CN100390198C - Technique for purifying recombined human interferon alpha 1b - Google Patents
Technique for purifying recombined human interferon alpha 1b Download PDFInfo
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Abstract
The present invention relates to a purification process of recombination human interferon alpha1b, which is used for extracting the recombination human interferon alpha1b from engineering bacteria used for expressing human interferon alpha1b. The purification process comprises the steps of bacteria fragmentation, expanded bed adsorption, affinity chromatography and gel filtration. The bacteria fragmentation is carried out in a Tris-HCl buffer solution. In the step of expanded bed adsorption, anionic gel is adopted, and Tris-HCl with the pH value of 7.4 is used as a buffer system; the engineering bacteria are treated by one-step elution by an eluent, wherein 0.4M of NaCl and 50mM of Tris-Hcl with the PH value of 7.4 are used as the eluent. In the step of affinity chromatography, gel coupled by a monoclonal antibody of the alpha1b interferon is adopted; PBS is used as a balancing solution and a cleaning solution, and 0.1M of Gly-HCl is used as an eluent; 0.1M of Tris-HCl is used as an actified solution, and 0.1M of NaAc-HAc is used as a buffer solution. In the step of gel filtration, Sephacryl gel is used, and the PBS is used as a buffer system. With the purification process of the recombination human interferon alpha1b, the productive efficiency can be greatly improved so that the production cost is greatly reduced.
Description
Technical field
The present invention relates to contain the pharmaceutical product of peptide, particularly relate to proteinic separation purification method, relate in particular to the separation purifying technique of recombinant human interferon alpha 1 b.
Background technology
Interferon, rabbit has antiviral activity widely, nineteen fifty-seven Isaacs and Lindenmann have found this material when the interference phenomenon that studies flu virus: after acting on cell with the influenza virus of deactivation, cell can produce a kind of soluble substance, make the breeding of live virus cell be interfered, so be referred to as Interferon, rabbit (Interferon writes a Chinese character in simplified form IFN).Interferon, rabbit has three big functions: suppress the viral proliferation activity, suppress cell fission activity and immunoregulatory activity.
Interferon, rabbit is the protein that a class is regulated cell function.Different according to antigen-specific and molecular structure are divided into types such as α, β, γ, ω.And in a specific Interferon, rabbit type,, mark off different hypotypes again according to the difference of aminoacid sequence or composition aspect, and such as: HuIFN (human interferon) α 1, α 2 etc.And in a specific Interferon, rabbit hypotype, can carry out mark in the hypotype back with a, b, c according to the variation of indivedual or a few amino acids again, and such as: HuIFN α 1a, 1b, 2b etc.
HuIFN α can carry out volume production by gene recombination and other biological engineering.And HuIFN α 1b relatively other 2, one important differences such as HuIFN α be that find according to Chinese science man years of researches: Chinese's white corpuscle is after the virus stimulation, topmost hypotype is α 1b in all Interferon, rabbit that induce.And test is proof also, and after Chinese used Interferon, rabbit, the probability that produces neutralizing antibody was minimum with α 1b, so α 1b more meets Chinese state of nature.At present China generally adopt by the applicant, the trade name that Kexing Bioengineering Co., Ltd, Shenzhen produces is that the HuIFN α 1b of Sai Ruojin is that professor Hou Yunde by Key State Laboratories Virogene Engineering For Virogene Engineering of Virology Inst., Chinese Academy of Preventive Medical Science equals nineteen eighty-two and utilizes Avian pneumo-encephalitis virus F strain (NDV-F) to stimulate the bleeding of the umbilicus white corpuscle first, extract mRNA, reverse transcription becomes cDNA, successfully cloned IFN α 1b cDNA clone, and in intestinal bacteria, obtained efficiently expressing.
Application present situation from Interferon, rabbit, HuIFN α type application case is many, with HuIFN α 1b is example, can be used for treating virus disease, malignant tumour, hemopathy etc., especially in the treatment of chronic type b, hepatitis C, route of administration adopts intramuscular injection more, and dosage is generally at 1,000,000-6,000,000 IU (international unit), efficiently reaches 60%.Clinical practice by domestic many hospitals is found, adopts the direct administration of HuIFN α 1b ultrasonic atomization, and is remarkable to viral upper respiratory tract infection, virus infected herpes, viral pharyngolaryngitis and chronic stomatocace result of treatment.
In the production process of people α 1b Interferon, rabbit, the engineering bacteria process goes down to posterity behind cultivation and the high density fermentation, very important technological process is proteinic separation and purification together: people α 1b Interferon, rabbit is extracted from the bacterial expression environment of complexity, remove various impurity, comprise non-target protein things such as particle, bacterium, intracellular toxin, nucleic acid and various foreign proteins, to reach the every index of medicine prison authorities of government, meet clinical needs to the requirement of this kind biological products.
The separation and purification key step of people α 1b Interferon, rabbit comprises bacteria breaking, preliminary purification and refining purifying at present.Because in above-mentioned engineering bacteria, people α 1b Interferon, rabbit is a soluble-expression in the born of the same parents, so the first step of separation and purification is broken bacterium, allows Interferon, rabbit discharge in born of the same parents, just can carry out further purifying.Specifically, preliminary purification comprises the steps: that centrifugal 1 → acidifying → centrifugal 2 → alkali neutralizes → saltout 1 → centrifugal 3 → 2 → centrifugal 4 totally 9 step process of saltouing, and comprise the centrifugal and 3 step precipitator method of 4 steps: 1 step salt Acid precipitation and 2 goes on foot (NH
4)
2SO4 salt precipitation, and Sephadex G-50 desalination.Refining purifying then comprises 3 step column chromatographies: CM-Sepharose F.F ion exchange chromatography, monoclonal antibody affinity chromatography, Sephacryl S-100 molecular sieve.As can be seen, whole purifying process is complexity, particularly preliminary purification part quite, and step is various.
Because step is various, complex process, and the strong acid and strong base processing just makes existing manufacturing technique have following problem: because in the protein purification process, all can there be loss in each step, thereby must cause the reduction of total yield; Handle for a long time, cause the low of production efficiency, add a large amount of uncovered operations in the preliminary purification, also cause product contamination easily, influence quality product; Too much and a large amount of manual operationss of step cause Material Cost and cost of labor too high, and process economy is poor.
On the other hand, except technology such as above-mentioned albumen precipitation and conventional post chromatographies, ExPANDED BED ADSORPTION TECHNIQUE begins to be applied to protein purification and handles.EBA system (Expanded Bed Adsorption Expanded Bed Adsorption) with Pharmacia is an example, it has adopted unique STREAMLINE gel and chromatography column: this gel is to combine quartzy composition on the basis of plain agar sugar gel, thereby has increased density and formed certain density gradient; Simultaneously, but designed the plunger free lifting and can guarantee the chromatography column that seals again.These unique design make the STREAMLINE gel can form stable expansion column bed under the effect of from bottom to top level pad, therefore, material without clarifying band impurity directly can be gone up sample, in last sample process, impurity such as cell debris are washed off between the gel particle, and biomolecules such as protein then are adsorbed on the gel.After cleaning, again gel is settled down, thereby the wash-out target protein reaches the purpose of purifying.
Can find out from the Expanded Bed Adsorption principle and it is advantageous that: can directly the material without refiner impurity directly be gone up sample, the gene engineering product of expressing in born of the same parents sample on can just can be directly after the cytoclasis for example, thereby the step of having exempted centrifugal or ultrafiltration by centrifugal or ultrafiltration removal cell debris.Therefore, Expanded Bed Adsorption has played clarification once going on foot, has concentrated and the effect of preliminary purification.
Can be applied to ExPANDED BED ADSORPTION TECHNIQUE the purifying process of Interferon, rabbit? how can ExPANDED BED ADSORPTION TECHNIQUE be applied to the purifying process of Interferon, rabbit, to reach the simplification technological process, the purpose of enhancing productivity? as if prior art is the not consideration of this respect, also do not provide corresponding solution.
Summary of the invention
The technical problem to be solved in the present invention is to avoid above-mentioned the deficiencies in the prior art part, improves the Interferon, rabbit purifying process that production cost reduces and propose a kind of production efficiency that can make.
The technical scheme that the present invention solves the problems of the technologies described above employing is, a kind of purifying process of recombinant human interferon alpha 1 b is proposed, be used for extracting recombinant human interferon alpha 1 b from the engineering bacteria that possesses the human interferon alpha 1 b expression, comprise bacteria breaking, and the catching of target protein, intermediate purification and treating process, and the acquisition procedure of described target protein adopts Expanded Bed Adsorption, and the intermediate purification process adopts affinity chromatography, and treating process adopts gel-filtration; Described Expanded Bed Adsorption adopts the ion-exchange gel sorbent material.
Described bacteria breaking is to carry out in the Tris-HCl damping fluid;
In the described Expanded Bed Adsorption step, select the negatively charged ion gel for use, specifically select STREAMLINE DEAE for use; Buffer system selects for use the pH value to be 7.0-8.0, and preferred value is 7.4 Tris-HCl, and adopts the method for salt concn wash-out; Described salt concn wash-out is selected for use and is contained 0.2-0.5, and preferred value is the pH7.4 50mMTris-Hcl elutriant one-step elution of 0.4 MNaCl.
In the described affinity chromatography step, select the monoclonal antibody link coupled gel of α 1b Interferon, rabbit for use; Balance liquid adopts PBS, and elutriant adopts 0.05-0.2, and preferred value is 0.1M Gly-HCl, and regenerated liquid adopts 0.05-0.2, and preferred value is 0.1M Tris-HCl and 0.05-0.2, and preferred value is 0.1MNaAc-Hac.
In the described gel-filtration step, buffer system is selected PBS for use; And select the Sephacryl gel for use.
Compare with old technology, have remarkable advantages with the improved new technology for purifying of Expanded Bed Adsorption among the present invention based on albumen precipitation.Be embodied in: process cycle shortens 40%, and the purifying yield improves 70%, and production cost reduces many-sides such as 40-50%.
Compare with prior art, the purifying process of recombinant human interferon alpha 1 b of the present invention, production efficiency can improve greatly, and production cost then reduces greatly.
Description of drawings
Fig. 1 is adopted Expanded Bed Adsorption chromatographic theory figure by the purifying process of recombinant human interferon alpha 1 b of the present invention.
Fig. 2-1 to 2-8 be Expanded Bed Adsorption operating system valve and pipeline schematic diagram.
Fig. 3 is an Expanded Bed Adsorption chromatography curve.
Fig. 4 is that STREAMLINE DEAE gel adsorptive capacity is measured curve.
Fig. 5 is that Expanded Bed Adsorption chromatographic theory stage number is measured curve.
Fig. 6 is the affinity chromatography curve.
Fig. 7 is the gel-filtration curve.
Embodiment
Below the present invention is further elaborated.
Why to select ExPANDED BED ADSORPTION TECHNIQUE for use?
The improvement of purifying process at first is the variation of purifying strategy and thinking.The conventional thought of recombinant protein purification is that purifying is divided into preliminary purification and highly purified two portions, the starting point of this thinking is to arrange the purifying production work from the angle of production management, its drawback is, angle from Technology, do not have clear and definite implication this cutting apart, is boundary there? which unit is a preliminary purification, and which unit is highly purified.Preliminary and highly purified notion is difficult to be used for determine an operational path, begins the target protein that progressively purifying acquisition meets various requirement from thalline.A kind of new purifying strategy is the angle from technology, and the purifying process of recombinant protein from thalline to pure product is divided into three parts: target protein catches → and intermediate purification → refining.Its thinking is at first target protein to be caught out from the initial material of complexity, does not require high purity, and requires high-recovery and significantly reduce to handle volume, and removes impurity particle.The second step intermediate purification is to improve the committed step of purity, with no particle and the material after significantly reducing volume adopt means of purification efficiently, remove most of impurity, increase substantially target protein purity.The 3rd one-step refining is a small amount of impurity such as the aggressiveness and the isomer that possible exist, and the new Impurity removal that may bring in the purifying early stage, to meet the requirements of purity.
Based on catching → problem that intermediate purification → purified purifying strategy and existing purifying process exist, we find importantly the preliminary purification technology of original poor efficiency complexity is replaced with efficient simple means.We have selected for use the EBA system (Expanded Bed Adsorption Expanded Bed Adsorption) of Pharmacia to carry out this improvement.
Since coming out in 1993, Expanded Bed Adsorption has been widely used in the research, development and production of gene engineering product.No matter be intestinal bacteria, yeast or mammalian cell, the example of extensive application is all arranged.1998, Japanese green cross company more was used for extensive Expanded Bed Adsorption the production of maximum up to now gene engineering product one recombinant albumin, as seen its use extensively.
After adopting the EBA system, we can be reduced to the purifying process of Interferon, rabbit:
Bacteria breaking → EBA → affinity chromatography → molecular sieve
After bacteria breaking, only need three step column chromatographies can finish the separation and purification work of Interferon, rabbit, obtain the pure product of Interferon, rabbit that conform to quality requirements.Whole purifying process was reduced to for 4 steps from original 13 steps, and wherein, the EBA1 step has replaced the step of 10 in the old technology: the 1 → centrifugal 3 → 2 → centrifugal 4 → desalination → ion-exchange of saltouing that neutralizes → saltout of centrifugal 1 → acidifying → centrifugal 2 → alkali.Whole purifying process is greatly simplified, and production efficiency is improved.
Consistent with our purifying strategy, three step column chromatographies are the three phases of corresponding purifying strategy respectively.Wherein, that EBA has replaced is centrifugal, acidifying, saltout, the step of desalination and ion-exchange, played the sample clarification, concentrated and the effect of preliminary purification, target is disturbed to have to imitate catches, remove particles such as cell debris and part foreign protein, nucleic acid, and significantly reduced volume of material.Affinity chromatography has been removed impurity such as most of foreign protein, nucleic acid, intracellular toxin as a kind of means of purification efficiently, and further concentrates the Interferon, rabbit sample.The final step molecular sieve then plays refining effect, the IgG that has removed the remaining a small amount of foreign protein of Interferon, rabbit, Interferon, rabbit dimer and may come off from affinity chromatography, and play the effect of exchange buffering liquid.
How specifically to use ExPANDED BED ADSORPTION TECHNIQUE?
We grope by two serial experiments, to determine relevant technical parameter and index.
Embodiment 1: the Expanded Bed Adsorption gel is selected and the chromatography parameter optimization
The special chromatography column of the manipulation require of EBA is to produce stable expanding bed.At the process exploitation initial stage, at first adopt the form of loading bed to experimentize and grope.Adopting the filling bed to carry out gel selects and parameter optimization, be because though the adsorption effect of expanding bed is relatively good, the required condition of dual mode gel absorption is consistent, for selection gel and the not influence of chromatography parameter, and can save the investment of expanding bed system on a small scale.Adopt the filling bed,, sample need be carried out centrifugation earlier, to obtain the clarification material owing to can not remove granule foreign.
Experimental program is (mm * cm) carry out column volume 20ml in the chromatography column in XK16 * 10.Select a kind of positively charged ion gel STREAMLINE SP and a kind of negatively charged ion gel STREAMLINE DEAE under different buffering systems and different pH and ionic strength conditions, to be optimized result such as following table respectively:
Wherein, STREAMLINE SP gel adopts citric acid and the acetate buffer solution of pH4.7, and the phosphoric acid buffer of pH6.0, pH6.4 has carried out repeatedly experiment.As seen: best result adopts the phosphoric acid buffer of pH6.4 as last sample and elution requirement, and secondary experiment yield is respectively 45.9% and 43.8%, average 44.8%.
STREAMLINE DEAE is at the phosphoric acid buffer of pH6.4, carried out repeatedly experiment under pH7.4 and 7.8 the Tris buffer conditions.As seen: twice experiment of pH7.8, yield is respectively 35% and 45%.Twice experiment of PH7.4, yield is respectively 54% and 50.2%, and is average 52.1%, and the rate of recovery is higher, and more stable.
Analyze above experimental result, as can be known: the STREAMLINE SP of pH6.4 and the STREAMLINE DEAE of pH7.4 can obtain result preferably, and both compare, and the STREAMLINE DEAE yield of pH7.4 exceeds 16% relatively.Select which kind of condition, a problem also will considering is front and back technology matching problems.Because bacterial cell disruption is to carry out in the Tris of pH7.8 damping fluid, the homogenate final pH just conform to STREAMLINE DEAE condition, and under the pH neutral environment, Interferon, rabbit should be the most stable about 7.4.Therefore, the STREAMLINE DEAE of pH7.4 is an ideal conditions.From another angle, if select the STREAMLINE SP of pH6.4 for use, then homogenate needs the exchange buffering system, under acidic conditions, albumen precipitation can occur, also may have other unexpected problem.Therefore, the DEAE-STEAMLINE gel is selected in decision for use, and the Tris-HCl of use pH7.4 is as the chromatography damping fluid.
Embodiment 2: use STREAMLINE DEAE filling bed pillar to carry out the Interferon, rabbit purifying process and grope
In laboratory scale, use the form of XK16 * 10 posts with the filling bed, select the Tris damping fluid of STREAMLINE DEAE and pH7.4 for use, use with the thalline homogenate of old technology same batch and produce to the pure product of Interferon, rabbit along new technology route.New old technology relatively in, the data of old technology are taken from actual production.
Experiment material:
1. equipment: chromatography column: XK16 * 10, gel: STREAMLINE DEAE, chromatographic system: Pharmacia GP-250 gradient controller
2. damping fluid: balance liquid: pH7.4 50mM Tris-HCl, elutriant: 0-0.5MNaCl pH7.4 50mM Tris-HCl
Experimental arrangement:
1. dress post: adopt the flow velocity of 20ml/min in the equalizing and buffering liquid system, to adorn post, dress column volume 20ml
2. balance: adopt the flow velocity of 10ml/min, the balance liquid balance columns bed of 10 column volumes
3. go up sample: thalline homogenate 1000ml sample under the 10ml/min flow velocity, collect stream and wear
4. clean: adopt balance liquid under the flow velocity of 10ml/min, to clean the post bed, be washed till uv-absorbing near baseline
5. wash-out: adopt 0-0.5MNaCl pH7.4 50mM Tris-HCl to carry out gradient elution, collect main peak
6. regeneration: adopt multiple CIP scavenging solution regeneration gel
7. preserve: adopt 20% ethanol to preserve gel
Experimental result sees the following form, and novel process on average improves yield 108% than old technology.
Wherein, Yn/Yo: novel process yield/old technology yield At: gross activity
Embodiment 3: the foundation of the manual purification system of Expanded Bed Adsorption
By experimental size in the process that industrial scale is amplified, be faced with the problem of setting up the Expanded Bed Adsorption purification system.Based on the consideration that reduces investment, we set up the manual purification system of Expanded Bed Adsorption with chromatography column supplier.
According to Fig. 2 Expanded Bed Adsorption principle of operation figure, the step of Expanded Bed Adsorption comprises that sample on balanced expansion, the sample, post clean, the sedimentation wash-out, column regeneration.For realizing expansion and two functions of chromatography simultaneously, must adopt double pump system.A pump is responsible for the expansion and the sedimentation of post bed, and another pump is responsible for balance, is gone up sample, wash-out, regeneration.Simultaneously, owing to there is the replacing of multiple liquid, therefore, need comparatively complicated valve and piping system.In addition, need basic detection and register system.
According to above analysis, at first be to determine under different chromatography states various fluidic schemas.To 2-8, comprise that original state, balanced expansion, plunger rise, go up the schema of different states such as sample, cleaning, plunger decline, wash-out, regeneration as Fig. 2-1 respectively.Wherein, 1-9 is a manual valve.A refers to initial buffer liquid, and B refers to elution buffer.In all schemas, the path that black line and arrow are represented is the flow path of liquid under this state.As Fig. 2-4, in the last sample schema, sample enters in the chromatography column through valve 3, valve 2, pump 1, valve 4, valve 5, valve 6, valve 8, the stream that last sample produces is worn from capital and is flowed out, enter Ultraviolet Detector through valve 7, valve 6, detect the uv-absorbing signal, be re-used as waste liquid and drain.
According to above flow process, selected relevant device for use: STREAMLINE200 chromatography column, two of WASTON-MARLOW peristaltic pumps, Ultraviolet Detector and datalogger, manually-operated gate and pipeline.With all component, install according to technical process, formed the manual purification system of Expanded Bed Adsorption.
Embodiment 4: the industrial scale of Expanded Bed Adsorption purifying alpha-interferon novel process is amplified
The condition of groping in the XK16/10 pillar with experimental size directly is amplified to the STREAMLINE200 expansion column, and column volume is amplified to 4.5L by 20ml, is to amplify in 225 times of one step.
Experiment material:
1. equipment: chromatography column: STREAMLINE 200, gel: STREAMLINE DEAE 4.5L,
Chromatographic system: the manual purification system of Expanded Bed Adsorption
2. damping fluid: balance liquid: pH7.4 50mM Tris-HCl,
Elutriant: 0.4MNaCl pH7.4 50mM Tris-HCl,
Regenerated liquid: 0.5MNaOH-1MNaCl, WFI, 30% Virahol, 25% acetic acid, 20% ethanol
Experimental arrangement:
1. dress post: in the equalizing and buffering liquid system, adorn post, in the chromatography column of directly gel being packed into, dress column volume 4.5L
2. balance: adopt the flow velocity of 300CM/H, the balance liquid balance columns bed of 10 column volumes
3. go up sample: thalline homogenate 100L sample under the 300CM/H flow velocity, collect stream and wear
4. clean: adopt balance liquid under the flow velocity of 300CM/H, to clean the post bed, be washed till uv-absorbing near baseline
5. wash-out: adopt 0.4MNaCl pH7.4 50mM Tris-HCl to carry out one-step elution, collect elution peak with 100CM/H
6. regeneration: adopt multiple CIP scavenging solution regeneration gel
7. preserve: adopt 20% ethanol to preserve gel
Experimental result: Expanded Bed Adsorption chromatography curve such as Fig. 3.Calibrating data such as following table, novel process on average improves yield 82% than old technology.
Wherein, Yn/Yo: novel process yield/old technology yield At: gross activity
Embodiment 5:STREAMLINE DEAE gel load testing
Experiment material:
Chromatography column XK16/6.9 adopts the form of filling bed to load 13.8ml STREAMLINE DEAE gel.
Balance liquid: pH7.4 50mM Tris damping fluid
Last sample: adopt the centrifugal removal particle of Interferon, rabbit thalline homogenate, as sample solution
Experimental arrangement:
1. with 10 post beds of damping fluid balance
2. with sample 1000ML on the speed of 270CM/H
3. spot sampling is worn in convection current, and stream wears that bleeding point is 0,100,200,300,400,500,600,700,800,900,1000ML
4. detect each stream and wear the Interferon, rabbit concentration C of bleeding point, remove Interferon, rabbit concentration C o in the above sample, obtain the C/Co value, C/Co is 1 to the maximum.
5. with of the collected volume mapping of C/Co value, as Fig. 4 to response.
Experimental result:
As can be seen from Figure 4, when last sample reached 400ml, Interferon, rabbit stream was worn C/Co=0.28, still can accept.During last sample 500ml, C/Co=0.44, unacceptable fully.Therefore, with the 400ml point as the gel maximal absorptive capacity.It is as follows to calculate STREAMLINEDEAE gel adsorptive capacity.
1. the maximum thalline load=400ml of gel * 50g cell/1000ml/13.8ml gel
＝1.45g?cell/ml?gel
2. the maximum thalline Interferon, rabbit of gel load=400ml * 0.0252mg IFN/ml/13.8ml gel
＝0.725mg?IFN/ml?gel
According to above gel load, the STEAMLINE200 post is adorned the 4.5L gel, and its maximum thalline applied sample amount is the 6.5Kg thalline, and by sample on the overall loading 80%, applied sample amount is a 5.2Kg Interferon, rabbit thalline.
Embodiment 6:STREAMLLINE200 post bed Detection of Stability
1. visual inspection: to the expansion column of glass, visual inspection is the important step of test column bed stability.Behind post bed balanced expansion, carry out visual inspection.In the stable post bed, the gel particle of suspension is only done little rotatablely moving in the original place.If turbulent flow or channel can cause adsorption effect undesirable.The cyclotron motion that post bed top is big may be that pillar is not in vertical position.Exist bubble or system of distribution partly to stop up in the sparger at the bottom of the channel prompting post of post bed bottom.
2. post bed expansion multiple detects
The expansion multiple is the ratio of the height H o of the height H of expansion rear pillar bed and sedimentation rear pillar bed, H/Ho.Under the perfect condition, expansion multiple and flow velocity, buffer system, temperature is relevant, and under the situation that these three indexs are determined, the expansion multiple is determined.Under the abnormal conditions, the decline of expansion multiple may mean and have bubble or obstruction in the grid distributor that gel is contaminated, problems such as pillar non-vertical.
In the STREAMLINE200 post, use STREAMLINE DEAE gel, the Tris damping fluid of 50mM pH7.4,20 ℃ working temperature and the equilibrium rate of 300CM/H, expansion multiple=H/Ho=43.6/14.5=3.0 is reasonable value.
3. theoretical plate number detects
Retention time distributes to test and can be used for determining theoretical plate number, with the situation of gel axial distribution in the assessment expanding bed.Sample on the acetone soln of dilution in the good post of expansion, is detected the uv-absorbing signal of acetone in the effluent, simultaneously according to chromatography collection of illustrative plates theory of computation stage number.Specific procedure is as follows:
(1). adopt level pad fully expansion under the 300CM/H flow velocity, record expanding bed height.
(2). reduce plunger and arrive from expanding bed glue face 0.5-1CM
(3). start registering instrument and Ultraviolet Detector.When baseline stability, the mixture of sample-loading buffer-acetone (0.25%V/V) also waits for that acetone uv-absorbing signal occurs.
(4). when UV signal is stabilized in obtained the maximum absorption (100%), change damping fluid into.Mark under doing on the recording paper.
(5). wait for UV signal decline and be stabilized in baseline values.
(6). according to shown in Figure 5, theory of computation stage number N.
N＝t
2/σ
2
The average retention time of t=,
Be that mark from step 4 is to the distance of 50% maximal ultraviolet signal
σ=standard deviation
Be from the maximal ultraviolet signal 15.85% to 84.15% half of distance
STREAMLINE DEAE gel at STREAMLINE200 post employing 14.5CM is expanded to 43.6CM, detects at last:
t＝120.6，σ＝25.9，
N=t
2/ σ
2=120.6
2/ 25.9
2=22 is rational theoretical plate number
Embodiment 7: affinity chromatography schedule of operation on the Expanded Bed Adsorption elutriant
Experiment material:
1. equipment: chromatography column 90 * 70, peristaltic pump, Ultraviolet Detector and registering instrument
2. material: monoclonal antibody glue MCAb-Sepharose F.F.,
Damping fluid PBS, 0.1M Gly-HCl, 0.1MTris-HCl, 0.1MNaAc, 0.5MNaOH
Experimental arrangement:
1. balance: use PBS balance columns bed
2. go up sample: the detected result according to the Expanded Bed Adsorption elutriant is determined applied sample amount, with sample on the elutriant to post
3. clean: use PBS to clean the impurity that does not adsorb in the post
4. wash-out: use elution buffer that the Interferon, rabbit that adsorbs on the post is washed down, collect elution peak, i.e. the 2nd peak among Fig. 6
5. regeneration: use impurity in the residual post of Tris-HCl and NaAc buffer solution for cleaning
The affinity chromatography collection of illustrative plates is seen Fig. 6, and X-coordinate is the time among the figure, and each lattice is 30 minutes, and ordinate zou is a 280nm ultraviolet radiation absorption relative value (percentage ratio) among the figure.
Embodiment 8: the affinity chromatography elutriant is handled in gel-filtration
Experiment material
1. equipment: chromatography column 130 * 70, peristaltic pump, Ultraviolet Detector and registering instrument
2. material: Sephacryl S-100,
Damping fluid PBS, 0.5MNaOH
Experimental arrangement
1. balance: use three column volumes of PBS damping fluid balance
2. go up sample: the affinity chromatography elutriant is pressed on the applied sample amount of column volume 4% in post
3. wash-out: use PBS buffer solution elution sample, collect the Interferon, rabbit main peak
4. regeneration: use 0.5MNaOH actifier column bed
The gel-filtration collection of illustrative plates is seen Fig. 7, and X-coordinate is the time among the figure, and each lattice is 60 minutes, and ordinate zou is a 280nm ultraviolet radiation absorption relative value (percentage ratio) among the figure.
Compared with prior art, adopt the purifying process of recombinant human interferon alpha 1 b of the present invention, production efficiency can improve greatly, and production cost then reduces greatly.Be embodied in: process cycle shortens 40%, and the purifying yield improves 70%, and production cost reduces many-sides such as 40-50%.
(1). process cycle is relatively
Last table is to compare new and old purifying process schema of Interferon, rabbit and duration.Therefrom as can be seen, in old technology, the whole purifying process cycle from the thalline to stoste is 5 day time, and novel process only is 3 day time, and the duration shortens 2 days.The purifying cycle shortens 40%, and corresponding purifying production capacity can improve 40%.And, just mean that the throughput of Interferon, rabbit raw material and preparation improves 40% because common purifying is the bottleneck place of whole production, and the purifying production capacity improves 40%.
(2). the purifying yield is relatively
From thalline,, obtain the pure product of Interferon, rabbit through whole purifying routes.The yield that under the different scales situation, has compared new old technology.
In laboratory scale, use with the thalline homogenate of old technology same batch and produce to the pure product of Interferon, rabbit along new technology route.Wherein, owing to there is not the laboratory scale expansion column, laboratory scale Expanded Bed Adsorption adopts the form of filling bed to carry out chromatography.And the data of old technology are taken from actual production.From the following table result, the ratio of the active yield of new old technology is 2.08, and promptly the novel process yield improves 1 times.
| Lot number | T1 | T2 | T3 | T4 | On average |
| The active yield Yn (%) of novel process | 25.8 | 30.2 | 16.9 | 30.4 | |
| The active yield Yo (%) of old technology | 11.9 | 13.9 | 9.6 | 14.1 | |
| Yn/Yo | 2.17 | 2.17 | 1.76 | 2.16 | 2.08 |
After technology is amplified,, adopt the STREAMLINE200 post to carry out Expanded Bed Adsorption, adopt identical thalline homogenate to be purified to pure product with old technology in industrial scale.Result such as following table, as can be seen, the industrial scale that coexists adopts the novel process yield of EBA to improve 82%.
| Lot number | P1 | P2 | P3 | On average |
| The active yield Yn (%) of novel process | 16.2 | 23.2 | 31.0 | |
| The active yield Yo (%) of old technology | 11.4 | 9.4 | 19.8 | |
| Yn/Yo | 1.42 | 2.46 | 1.57 | 1.82 |
The amplitude that the industrial scale yield improves is lower than experimental size, major cause has 2 points: 1. since the EBA yield far above corresponding pure and mild ion-exchange step just in the old technology, therefore the excessive sample problem that goes up often occurs during monoclonal antibody on the EBA gleanings, it is many to cause Interferon, rabbit stream to pass.And there is not this problem in old technology, in the loss of monoclonal antibody step seldom.2.EBA gleanings is owing to only through single step purification, may exist the enzymolysis problem in the sample retention process.
(3). quality product is relatively
New old technology no significant difference on quality index all satisfies the requirement of biological products rules fully.All indexs comprise: than live, HPLC and electrophoresis purity, intracellular toxin, foreign DNA, host protein, IgG content is all qualified, it is all qualified that the experimentation on animalies of finished product etc. require.
(4). production cost is relatively
A. the novel process investment in fixed assets reduces.Owing to removed steps such as centrifugation, multistep precipitation, column chromatography desalination and ion-exchange, thereby reduced the wander about as a refugee investment of scheming, a plurality of retort and chromatographic system of Large Scale and Continuous.In the industrial scale of producing 1000 ten thousand Interferon, rabbit per year, the investment of purifier apparatus can reduce by 2,600,000 Renminbi, and the minimizing amplitude is 41%.
B. Material Cost reduces.The minimizing of steps such as acid precipitation, sulphur ammonia are saltoutd, desalination makes Material Cost reduce.In the industrial scale of producing 1000 ten thousand Interferon, rabbit per year, Material Cost reduces by 180,000, and the reduction amplitude is 58%.Also be accompanied by the decline of costs such as water power simultaneously.
C. personnel cost descends.Since the simplification of technology, the reduction of labor capacity.Make that finishing the required number of same task reduces greatly.In the industrial scale of producing 1000 ten thousand Interferon, rabbit per year, the purifying number is kept to 8 people from 14 people, and personnel cost reduces 43%.
Comprehensive above factor, as can be seen, owing to adopting new technology, the production cost of the whole purifying process 40-50% that descended.
More than each embodiment be intended to specify the present invention's mentality of designing: in the purifying process of recombinant human interferon alpha 1 b, adopt ExPANDED BED ADSORPTION TECHNIQUE to replace traditional column chromatography technology, and how to realize this technology.The present invention's enforcement is not limited to the disclosed mode of above each embodiment, and all mentalities of designing based on the present invention are simply deduced and replaced, and the purifying process of the concrete recombinant human interferon alpha 1 b that obtains all belongs to enforcement of the present invention.
Claims (13)
1. the purification technique of a recombinant human interferon alpha 1 b is used for extracting recombinant human interferon alpha 1 b from the engineering bacteria of expressing human interferon alpha 1 b, comprises bacteria breaking, and the catching of target protein, intermediate purification and treating process, it is characterized in that:
The acquisition procedure of described target protein adopts Expanded Bed Adsorption, and the intermediate purification process adopts affinity chromatography, and treating process adopts gel-filtration; Described Expanded Bed Adsorption adopts the ion-exchange gel sorbent material.
2. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 1 is characterized in that: the monoclonal antibody link coupled gel of described affinity chromatography employing α 1b Interferon, rabbit.
3. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 1 is characterized in that: described affinity chromatography adopts that PBS cooks balance liquid, 0.05-0.2M Gly-HCl does elutriant and 0.05-0.2M Tris-HCl and 0.05-0.2M NaAc and does regenerated liquid.
4. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 3 is characterized in that: described elutriant employing 0.1M Gly-HCl; Described regenerated liquid adopts 0.1M Tris-HCl and 0.1M NaAc.
5. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 1 is characterized in that: described gel-filtration employing Sephacryl gel.
6. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 1 is characterized in that: described gel-filtration adopts PBS to cook damping fluid.
7. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 1, it is characterized in that: described bacteria breaking process is to carry out in the Tris-HCl damping fluid.
8. as the purification technique of claim 1 to 3,5 to 7 arbitrary described recombinant human interferon alpha 1 bs, it is characterized in that: described Expanded Bed Adsorption adopts the negatively charged ion gel adsorber.
9. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 8, it is characterized in that: described negatively charged ion gel adsorber is STREAMLINE DEAE.
10. as the purification technique of claim 1 to 3,5 to 7 arbitrary described recombinant human interferon alpha 1 bs, it is characterized in that: described Expanded Bed Adsorption adopts the Tris-HCl damping fluid of pH value for 7.0-8.0, and adopts the method for salt concn wash-out.
11. the purification technique of recombinant human interferon alpha 1 b as claimed in claim 10 is characterized in that: described damping fluid employing pH value is 7.4 Tris-HCl.
12. the purifying process of recombinant human interferon alpha 1 b as claimed in claim 10 is characterized in that: described salt concn wash-out adopts the pH7.4 50mM Tris-HCl that contains 0.2-0.5M NaCl to do elutriant.
13. the purifying process of recombinant human interferon alpha 1 b as claimed in claim 12 is characterized in that: it is that 7.4 50m MTris-HCl do elutriant that described salt concn wash-out adopts the pH value that contains 0.4M NaCl.
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| CN1408873A (en) * | 2002-07-30 | 2003-04-09 | 复旦大学 | process for efficiently purifying gene engineering bacteria recombination expression product and its use |
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