CN105504005A - Method for purifying and preparing polypeptide - Google Patents
Method for purifying and preparing polypeptide Download PDFInfo
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- CN105504005A CN105504005A CN201610024812.9A CN201610024812A CN105504005A CN 105504005 A CN105504005 A CN 105504005A CN 201610024812 A CN201610024812 A CN 201610024812A CN 105504005 A CN105504005 A CN 105504005A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention relates to a method for purifying and preparing polypeptide. The technical problems that in an existing polypeptide purification and preparation method, the cost is high, and volatile organic compounds are discharged, and salt transfer is difficult are solved. According to the technical scheme, separation and purification, concentration, salt transfer and re-concentration are conducted on the polypeptide, produced high-concentration acetonitrile waste fluid is recycled by a system after distillation recovery is conducted, and the high-concentration acetonitrile waste fluid is synchronously applied to a polypeptide separation and purification method. The method for purifying and preparing the polypeptide greatly reduces the cost of polypeptide purification and preparation, and discharging of the acetonitrile waste fluid is reduced.
Description
Technical field
The present invention relates to a kind of peptide purification preparation method, be specially a kind of reversed-phased high performace liquid chromatographic that utilizes in conjunction with the recycle and reuse of acetonitrile, polypeptide carried out to the method for purification.
Background technology
Along with the development of modern biotechnology, occurred various polypeptide drug, polypeptide has huge using value in clinical medicine.Since invention solid phase method chemically synthesized polypeptide, the chemosynthesis about various active polypeptide has had and has developed on a large scale very much, but product component more complicated, be generally the polypeptide mixture of several structural similitudies based on target polypeptides.Therefore, be an important research topic to the separation and purification of this mixture always, the optimization study of its separation and purification is just seemed especially important.In addition, provide highly purified polypeptide sample, the research for its physico-chemical property, biological activity and medical function thereof has important effect.Draw from modern separation science theory, chromatogram and electrophoresis are two kinds of methods of separating effect the best known today.Electrophoresis only can for separating of and the separation and purification of polypeptide can not be used for.And high performance liquid chromatography was through the fast development of more than 30 years, in chromatographic theory research, instrument research level, analyzes in practical application, quality approach, industrialized production etc., achieve significant progress.Wherein RPLC is because of its good selectivity, and have good separating effect, resolving power is high, the rate of recovery is high feature, in the separation and purification of polypeptide, enjoy favor, its range of application constantly expands, and has bright prospects.
When utilizing RPLC to carry out separation and purification to polypeptide, reverse chromatograms column packing is often take silica gel as matrix, and surface bond has the Bonded Phase of the relatively weak functional group of polarity, usually uses octadecylsilane chemically bonded silica.The moving phase polarity that reverse chromatograms uses is comparatively strong, usually with water, damping fluid for aqueous phase, methyl alcohol, acetonitrile, Virahol etc. be organic phase, and aqueous phase and organic phase form mix reagent and carry out binary gradient wash-out.It is that the component that polarity is stronger is rinsed out at first that sample flows out the order of chromatographic column, and the weak component of polarity can have stronger reservation on a column.
In recent years, acetonitrile becomes the conventional organic modifiers of RPLC and solvent, particularly in the application of peptide separation purification art widely.When RPLC carries out separation and purification to polypeptide, in the moving phase (acetonitrile and aqueous phase) used, the ratio shared by acetonitrile is 30%-70%(V/V), acetonitriles a large amount of is not like this recycled after using, unfortunately, not only can cause the waste of resource, also pollute the environment.
Along with haze sky is frequently existing, people more and more pay close attention to topsoil.Volatile organic matter, as one of four large population's pollutents, is the front extract forming haze and PM2.5, and be main " arch-criminal " that cause environmental pollution, damage HUMAN HEALTH, therefore, country is imperative to the improvement of volatile organic matter.Volatile organic contaminant is as far back as the eighties in last century, and the developed countries such as the U.S. just start the reduction of discharging problem paying attention to volatile organic contaminant.In May, 2010, State Council issued " instruction about advancing the work of topsoil groupcontrol to improve region Air quality ", first volatile organic matter is classified as together with sulfurous gas, oxynitride, particulate matter etc. the priority pollutant of topsoil groupcontrol, proposes to strengthen volatile organic matter prevention and cure of pollution." 12 " period, volatile organic matter Pollution abatement and prevention and control policy then enter the intensive issue phase, and environmental protection " 12 " planning proposition will carry out volatile organic matter and poisonous fume monitoring, improves key industry pollutant emission standard.At present, volatile organic matter is administered and has been entered China's air contaminant treatment schedule.The first half of the year in 2015, Chinese Ministry of Environmental Protection has printed and distributed " volatile organic matter Pollution Charges Site way ", on December 17th, 2015, " Shanghai City volatile organic matter Pollution Charges Site implementing method " is issued in Shanghai City, determines that pilot collects volatile organic matter pollution charge.Pilot divides three phases to implement, and progressively improves expenses standard.For this situation, many enterprises, in order to reduce organism blowdown, by the volatile organic matter produced, as acetonitrile waste liquid etc., after reclaiming, refine it, then again supply manufacturing enterprise and use.
Traditional acetonitrile reclaims variable-pressure rectification method, constant boiling rectification method, salting-out method, dewatering agent evaporation, this several method all uses thick steaming and rectifying two steps, special rectificating method is used to carry out separating treatment, there is treatment process complexity, equipment cost is high and separation efficiency is low, the problems such as energy consumption is high.
Traditional salt method that turns utilizes inside glass column or polyethylene post to load zwitterion resin, and what utilize the principle of ion-exchange to realize polypeptide products turns salt process.Utilize traditional cation and anion exchange principle in addition, salt is turned to polypeptide products, step 1) is after the PH of adjustment polypeptide products liquid, loading hanging column 2) rinse sample 3) utilize soda acid adjust washing fluid PH after polypeptide products is rinsed, in the process, each link will be got a little effluent liquid and be carried out HPLC detection, and confirmation can carry out further work after rinsing well.In this approach, workflow is many in institute, and time loss is large, and can produce a large amount of waste liquids, the more important thing is that the last polypeptide products qualifying liquid volume collected is large, is unfavorable for concentrated and freeze-drying work below.Need in addition to purchase plant and instrument regulating YIN and YANG ion filler, also need other experiment arrangement place and personnel, consume a large amount of manpower and materials.
Is no lack of the document about RPLC application both at home and abroad, also many documents and materials reclaiming about acetonitrile, refine are had, but the rare reversed-phased high performace liquid chromatographic that utilizes, in conjunction with the recycle and reuse of acetonitrile, carries out the method pertinent literature of separation and purification to polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of reversed-phased high performace liquid chromatographic that utilizes in conjunction with the recycle and reuse of acetonitrile, polypeptide carried out to the method for purification, mainly solve existing peptide purification preparation method and have that cost is high, Volatile organic emissions, turn the technical problems such as salt difficulty.Its technical thought is the while of utilizing reversed-phased high performace liquid chromatographic to carry out purification to polypeptide, and by the high density acetonitrile waste liquid produced, through reclaiming, again utilized by system, synchronous applications is in the method for peptide separation purifying.This method has that technique is simple, easy to operate, low cost and other advantages.More meaningfully this method greatly reduces cost prepared by peptide purification, and decreases the discharge of acetonitrile waste liquid, has not only saved money but also environmental protection.Adopting said method carries out purification to polypeptide, and industrial scale is larger, and the cost saved is more considerable.
Technical scheme of the present invention is as follows:
A method prepared by peptide purification, comprises the following steps:
1) polypeptide dissolving crude product is in acetic acid, acetonitrile, the aqueous solution, supersound process, membrane filtration, and it is stand-by to get filtrate;
2) rp-hplc system is utilized, separation and purification is carried out to the polypeptide crude product after filtering, prepare dynamic axial compression column, mobile phase A is mass percentage concentration 100% acetonitrile solution, Mobile phase B is mass percentage concentration 0.1%-0.3% aqueous acetic acid, carry out gradient elution separation purifying, adopt UV-detector to detect sample, the peptide solution of Fractional Collections object peak value; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
3) the high purity object peptide solution obtained after separation and purification is carried out vacuum rotary steam to concentrate, concentrated solution is collected stand-by; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
4) by concentrated solution through anti-phase dynamic axial compression column, adopt ion exchange method change into acetate; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
5) the acetate qualified product that will take a turn for the better carry out that vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, are qualifiedly after testing the finished product; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme.
Step 2), 3), 4), 5) described in acetonitrile devil liquor recovery treatment scheme be: acetonitrile waste liquid enters waste tank, by the acetonitrile waste liquid in waste tank by vacuumizing, be transferred in the tower reactor of rectifying tower, utilize rectifying tower, rectifying is carried out to acetonitrile waste liquid, the qualified recovery acetonitrile of detection, in storage tank, is transferred in qualified recovery acetonitrile storage tank through pipeline by magnetic drive pump, recycles for rp-hplc system by the recovery acetonitrile collecting high density.
Its dissolution conditions volume ratio of polypeptide crude product described in step 1): acetic acid: acetonitrile: water=1: 10: 40.
Step 2) described in the in-built stationary phase of dynamic axial compression column of preparing be the reverse phase silica gel of octadecylsilane chemically bonded silica.
Anti-phase dynamic axial compression column described in step 4) turns acetate and uses moving phase to be 20 ~ 50mmol Ammoniom-Acetate buffer salt system.
Described rectifying tower, loads the glass spring filler that diameter is 4mm in tower body.The external heating cycle device of tower body, inside connects reboiler, realizes the heating of acetonitrile waste liquid internal recycling and the automatic adjustment of Heating temperature and monitoring.
The recovery acetonitrile that described detection is qualified, purity is greater than 99%, and mass percentage concentration is greater than 85%.
beneficial effect of the present invention:the present invention proposes a kind of utilize reversed-phased high performace liquid chromatographic in conjunction with acetonitrile return
Receive recycle, polypeptide is carried out to the method for purification.Acetonitrile of the present invention reclaims has following feature: feature 1: the acetonitrile waste liquid of generation when rp-hplc system carries out purification to polypeptide, through rectifying tower rectifying, the qualified recovery acetonitrile of detection is transferred in organic phase storage tank by magnetic drive pump through tetrafluoro pipeline, directly for rp-hplc system, such an approach achieves the interlock of two individual system, make acetonitrile obtain recycle in systems in which.Feature 2: because the amount of liquid of acetonitrile waste liquid and recovery acetonitrile is very large, and have certain volatility, if pass through the mode transfer liquid of artificial carrying, both labor intensive also had potential safety hazard.Rectifying tower and rp-hplc system are integrated by this method, and whole system is connected by tetrafluoro flexible pipe, with vacuum pump and magnetic drive pump for propulsion source, avoid artificial contact.Feature 3: load the glass spring filler that diameter is 4mm in rectifying tower tower body, the external heating cycle device of tower body, inside connects reboiler, realizes the heating of acetonitrile waste liquid internal recycling and the automatic adjustment of Heating temperature and monitoring.This rectifier unit is all-glass construction, and cost is low, efficiency is high, simple to operate, outfit is convenient, under lab can be equipped with in face, also can be equipped with in scale operation workshop.Use RPLC purification polypeptide, and turn salt link by with chromatographic system own, without the need to buying instrument and chromatographic column in addition, the moving phase of use is also reagent very conventional in RPLC.In whole separation and purification, concentrate, turn in salt, reconcentration process, collect the acetonitrile waste liquid of high density, utilize rectifying tower, rectifying recovery is carried out to acetonitrile waste liquid, purity is greater than 99%, the recovery acetonitrile that concentration is greater than 85%, is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.Turn compared with salt method with traditional, the present invention has following feature: feature 1: adopt axial compression column CXTH-DAC200 preparation system to turn salt to polypeptide products.After purification step terminates, immediately salt is turned to the qualified product liquid of purified pool, utilize same set of system to do same polypeptide, avoid the crossed contamination between different sample.Feature 2: by the concentration of adjustment buffered soln and the flushing gradient realization that salt is moving phase can be turned in whole turning in salt process, while be separated, while turn salt two steps, both ensure that the requirement of polypeptide products to ion content also ensure that the final purity of polypeptide products.Thus ensure that whole polypeptide products specification of quality, greatly reduce the generation of unacceptable product.Feature 3: utilize axial compression column CXTH-DAC200 preparation system to turn salt to polypeptide products, without the need to purchasing other plant and instrument in addition, without the need to other experiment arrangement place.Same production technology personnel can complete purifying and turn salt two steps in set of system, greatly save time, space, human and material resources.Because the waste liquid exhausted air quantity produced in process of production is far less than traditional method, so be also greatly better than traditional method in energy-saving and emission-reduction.By this approach significantly increasing product production and yield, shortening the production cycle, greatly reducing the cost of peptide separation purifying, and decrease the discharge of acetonitrile waste liquid, not only saved money but also environmental protection.Adopting said method can carry out heavy industrialization purification to polypeptide, and industrial scale is larger, and the cost saved is more considerable.
Accompanying drawing explanation
Fig. 1 is present invention process schema.
Embodiment
embodiment 1:with reference to Fig. 1,
1, sample preparation: take appropriate polypeptide dissolving crude product in volume ratio: acetic acid: acetonitrile: water=1: in the acetate acetonitrile aqueous solution of 10: 40, supersound process, treats that sample is clarified completely is 0.45 μm of membrane filtration with aperture, and collection filtrate is stand-by.
2, separation and purification:
Purification condition: chromatographic column: the CXTH-DAC200(Beijing Chuangxin Tongheng Science and Technology Co., Ltd.-dynamic axial compression column 200 taking octadecylsilane chemically bonded silica as stationary phase) dynamic axial compression column, pillar diameter and filling length are: 20cm × 28cm.Moving phase: A: mass percentage concentration 100% acetonitrile solution; B: mass percentage concentration 0.1% aqueous acetic acid.Flow velocity: 1000ml/min.Determined wavelength: 230nm.Gradient is set, loading.
Purge process: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes accounting for this flush volume total amount 5%, loading.Linear gradient elution, collects object peak, stand-by after the rotary evaporation to about 10-50mg/ml that reduced pressure by the object peptide solution collected.Acetonitrile waste collection separation and purification produced is in waste tank.
3, salt is turned: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with accounting for this flush volume total amount 5% mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes, loading, rinses pillar 3 column volumes with the 20mmol Ammoniom-Acetate aqueous solution; 2 column volumes are rinsed with mass percentage concentration 100% acetonitrile solution accounting for this volume total amount 5% afterwards with accounting for this flush volume total amount 95% ultrapure water; Finally with mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80%, sample is gone out from dynamic axial compression column, will the acetonitrile waste collection of salt generation be turned in waste tank.The qualified product of the acetate that takes a turn for the better are carried out vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, be qualifiedly after testing the finished product.
4, acetonitrile is recycled: open vacuum pump, pump air in tower bottom of rectifying tower, make in tower reactor in negative pressure state, then tower reactor liquid feed valve is opened, by separation and purification, turn salt, revolve the acetonitrile waste liquid of steaming process generation by tetrafluoro flexible pipe, be extracted in waste tank tower reactor, close vacuum pump, open purging valve, tower bottom of rectifying tower is made to be atmospheric pressure state, close purging valve, keep tower body sealing, open coolant circulation pump and heating cycle device, the cooling temperature of coolant circulation pump is set to-10 DEG C, heating cycle device Heating temperature is set to 76 DEG C, start distillation, whole boiling range temperature is 76 DEG C-80 DEG C, the purity being finally recycled acetonitrile is 99.42%, concentration is 86.56%.Close heating cycle device and coolant circulation pump, open the tap valve collected on storage tank, recovery acetonitrile is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.
embodiment 2:with reference to Fig. 1,
1, sample preparation: take appropriate polypeptide dissolving crude product in volume ratio: acetic acid: acetonitrile: water=1: in the acetate acetonitrile aqueous solution of 10: 40, supersound process, treats that sample is clarified completely is 0.45 μm of membrane filtration with aperture, and collection filtrate is stand-by.
2, separation and purification:
Purification condition: chromatographic column: the CXTH-DAC200 dynamic axial compression column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and filling length are: 20cm × 28cm.Moving phase: A: mass percentage concentration 100% acetonitrile solution; B: mass percentage concentration 0.1% aqueous acetic acid.Flow velocity: 1000ml/min.Determined wavelength: 230nm.Gradient is set, loading.
Purge process: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes accounting for this flush volume total amount 5%, loading.Linear gradient elution, collects object peak, stand-by after the rotary evaporation to about 10-50mg/ml that reduced pressure by the object peptide solution collected.Acetonitrile waste collection separation and purification produced is in waste tank.
3, salt is turned: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with accounting for this flush volume total amount 5% mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes, loading, rinses pillar 3 column volumes with the 50mmol Ammoniom-Acetate aqueous solution; 2 column volumes are rinsed with mass percentage concentration 100% acetonitrile solution accounting for this volume total amount 5% afterwards with accounting for this flush volume total amount 95% ultrapure water; Finally with mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80%, sample is gone out from dynamic axial compression column, will the acetonitrile waste collection of salt generation be turned in waste tank.The qualified product of the acetate that takes a turn for the better are carried out vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, be qualifiedly after testing the finished product.
4, acetonitrile is recycled: open vacuum pump, pump air in tower bottom of rectifying tower, make in tower reactor in negative pressure state, then tower reactor liquid feed valve is opened, by separation and purification, turn salt, revolve the acetonitrile waste liquid of steaming process generation by tetrafluoro flexible pipe, be extracted in waste tank tower reactor, close vacuum pump, open purging valve, tower bottom of rectifying tower is made to be atmospheric pressure state, close purging valve, keep tower body sealing, open coolant circulation pump and heating cycle device, the cooling temperature of coolant circulation pump is set to-10 DEG C, heating cycle device Heating temperature is set to 76 DEG C, start distillation, whole boiling range temperature is 76 DEG C-85 DEG C, the purity being finally recycled acetonitrile is 99.26%, concentration is 86.32%.Close heating cycle device and coolant circulation pump, open the tap valve collected on storage tank, recovery acetonitrile is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.
embodiment 3:with reference to Fig. 1,
1, sample preparation: take appropriate polypeptide dissolving crude product in volume ratio: acetic acid: acetonitrile: water=1: in the acetate acetonitrile aqueous solution of 10: 40, supersound process, treats that sample is clarified completely is 0.45 μm of membrane filtration with aperture, and collection filtrate is stand-by.
2, separation and purification:
Purification condition: chromatographic column: the CXTH-DAC200 dynamic axial compression column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and filling length are: 20cm × 28cm.Moving phase: A: mass percentage concentration 100% acetonitrile solution; B: mass percentage concentration 0.3% aqueous acetic acid.Flow velocity: 1000ml/min.Determined wavelength: 230nm.Gradient is set, loading.
Purge process: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes accounting for this flush volume total amount 5%, loading.Linear gradient elution, collects object peak, stand-by after the rotary evaporation to about 10-50mg/ml that reduced pressure by the object peptide solution collected.Acetonitrile waste collection separation and purification produced is in waste tank.
3, salt is turned: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with accounting for this flush volume total amount 5% mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes, loading, rinses pillar 3 column volumes with the 30mmol Ammoniom-Acetate aqueous solution; 2 column volumes are rinsed with mass percentage concentration 100% acetonitrile solution accounting for this volume total amount 5% afterwards with accounting for this flush volume total amount 95% ultrapure water; Finally with mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80%, sample is gone out from dynamic axial compression column, will the acetonitrile waste collection of salt generation be turned in waste tank.The qualified product of the acetate that takes a turn for the better are carried out vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, be qualifiedly after testing the finished product.
4, acetonitrile is recycled: open vacuum pump, pump air in tower bottom of rectifying tower, make in tower reactor in negative pressure state, then tower reactor liquid feed valve is opened, by separation and purification, turn salt, revolve the acetonitrile waste liquid of steaming process generation by tetrafluoro flexible pipe, be extracted in waste tank tower reactor, close vacuum pump, open purging valve, tower bottom of rectifying tower is made to be atmospheric pressure state, close purging valve, keep tower body sealing, open coolant circulation pump and heating cycle device, the cooling temperature of coolant circulation pump is set to-10 DEG C, heating cycle device Heating temperature is set to 76 DEG C, start distillation, whole boiling range temperature is 76 DEG C-85 DEG C, the purity being finally recycled acetonitrile is 99.18%, concentration is 85.26%.Close heating cycle device and coolant circulation pump, open the tap valve collected on storage tank, recovery acetonitrile is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.
embodiment 4:with reference to Fig. 1,
1, sample preparation: take appropriate polypeptide dissolving crude product in volume ratio: acetic acid: acetonitrile: water=1: in the acetate acetonitrile aqueous solution of 10: 40, supersound process, treats that sample is clarified completely is 0.45 μm of membrane filtration with aperture, and collection filtrate is stand-by.
2, separation and purification:
Purification condition: chromatographic column: the CXTH-DAC200 dynamic axial compression column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and filling length are: 20cm × 28cm.Moving phase: A: mass percentage concentration 100% acetonitrile solution; B: mass percentage concentration 0.2% aqueous acetic acid.Flow velocity: 1000ml/min.Determined wavelength: 230nm.Gradient is set, loading.
Purge process: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes accounting for this flush volume total amount 5%, loading.Linear gradient elution, collects object peak, stand-by after the rotary evaporation to about 10-50mg/ml that reduced pressure by the object peptide solution collected.Acetonitrile waste collection separation and purification produced is in waste tank.
3, salt is turned: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with accounting for this flush volume total amount 5% mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes, loading, rinses pillar 3 column volumes with the 30mmol Ammoniom-Acetate aqueous solution; 2 column volumes are rinsed with mass percentage concentration 100% acetonitrile solution accounting for this volume total amount 5% afterwards with accounting for this flush volume total amount 95% ultrapure water; Finally with mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80%, sample is gone out from dynamic axial compression column, will the acetonitrile waste collection of salt generation be turned in waste tank.The qualified product of the acetate that takes a turn for the better are carried out vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, be qualifiedly after testing the finished product.
4, acetonitrile is recycled: open vacuum pump, pump air in tower bottom of rectifying tower, make in tower reactor in negative pressure state, then tower reactor liquid feed valve is opened, by separation and purification, turn salt, revolve the acetonitrile waste liquid of steaming process generation by tetrafluoro flexible pipe, be extracted in waste tank tower reactor, close vacuum pump, open purging valve, tower bottom of rectifying tower is made to be atmospheric pressure state, close purging valve, keep tower body sealing, open coolant circulation pump and heating cycle device, the cooling temperature of coolant circulation pump is set to-10 DEG C, heating cycle device Heating temperature is set to 76 DEG C, start distillation, whole boiling range temperature is 76 DEG C-90 DEG C, the purity being finally recycled acetonitrile is 98.21%, concentration is 82.08%.Close heating cycle device and coolant circulation pump, open the tap valve collected on storage tank, recovery acetonitrile is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.
embodiment 5:with reference to Fig. 1,
1, sample preparation: take appropriate polypeptide dissolving crude product in volume ratio: acetic acid: acetonitrile: water=1: in the acetate acetonitrile aqueous solution of 10: 40, supersound process, treats that sample is clarified completely is 0.45 μm of membrane filtration with aperture, and collection filtrate is stand-by.
2, separation and purification:
Purification condition: chromatographic column: the CXTH-DAC200 dynamic axial compression column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and filling length are: 20cm × 28cm.Moving phase: A: mass percentage concentration 100% acetonitrile solution; B: mass percentage concentration 0.3% aqueous acetic acid.Flow velocity: 1000ml/min.Determined wavelength: 230nm.Gradient is set, loading.
Purge process: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes accounting for this flush volume total amount 5%, loading.Linear gradient elution, collects object peak, stand-by after the rotary evaporation to about 10-50mg/ml that reduced pressure by the object peptide solution collected.Acetonitrile waste collection separation and purification produced is in waste tank.
3, salt is turned: dynamic axial compression column mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80% is rinsed 3 column volumes (column volume about 8 minutes), then with accounting for this flush volume total amount 5% mass percentage concentration 100% acetonitrile solution balance pillar 2 column volumes, loading, rinses pillar 3 column volumes with the 50mmol Ammoniom-Acetate aqueous solution; 2 column volumes are rinsed with mass percentage concentration 100% acetonitrile solution accounting for this volume total amount 5% afterwards with accounting for this flush volume total amount 95% ultrapure water; Finally with mass percentage concentration 100% acetonitrile solution accounting for this flush volume total amount 80%, sample is gone out from dynamic axial compression column, will the acetonitrile waste collection of salt generation be turned in waste tank.The qualified product of the acetate that takes a turn for the better are carried out vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, be qualifiedly after testing the finished product.
4, acetonitrile is recycled: open vacuum pump, pump air in tower bottom of rectifying tower, make in tower reactor in negative pressure state, then tower reactor liquid feed valve is opened, by separation and purification, turn salt, revolve the acetonitrile waste liquid of steaming process generation by tetrafluoro flexible pipe, be extracted in waste tank tower reactor, close vacuum pump, open purging valve, tower bottom of rectifying tower is made to be atmospheric pressure state, close purging valve, keep tower body sealing, open coolant circulation pump and heating cycle device, the cooling temperature of coolant circulation pump is set to-10 DEG C, heating cycle device Heating temperature is set to 76 DEG C, start distillation, whole boiling range temperature is 76 DEG C-95 DEG C, the purity being finally recycled acetonitrile is 96.21%, concentration is 78.18%.Close heating cycle device and coolant circulation pump, open the tap valve collected on storage tank, recovery acetonitrile is transferred in organic phase storage tank through tetrafluoro pipeline by magnetic drive pump, recycles for rp-hplc system.
Claims (5)
1. the method prepared of peptide purification, is characterized in that comprising the following steps:
1) polypeptide dissolving crude product is in acetic acid, acetonitrile solution, supersound process, membrane filtration, and it is stand-by to get filtrate;
2) rp-hplc system is utilized, separation and purification is carried out to the polypeptide crude product after filtering, prepare dynamic axial compression column, mobile phase A is mass percentage concentration 100% acetonitrile solution, Mobile phase B is mass percentage concentration 0.1%-0.3% aqueous acetic acid, carry out gradient elution separation purifying, adopt UV-detector to detect sample, the peptide solution of Fractional Collections object peak value; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
3) the high purity object peptide solution obtained after separation and purification is carried out vacuum rotary steam to concentrate, concentrated solution is collected stand-by; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
4) by concentrated solution through anti-phase dynamic axial compression column, adopt ion exchange method change into acetate; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
5) the acetate qualified product that will take a turn for the better carry out that vacuum rotary steam is concentrated, lyophilize, obtain white flock powder-product, are qualifiedly after testing the finished product; Acetonitrile waste liquid enters acetonitrile devil liquor recovery treatment scheme;
Step 2), 3), 4), 5) described acetonitrile devil liquor recovery treatment scheme: acetonitrile waste liquid enters waste tank, by the acetonitrile waste liquid in waste tank by vacuumizing, be transferred in the tower reactor of rectifying tower, utilize rectifying tower, rectifying is carried out to acetonitrile waste liquid, the qualified recovery acetonitrile of detection, in storage tank, is transferred in qualified recovery acetonitrile storage tank through pipeline by magnetic drive pump, recycles for rp-hplc system by the recovery acetonitrile collecting high density.
2. the method prepared of a kind of peptide purification according to claim 1, is characterized in that: the in-built stationary phase of dynamic axial compression column is the reverse phase silica gel of octadecylsilane chemically bonded silica.
3. the method prepared of a kind of peptide purification according to claim 1, is characterized in that: the volume ratio during configuration of the step 1) acetate acetonitrile aqueous solution: acetic acid: acetonitrile: water=1: 10: 40.
4. the method prepared of a kind of peptide purification according to claim 1, it is characterized in that: in step 4), concentrated solution is completed ion-exchange through anti-phase dynamic axial compression column and change into acetate, used moving phase is 20 ~ 50mmol acetate buffer salt system.
5. the method prepared of a kind of peptide purification according to claim 1, it is characterized in that: load the glass spring filler that diameter is 4mm in rectifying tower tower body, the external heating cycle device of tower body, inside connects reboiler.
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