CN111072765A - Solid-liquid separation method of ethanol/protein suspension in pregnant mare serum gonadotropin crude product extraction process - Google Patents
Solid-liquid separation method of ethanol/protein suspension in pregnant mare serum gonadotropin crude product extraction process Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 249
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 105
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 105
- 239000007788 liquid Substances 0.000 title claims abstract description 86
- 239000000725 suspension Substances 0.000 title claims abstract description 53
- 238000000926 separation method Methods 0.000 title claims abstract description 43
- 210000002966 serum Anatomy 0.000 title claims abstract description 30
- 239000012043 crude product Substances 0.000 title claims abstract description 25
- 108010086677 Gonadotropins Proteins 0.000 title claims abstract description 13
- 102000006771 Gonadotropins Human genes 0.000 title claims abstract description 13
- 238000000605 extraction Methods 0.000 title claims abstract description 13
- 239000002622 gonadotropin Substances 0.000 title claims abstract description 13
- 239000011087 paperboard Substances 0.000 claims abstract description 43
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 40
- 238000001914 filtration Methods 0.000 claims abstract description 29
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000011780 sodium chloride Substances 0.000 claims abstract description 20
- 238000011068 loading method Methods 0.000 claims abstract description 11
- 238000005086 pumping Methods 0.000 claims abstract description 10
- 238000002791 soaking Methods 0.000 claims abstract description 9
- 239000000123 paper Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 27
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 description 71
- 230000008569 process Effects 0.000 description 20
- 230000000694 effects Effects 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 9
- 238000005265 energy consumption Methods 0.000 description 8
- 210000002381 plasma Anatomy 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000004062 sedimentation Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 3
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000012173 estrus Effects 0.000 description 3
- 229940028334 follicle stimulating hormone Drugs 0.000 description 3
- 238000000703 high-speed centrifugation Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229940040129 luteinizing hormone Drugs 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010011631 Equine Gonadotropins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010042573 Superovulation Diseases 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 230000000920 spermatogeneic effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- Gastroenterology & Hepatology (AREA)
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- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Peptides Or Proteins (AREA)
Abstract
A solid-liquid separation method of ethanol/protein suspension in the extraction process of pregnant mare serum gonadotropin crude products comprises the following steps: s1, taking pregnant mare serum, adding a metaphosphoric acid solution to adjust the pH to 3.0-3.8, and carrying out solid-liquid separation on the obtained metaphosphoric acid/protein suspension to obtain a protein clear liquid A; s2, concentrating the protein clear liquid A, adding ethanol to enable the ethanol concentration of the solution system to be 45-60% (v/v) to obtain ethanol/protein suspension, pumping the ethanol/protein suspension into a plate-and-frame filter press, loading the pretreated filter paper board into the plate-and-frame filter press, and performing solid-liquid separation to obtain a protein clear liquid B. The pretreatment of the filter paper sheet in step S2 includes the steps of: soaking the filtering paperboard in 45-60% ethanol solution of 0.5-2% (w/v) sodium chloride for 50-100min, draining, and placing into a plate-and-frame filter press.
Description
Technical Field
The invention belongs to the technical field of pregnant mare serum gonadotropin crude product production, and particularly relates to a solid-liquid separation method of ethanol/protein suspension in a pregnant mare serum gonadotropin crude product extraction process.
Background
Pregnant mare serum gonadotropin (PMSG for short) is glycoprotein gonadotropin secreted by chorioallantoic cells of pregnant mare. PMSG has double activities similar to Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), but mainly takes the effect of FSH as the main part and has obvious follicle development promoting effect; meanwhile, the compound has certain functions of ovulation promotion and corpus luteum formation, and has the functions of promoting the development of sperm tubes and sexual cell differentiation for male animals. In practice, the PMSG can regulate and control the estrus of the female animals at the same time, realize controllable synchronous batch production, shorten the mating time, reduce the infertility probability and improve the propagation efficiency. At present, PMSG has been widely used for inducing estrus, synchronous estrus, superovulation or increasing ovulation rate of animals; meanwhile, the medicine also has good curative effect on ovarian hypoplasia or male spermatogenic decline and the like.
The raw material for producing PMSG is mare serum collected from 40-120 days of pregnancy, and the classical extraction method is as follows: first with metaphosphoric acid (HPO)3) Performing metaphosphate-protein reaction with blood serum to precipitate impure protein in blood plasma, and performing solid-liquid separation to obtain clear liquid; then, ethanol and clear liquid are used for carrying out 'ethanol/protein' classification to ensure that partial impure protein in the blood plasma is precipitated, then, the clear liquid is obtained by carrying out solid-liquid separation, and finally, the protein is precipitated by the ethanol to obtain a PMSG crude product.
In the small-scale PMSG production process, the solid-liquid separation method adopts a sedimentation type centrifugal separation method. The solid content of the suspension formed by the ethanol/protein classification is up to more than 10 percent. In practice, if a tubular centrifuge is used for separation, due to the fact that the drum size is small and the solid content is limited, materials with high solid content need to be frequently unloaded and cleaned in the production operation process, and the working efficiency is low; in addition, due to the particularity of particles in suspension formed by plasma protein, the disk centrifuge cannot solve the solid-liquid separation problem of the suspension formed by ethanol/protein classification, and the obtained clear liquid is still very turbid. With the development of intensive and large-scale high-efficiency animal husbandry, the demand of the market for PMSG is greatly increased, the daily processing capacity of pregnant horse serum needs to be increased from 100kg to 2000kg, the traditional sedimentation type centrifugation method cannot meet the solid-liquid separation in plasma extraction, and the large-scale solid-liquid separation method and equipment are inevitably sought.
Disclosure of Invention
Aiming at the problems in the preparation process of PMSG in the prior art, the invention provides a method for carrying out large-scale solid-liquid separation of feed liquid of ethanol/protein suspension by using a plate-and-frame filter press instead of a sedimentation type centrifuge, which solves the bottleneck of carrying out solid-liquid separation of the ethanol/protein suspension in the process of producing PMSG from pregnant mare serum, greatly improves the working efficiency, reduces the energy consumption, saves the process time, and plays an active role in maintaining the bioactivity of PMSG.
The above object of the present invention is achieved by the following technical solutions:
a solid-liquid separation method of ethanol/protein suspension in the extraction process of pregnant mare serum gonadotropin crude products comprises the following steps:
s1, taking pregnant mare serum, adding a metaphosphoric acid solution to adjust the pH to 3.0-3.8, and carrying out solid-liquid separation on the obtained metaphosphoric acid/protein suspension to obtain a protein clear liquid A;
s2, concentrating the protein clear liquid A, adding ethanol to enable the ethanol concentration of the solution system to be 45-60% (v/v) to obtain ethanol/protein suspension, pumping the ethanol/protein suspension into a plate-and-frame filter press, loading the pretreated filter paper board into the plate-and-frame filter press, and performing solid-liquid separation to obtain a protein clear liquid B.
Preferably, the pretreatment of the filter paper sheet in step S2 includes the steps of: soaking the filter paper board in 45-60% ethanol solution of 0.5-2% (w/v) sodium chloride for 50-100 min.
Preferably, the concentration of the metaphosphoric acid solution in step S1 is 3 to 8% (m/v) by mass.
Preferably, the pH is adjusted to 3.5 by adding a metaphosphoric acid solution.
Preferably, the plate-and-frame filter press is loaded with the pretreated filter paper board, and then circularly cleaned with a 45-60% ethanol solution of 0.2-1% (w/v) sodium chloride for 20-40min, drained and then loaded into the plate-and-frame filter press.
Preferably, the filtering pressure of the plate-and-frame filter press is 0.05-0.12 MPa.
Preferably, the pore size of the filter paper board is 1-20 μm.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts the plate-and-frame filter press to replace the traditional decanter centrifuge, solves the bottleneck of large-scale solid-liquid separation of ethanol/protein suspension in the PMSG (permanent magnet synchronous generator) production process by pregnant mare serum, and has the advantages of low labor intensity, high working efficiency, low energy consumption and the like compared with a centrifugal method. And the filter material is pretreated to eliminate the charges carried in the filter paper plate, thereby reducing the adsorption of target Protein (PMSG), keeping the bioactivity of PMSG, and effectively improving the yield and activity of PMSG crude product.
Detailed Description
In the following, some embodiments are described for the inventive method for solid-liquid separation of ethanol/protein suspension during extraction of crude pregnant mare serum gonadotropins, however, these embodiments are exemplary and the present disclosure is not limited thereto.
In some embodiments of the present invention, a method for separating solid and liquid from ethanol/protein suspension during extraction of crude pregnant mare serum gonadotropin comprises the following steps:
s1, taking pregnant mare serum, adding a metaphosphoric acid solution to adjust the pH to 3.0-3.8, and carrying out solid-liquid separation on the obtained metaphosphoric acid/protein suspension to obtain a protein clear liquid A;
s2, concentrating the protein clear liquid A, adding ethanol to enable the ethanol concentration of the solution system to be 45-60% (v/v) to obtain ethanol/protein suspension, pumping the ethanol/protein suspension into a plate-and-frame filter press, loading the pretreated filter paper board into the plate-and-frame filter press, and performing solid-liquid separation to obtain a protein clear liquid B.
Pregnant horse serum contains a lot of proteins and is various, and PMSG has low concentration in pregnant horse serum, so that a large amount of impure proteins need to be removed firstly, and PMSG is concentrated and enriched. A large amount of polymers with high charges and large molecular weights exist in metaphosphoric acid solution, so that the charges on the surfaces of protein colloidal particles can be effectively neutralized, hydration membranes on the surfaces of the protein colloidal particles are damaged, the protein colloidal particles are coagulated and precipitated, the pH value is adjusted to be between 3.0 and 3.8, most of hetero-proteins in pregnant mare plasma are precipitated, and protein clear liquid A is obtained after solid-liquid separation. After the protein clear liquid A is concentrated, ethanol is added, and the ethanol changes the dielectric constant to reduce the solubility of impurity proteins, so that precipitation is generated. The solid content of the ethanol/protein suspension after the protein clear liquid A and the ethanol react can reach more than 10 percent, and if the large-scale production of PMSG is involved, the working efficiency of processing the suspension by adopting a decanter centrifuge can be greatly reduced, and the cost is greatly increased. The invention adopts the plate-and-frame filter press to process the ethanol/protein suspension, and utilizes the characteristics of large filtering area, good filter material permeability and large filtering capacity and solid content of the plate-and-frame filter press to replace a common sedimentation centrifuge to carry out solid-liquid separation of large-scale feed liquid. And the filter paper board in the plate-and-frame filter press needs to be treated in advance before use: soaking commercially available filter paper board in 45-60% (v/v) ethanol solution of 0.5-2% (w/v) sodium chloride for 50-100 min. The filtering paper board is soaked in the ethanol solution of sodium chloride, the high-conductivity positive and negative charges of the sodium chloride are utilized to eliminate the charges carried in the filtering paper board, and the filtering environment is close to a feed liquid system, so that the adsorption of target Protein (PMSG) is reduced, and the bioactivity of the PMSG is kept higher.
The invention adopts a plate-and-frame filter press containing special pretreatment filter paper boards to treat suspension after ethanol/protein reaction, and overcomes the following defects of a centrifugal separation method: firstly, the shearing force influences the protein, and the shearing force generated by high-speed centrifugation can cause the change of the three-dimensional structure of the protein; secondly, the influence of heat production on the protein, the machine can generate heat in the high-speed centrifugation process, so that the protein is heated and denatured; in the actual production process, in order to reduce heat generated by high-speed centrifugation, the centrifuge needs to be matched with a high-performance compressor unit to provide a cold environment so as to reduce heat generated by high-speed rotation of a rotor in a cavity, the energy consumption generated by the compressor is added with the high power consumption of the centrifuge, and the energy consumption generated in the centrifugation process is very large; and thirdly, in the centrifugal operation, because the capacity of a single centrifugal machine is limited, a plurality of centrifugal machines are required to be started simultaneously, the labor intensity of production personnel is high, the safety risk is high, and in the operation process, the dripping, leaking and sprinkling of materials cannot be avoided. Therefore, the invention adopts the plate-and-frame filter press to process the suspension, solves the bottleneck of solid-liquid separation in the PMSG production process from pregnant mare plasma, greatly improves the working efficiency, reduces the energy consumption, saves the process time, plays an active role in maintaining the bioactivity of PMSG, and improves the yield of the bioactivity of PMSG crude products to a certain extent.
The filtering paper board is a deep filtering medium compounded by natural plant fibers, high-efficiency filter aid and cationic resin, and liquid impurities are removed by two mechanisms of mechanical interception and electrostatic adsorption. The present invention prefers filter paper sheets with a pore size of 1-20 μm. The paper board can be selected from HS series paper boards of Beijing Forster Ruite filtration technology limited or other commercially available filtration paper boards, and one or more of HS600, HS800, HS1000, HS1600, HS2000, HS4000, HS6000, HS7000, HS8000, HS9000 series paper boards or similar varieties are selected.
In other embodiments of the present invention, the concentration of the metaphosphoric acid solution in step S1 is 3 to 8% (m/v) by mass.
In other embodiments of the invention, a solution of metaphosphoric acid is added to adjust the pH to 3.5.
In other embodiments of the present invention, the plate and frame filter press operation comprises the steps of:
loading the pretreated filter paper board into a plate-and-frame filter press, and ensuring the sealing of a sealing ring in the loading process; maintaining the pressure of the plate-and-frame filter press at 1.5-2.0 MPa; starting the equipment, circularly cleaning the filter paperboard for 20-40min by using a 45-60% ethanol solution of 0.2-1% (w/v) sodium chloride, and carefully checking whether the pipeline has a liquid leakage phenomenon when the equipment runs; opening an inlet valve, starting a plate-and-frame filter press, pumping ethanol/protein suspension, and collecting the filtered protein clear liquid B into a storage tank, wherein the filtering pressure is not more than 0.12MPa, preferably 0.05-0.12MPa, in the running process of equipment; and when the filtering of the ethanol/protein suspension is finished, stopping the equipment, loosening the plate frame, and then removing the slag and cleaning the filter press.
The whole extraction process of pregnant mare serum gonadotropin crude products comprises the following steps:
s1, taking pregnant mare plasma, adding a metaphosphoric acid solution to adjust the pH value to 3.0-3.8, and carrying out solid-liquid separation on the obtained metaphosphoric acid/protein suspension to obtain a protein clear solution A;
s2, concentrating the protein clear liquid A, adding ethanol to enable the ethanol concentration of the solution system to be 45-60% (v/v) to obtain ethanol/protein suspension, pumping the ethanol/protein suspension into a plate-and-frame filter press, loading the pretreated filter paper board into the plate-and-frame filter press, and performing solid-liquid separation to obtain a protein clear liquid B. The filter board pretreatment comprises the following steps: soaking the filter paper board in 45-60% ethanol solution of 0.5-2% (w/v) sodium chloride for 50-100 min.
S3, adding ethanol into the protein clear liquid B to enable the ethanol concentration of the solution system to be 75-85% (v/v), standing for layering, siphoning to remove the supernatant, collecting the lower-layer precipitate, and drying in vacuum to obtain the pregnant mare serum gonadotropin crude product.
As for the solid-liquid separation technique in step S1, there is no limitation, and a decanter centrifuge or a plate and frame filter press may be used. And the standing and layering time in the step S3 is 10-24h, so that the upper layer and the lower layer are sufficiently separated, and the subsequent siphoning is facilitated.
Hereinafter, the technical solution of the present invention will be further described and illustrated by specific examples. Unless otherwise specified, the raw materials used in the following specific examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art.
The centrifuge used in the following specific examples was a DL-8M ultra-large capacity refrigerated centrifuge (shanghai luxiang instrument centrifuge, ltd); the plate-and-frame filter press is a Niro 600 paperboard filter press of Beijing Forster Ruite filtration technology GmbH; metaphosphoric acid is chemically pure and purchased from chemical reagents of national medicine group, Inc.; pregnant mare serum is obtained by processing plasma collected from pregnant mare for 40-120 days.
Example 1
The process for extracting the PMSG crude product comprises the following steps:
120Kg of pregnant mare serum is taken and 5% (m/v) HPO is dripped3Adjusting pH to 3.5, suspending for 5min to obtainPouring the metaphosphoric acid/protein suspension into a centrifuge, wherein the centrifugation speed is 3000rpm, the centrifugation time is 30min, and separating to obtain a protein clear liquid A; concentrating the protein clear liquid A to 30Kg with an ultrafiltration machine, then dropwise adding absolute ethyl alcohol to make the ethanol concentration of the system reach 50%, and carrying out centrifugal separation on the 50% ethanol/protein suspension at the centrifugal speed of 3000rpm for 10min to obtain protein clear liquid B55.3Kg; and (3) dropwise adding absolute ethyl alcohol into the protein clear liquid B to enable the concentration of the ethyl alcohol in the system to reach 80%, standing for 12h for layering, siphoning to remove the supernatant, collecting the lower-layer precipitate, and drying in vacuum to obtain 96.8g of PMSG crude product.
Example 2
The process for extracting the PMSG crude product comprises the following steps:
120Kg of pregnant mare serum is taken and 5% (m/v) HPO is dripped3Adjusting pH to 3.5, suspending for 5min, pouring the obtained metaphosphoric acid/protein suspension into a centrifuge, centrifuging at 3000rpm for 30min, and separating to obtain protein clear liquid A; concentrating the protein clear liquid A to 30Kg with an ultrafilter, and then dropwise adding anhydrous ethanol to make the ethanol concentration of the system reach 50% to obtain a 50% ethanol/protein suspension; selecting HS4000 filter paper board, soaking the filter paper board for 60min by 50% (v/v) ethanol solution containing 1% (w/v) sodium chloride; loading the pretreated filter paper board into a plate-and-frame filter press; performing pressure maintaining on a plate-and-frame filter press, wherein the pressure maintaining pressure is 2.0 MPa; opening the device, and circularly washing the pretreated filter paper board with 50% (v/v) ethanol of 0.5% (w/v) sodium chloride for 30 min; opening the inlet valve, starting the plate-and-frame filter press, pumping 50% ethanol/protein suspension, setting the filtration pressure to 0.10MPa in the running process of the equipment, and filtering to obtain 56.2Kg of protein clear liquid B. And (3) dropwise adding absolute ethyl alcohol into the protein clear liquid B to enable the concentration of the ethyl alcohol in the system to reach 80%, standing for 12h for layering, siphoning to remove the supernatant, collecting the lower-layer precipitate, and drying in vacuum to obtain 97.2g of PMSG crude product.
Example 3
The process for extracting the PMSG crude product comprises the following steps:
300Kg of pregnant mare serum is taken and 4% (m/v) HPO is dripped3Adjusting pH to 3.5, suspending for 6min, and pouring the obtained metaphosphoric acid/protein suspension intoIn a centrifugal machine, the centrifugal speed is 3000rpm, the centrifugal time is 30min, and protein clear liquid A is obtained through separation; concentrating the protein clear liquid A to 75Kg with an ultrafilter, and then dropwise adding anhydrous ethanol to make the ethanol concentration of the system reach 48% to obtain 48% ethanol/protein suspension; selecting HS6000 filtering paper board, soaking the filtering paper board in 48% (v/v) ethanol solution containing 1.2% (w/v) sodium chloride for 65 min; loading the pretreated filter paper board into a plate-and-frame filter press; performing pressure maintaining on a plate-and-frame filter press, wherein the pressure maintaining pressure is 2.0 MPa; opening the device, and circularly washing the pretreated filter paper board with 48% (v/v) ethanol of 0.6% (w/v) sodium chloride for 25 min; opening the inlet valve, starting the plate-and-frame filter press, pumping 48% ethanol/protein suspension, setting the filtration pressure to 0.11MPa in the running process of the equipment, and filtering to obtain protein clear liquid B137.2Kg. And (3) dropwise adding absolute ethyl alcohol into the protein clear liquid B to enable the concentration of the ethyl alcohol in the system to reach 78%, standing for 12h for layering, siphoning to remove the supernatant, collecting the lower-layer precipitate, and drying in vacuum to obtain 229.5g of PMSG crude product.
Example 4
The process for extracting the PMSG crude product comprises the following steps:
900Kg of pregnant mare serum is taken and 6% (m/v) HPO is dripped3Adjusting pH to 3.5, suspending for 6min, pouring the obtained metaphosphoric acid/protein suspension into a centrifuge, centrifuging at 3000rpm for 30min, and separating to obtain protein clear liquid A; concentrating the protein clear liquid A to 225Kg with an ultrafilter, and then dropwise adding anhydrous ethanol to make the ethanol concentration of the system reach 52% to obtain a 52% ethanol/protein suspension; selecting HS7000 filter paper board, soaking the filter paper board for 70min by using 52% (v/v) ethanol solution containing 0.9% (w/v) sodium chloride; loading the pretreated filter paper board into a plate-and-frame filter press; performing pressure maintaining on a plate-and-frame filter press, wherein the pressure maintaining pressure is 1.9 MPa; opening the device, and circularly washing the pretreated filter paper board with 52% (v/v) ethanol of 0.4% (w/v) sodium chloride for 35 min; opening the inlet valve, starting the plate-and-frame filter press, pumping 52% ethanol/protein suspension, setting the filtration pressure to 0.09MPa in the running process of the equipment, and filtering to obtain protein clear liquid B421.7 Kg. Adding anhydrous ethanol dropwise into the protein clear solution B to make the ethanol concentration of the system reach 76%, standing for 12h for layering, siphoning, and discardingSupernatant, collecting lower precipitate and vacuum drying to obtain crude PMSG 640.5 g.
Example 5
The process for extracting the PMSG crude product comprises the following steps:
1500Kg of pregnant mare serum is taken and 5% (m/v) HPO is dripped3Adjusting pH to 3.5, suspending for 6min, pouring the obtained metaphosphoric acid/protein suspension into a centrifuge, centrifuging at 3000rpm for 30min, and separating to obtain protein clear liquid A; concentrating the protein clear liquid A to 300Kg with an ultrafilter, and then dropwise adding anhydrous ethanol to make the ethanol concentration of the system reach 55%, to obtain 55% ethanol/protein suspension; selecting HS8000 filter paper board, soaking the filter paper board in 55% (v/v) ethanol solution containing 0.9% (w/v) sodium chloride for 65 min; loading the pretreated filter paper board into a plate-and-frame filter press; performing pressure maintaining on the plate-and-frame filter press, wherein the pressure maintaining pressure is 1.8 MPa; opening the device, and circularly washing the pretreated filter paper board with 55% (v/v) ethanol of 0.3% (w/v) sodium chloride for 40 min; opening the inlet valve, starting the plate-and-frame filter press, pumping 52% ethanol/protein suspension, setting the filtration pressure to 0.09MPa in the running process of the equipment, and filtering to obtain protein clear liquid B556.2 Kg. And (3) dropwise adding absolute ethyl alcohol into the protein clear liquid B to enable the concentration of the ethyl alcohol in the system to reach 77%, standing for 12h for layering, siphoning to remove the supernatant, collecting the lower-layer precipitate, and drying in vacuum to obtain 1020.2g of PMSG crude product.
Example 6
Example 6 differs from example 2 in that the HS4000 filter board of example 6 was not soaked with a 50% (v/v) ethanol solution containing 1% (w/v) sodium chloride and the filter board was not washed with 50% (v/v) ethanol containing 0.5% (w/v) sodium chloride after the plate and frame filter press was opened. Other process parameters are the same as those in example 2, and 91.5g of PMSG crude product is obtained.
Aiming at ethanol/protein suspension produced by ethanol-protein classification, in the production, each ton of serum is fed, if a plate-and-frame filter press is used, the filtering can be finished in about 80 minutes by one plate-and-frame filter press with the filtering area of 8 square, and the working capacity of 15 DL-8M ultra-large-capacity refrigerated centrifuges in 80 minutes is equivalent. The energy consumption of the present invention using a plate and frame filter press can be greatly reduced as compared to the energy consumption of table 1 below.
TABLE 1 energy consumption comparison of solid-liquid separation apparatus
| Plate frame filter press | Ultra-large capacity centrifuge | |
| Number of | 1 table | 15 tables |
| Electric power | 3 kilowatt/station | 7.5 kilowatt/station |
| Total power | 3 kilowatt | 112.5 kilowatts |
The activity data for the crude PMSG of inventive examples 1-6 are shown in Table 2. In table 2, the PMSG yield was calculated in the following manner:
yield% (% crude total activity/total plasma activity)% 100%
Table 2 bioactivity data for examples 1-6
As can be seen from the data in Table 2, the solid-liquid separation of the large-volume suspension can be processed with high efficiency and low consumption in examples 2-5 by using the plate-and-frame filter press for filtration, and the activity and yield of the obtained PMSG crude product are higher than those of the PMSG crude product (obtained in example 1) obtained by the centrifuge filtration. While the pretreatment of the filter board has a great effect on the crude extraction of PMSG, example 6 adopts the filter board without pretreatment, and the yield and activity of the extracted crude product are reduced although the efficiency is higher and the consumption is lower compared with the centrifugal method.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (7)
1. A solid-liquid separation method of ethanol/protein suspension in the extraction process of pregnant mare serum gonadotropin crude products is characterized by comprising the following steps:
s1, taking pregnant mare serum, adding a metaphosphoric acid solution to adjust the pH to 3.0-3.8, and carrying out solid-liquid separation on the obtained metaphosphoric acid/protein suspension to obtain a protein clear liquid A;
s2, concentrating the protein clear liquid A, adding ethanol to enable the ethanol concentration of the solution system to be 45-60% (v/v) to obtain ethanol/protein suspension, pumping the ethanol/protein suspension into a plate-and-frame filter press, loading the pretreated filter paper board into the plate-and-frame filter press, and performing solid-liquid separation to obtain a protein clear liquid B.
2. The solid-liquid separation method according to claim 1, wherein the pretreatment of the filter paper sheet in step S2 comprises the steps of: soaking the filter paper board in 45-60% ethanol solution of 0.5-2% (w/v) sodium chloride for 50-100 min.
3. The solid-liquid separation method according to claim 1, wherein the concentration by mass of the metaphosphoric acid solution in step S1 is 3 to 8% (m/v).
4. The solid-liquid separation method according to claim 1, wherein a metaphosphoric acid solution is added to adjust the pH to 3.5.
5. The solid-liquid separation method according to claim 1, wherein the plate and frame filter press is used for circularly cleaning the filter paperboard with a 45-60% ethanol solution of 0.2-1% (w/v) sodium chloride for 20-40min after being filled with the pretreated filter paperboard.
6. The solid-liquid separation method according to claim 1, wherein the filtration pressure of the plate-and-frame filter press is 0.05 to 0.12 MPa.
7. The method for solid-liquid separation of ethanol/protein suspension during extraction of crude pregnant mare serum gonadotropin as claimed in claim 1, wherein the pore size of the filter paper board is 1-20 μm.
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