autolyzing for 10 hr;
Step three: activating in water bath at 20 ℃ for 2 hours, and then adding isopropanol serving as an organic solvent for activating and quenching;
step four: filtering insoluble substances by using a screen to obtain filtrate;
step five: adding an organic solvent isopropanol into the filtrate to precipitate pancreatin, stirring for half an hour, and standing for half an hour to remove the upper liquid;
Step six: continuously adding an organic solvent isopropanol into the wet precipitate in the fifth step for washing for half an hour, standing for solid-liquid separation, and removing upper liquid;
Step seven: adding an organic solvent isopropanol into the wet precipitate in the step six to dehydrate, press-filtering after dehydration, and drying a filter cake at 65 ℃ for 16 hours;
step eight: crushing to obtain the pancreatin with virus removed.
Examples 1-4, comparative examples 1-9, pancreatin trisaccharide content, microbiological and viral detection results are shown in tables 1, 2:
TABLE 1
TABLE 2
From the results in tables 1 and 2, it can be seen that:
The combination of peracetic acid, isopropanol and high temperature sterilization of the present invention not only reduces or inactivates viruses and microorganisms during the production of pancreatin, but also does not compromise the purity, composition or potency as measured by enzyme activity or enzyme activity ratio.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A method for preparing pancreatin by removing viruses, comprising the steps of:
step one: soaking the pig pancreas in a peroxyacetic acid solution and then washing;
Step two: mincing and pulping the washed pig pancreas, and adding a calcium chloride solution for autolysis to obtain slurry;
Step three: sequentially activating and quenching the slurry obtained in the step two;
step four: carrying out solid-liquid separation on the quenched slurry to obtain filtrate;
step five: adding an organic solvent effective for the lipid viruses into the filtrate obtained in the step four to precipitate pancreatin;
Step six: washing and separating wet precipitate obtained in the fifth step;
step seven: the precipitate is obtained by dehydration and dried at high temperature.
2. The method for preparing pancreatin by removing viruses according to claim 1, wherein in the first step, the concentration of the peracetic acid solution is 10000 to 40000ppm.
3. The method for preparing pancreatin by removing viruses according to claim 1, wherein in the first step, the soaking time is 30min or longer.
4. The method for preparing pancreatin by removing viruses according to claim 1, wherein in the second step, the mass fraction of calcium chloride in the calcium chloride solution is 1-2%.
5. The method for removing viruses from pancreatic enzymes produced by said method according to claim 1, wherein in said second step, said autolysis is carried out for 10 hours or longer.
6. The method for preparing pancreatin by removing viruses according to claim 1, wherein in the third step, the specific process of activation is:
activating in water bath at 20-25deg.C for 2-3 hr.
7. The method for removing viruses from pancreatic enzymes as claimed in claim 1, wherein in step three, quenching is performed by adding an organic solvent to the activated pulp.
8. The method for preparing pancreatin by removing viruses according to claim 1, wherein in the fifth step, the organic solvent is isopropyl alcohol.
9. The method for preparing pancreatin by removing viruses according to claim 1, wherein the high temperature drying in the fifth step is 80-100 ℃.
10. The method for removing viruses for preparing pancreatin according to any one of claims 1 to 9, further comprising:
Step eight: and D, crushing the precipitate obtained in the step seven to obtain pancreatin after virus removal.
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