CN101003564A - Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast - Google Patents
Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast Download PDFInfo
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Abstract
This invention relates to a method for separating and purifying recombinant hepatitis B virus surface antigen expressed in Hansenula polymorpha. The method comprises: (1) crushing culture solution of recombinant B virus surface antigen expressed in Hansenula polymorpha till 50-90% cells are crushed; (2) centrifuging to remove cell debris, adjusting pH value and electrical conductivity, performing anion exchange chromatography, and collecting the eluates with activity higher than 20%; (3) incorporating the eluates, adjusting the pH value and electrical conductivity, and performing hydrophobic chromatography; (4) concentrating the eluate with ultrafiltration membrane; (5) separating by a gel filtration column to obtain recombinant B virus surface antigen expressed in Hansenula polymorpha with purity higher than 99%. The method has such advantages as high recovery rate, high product purity, few steps, and short time.
Description
Technical field
The invention belongs to the protein separation field, specifically relate to a kind of method of recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast.
Background technology
Hepatitis B virus serious threat human health.At present, the inoculation Hepatitis B virus vaccine is still the main means that the control hepatitis B virus spreads.Owing to have viruses such as carrying HIV and originate problem such as limited, come from the blood source Hepatitis B virus vaccine in hepatitis B virus carriers's blood---hepatitis B surface antigen (Hepatitis B Surface Antigen, hereinafter to be referred as HBsAg)---banned by the World Health Organization, the substitute is reconstituted hepatitis B vaccine.The system that routine is used for HBsAg expression mainly contains mammalian cell, yeast cell and bovine vaccine system etc.Wherein, yeast cell extremely favor of manufacturer owing to have far above the expression amount of zooblast is by the Hepatitis B virus vaccine occuping market dominant position of yeast cell to express.The yeast cell that is usually used in expressing Hepatitis B virus vaccine mainly contains brewing yeast cell (Saccharomyces cerevisiae), pichia spp cell (Pichia pastorius) and debaryomyces hansenii cell (Hansenula polymorpha).Though the HBsAg of various yeast cell to express all belongs to intracellular product, before separation and purification, all need at first carry out cytoclasis, but the hepatitis B surface antigen that different strains of Yeast is expressed is different on physics, chemistry and immunological properties, adapt with it, its purifying process is different, in addition, the dominant company that produces Hepatitis B virus vaccine differs from one another on the purifying process of HBsAg and production cost.
Compare with pichia spp with yeast saccharomyces cerevisiae, it is higher that debaryomyces hansenii is used for the HBsAg expression expression amount.In general, the yeast saccharomyces cerevisiae expression amount is minimum, and the expression amount of pichia spp is also less than 100mg/L, and the employing expressed by Hansenula yeast, its expression amount can reach 200～700mg/L.Nineteen ninety-five, Argentina's approval Laboratorio Pablo Cassara (LPC) manufacturer entitling is AgB
The debaryomyces hansenii recombinant HBsAg, after 2 years, WHO approval Korea S Green Cross (KGCC) manufacturer of company entitling is HepavaxGene
The debaryomyces hansenii recombinant HBsAg.
The separation and purification process of the HBsAg of existing expressed by Hansenula yeast is mainly taked PEG precipitation, diatomite adsorption, ion exchange chromatography, ultracentrifugation and gel permeation chromatography technology.Wherein, specific adsorption is the method for the most effective removal host cell proteins, and this process realizes adsorption and desorption by control pH and temperature variation, makes the single step yield reach 80%, obtains the purifying multiple about 10 simultaneously.Ion exchange chromatography and ultrafiltration are used for further reduced volume and remove the lipid in most of host source.The CsCl ultracentrifugation is used for removing at last the impurity in host source, and follow-up gel-filtration then is used to remove CsCl.But all there is certain problem in above-mentioned technology.For example, the absorption carrier life-span is limited, and operating process is extremely loaded down with trivial details, and the rate of recovery is lower.And the ultracentrifugation method is utilized target protein and the difference of impurity on density, surface charge and size, completely different with the chromatography technological principle, the influence that protein receptor arrives in the purge process is less, does not relate to the protein subunit depolymerization substantially, and activity recovery is higher relatively, proterties is good and more stable, but it is less that its shortcoming is a treatment capacity, and the purifying cycle is longer, and the labor intensive material resources are more, restrict industrial scale, and on purity and DNA residual quantity, be not so good as chromatography technology; And the employing chromatographic technique; processing step is more relatively; hydrophobic chromatography that is utilized or ion exchange chromatography process are for want of to polymerized methylene target protein particulate sfgd.; all can be influential in the absorption-desorption process to proteinic structure, state; may cause the subunit depolymerization to a certain extent and change proteic three, quaternary structure; make the albumen instability of purifying, be easy to occur protein denaturation, polymerization, finally cause activity recovery very low.Therefore, disadvantages such as (being generally less than 20%) that above-mentioned purifying process ubiquity step is various, the cycle is long, activity recovery is on the low side.
Summary of the invention
The objective of the invention is to overcome defective such as the step that exists in the separation and purification process of HBsAg of existing expressed by Hansenula yeast is various, the cycle is long, activity recovery is on the low side, thereby the method for the recombination hepatitis B surface antigen of the separation and purification expressed by Hansenula yeast that a kind of technology is simple, the cycle is short, cost is low, purity is high, activity recovery is higher is provided.
Purpose of the present invention is achieved through the following technical solutions:
The method of the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast provided by the invention as shown in Figure 1, is the method for technology such as a kind of integrating cell fragmentation, chromatography and membrane sepn, specifically comprises following step:
1) cytoclasis
Get the recombination hepatitis B surface antigen nutrient solution of the expressed by Hansenula yeast that the common process fermentation culture obtains, by debaryomyces hansenii cell centrifugal and that washing is used to express hepatitis B surface antigen, place high pressure homogenizer or ball mill then, add and to account for the enzyme inhibitors (as PMSF) that the TritonX-100 that treats broken liquid weight ratio 0.05～0.3% and final concentration are 0.1～10mM, broken 1～6 time repeatedly, reach 50～90% until cell crashing ratio;
2) ion exchange chromatography
Cell after the fragmentation with the centrifugal cell debris of removing of the rotating speed of 5000～20000g, regulate pH value to 5.5～8.5 with 1.0M NaOH and 1.0MHCl, and specific conductivity is carried out anion-exchange chromatography then less than 5.0～20mS/cm;
The aglucon of described anion-exchange chromatography medium is DEAE (DEAE) or quaternary ammonium salt (Q), elutriant is for containing 0.1～1.0M inorganic salt (ammonium sulfate, sodium-chlor or sodium sulfate, 10～50mM damping fluid (pH5.5～8.5) down together), flow velocity is 1.0～20ml/ (hml gel), finish the whole process of ion exchange chromatography in proper order according to balance, charging, drip washing, wash-out, the regeneration of routine, the monitoring of ELISA method, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%;
3) hydrophobic chromatography
The recombination hepatitis B surface antigen component of the active expressed by Hansenula yeast that the merging anion-exchange chromatography obtains, adding inorganic salt (ammonium sulfate, sodium sulfate or sodium-chlor) is 50～200ms/cm until its specific conductivity, regulate pH to 5.5～8.5 with 1.0M NaOH and 1.0M HCl then, carry out hydrophobic chromatography then;
The aglucon of the medium of described hydrophobic chromatography is butyl (Butyl), phenyl (Phenyl) or octyl group (Octyl), use the buffer A (10～50mM damping fluid that contains 5～20wt% ammonium sulfate successively, pH5.5～8.5) balance, charging, drip washing, re-use 100% buffer B (10～50mM damping fluid, pH5.5～8.5) gradient elution, use the regeneration of 0.5～1.0M NaOH or 20～40wt% Virahol at last, the whole process flow rate control is at 1.0～20ml/ (hml gel), the monitoring of ELISA method, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%;
4) ultrafiltration and concentration
To the hydrophobic chromatography obtained component, the employing molecular weight cut-off is that the ultra-filtration membrane of 100～500KD repeats the sample one or many to concentrate, and protein concn is 0.1～1.0mg/ml in sample, and flow velocity is 10～100cm/s during ultrafiltration, and pressure is below 0.3MPa;
5) gel-filtration
The concentrated solution that step 4) is obtained uses gel-filtration column, carries out gel-filtration according to balance (1～2 column volume), charging, wash-out, the reequilibrate of routine, obtains the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% at last;
Described gel-filtration column is Sepharose 4FF or 6FF, feed volume is 0.5～5% of a gel column volume, moving phase is the damping fluid of pH5.5～7.5 that contain 0.05～0.5M ammonium sulfate, sodium sulfate or sodium-chlor, and it is constant that the whole process flow velocity keeps, and is 0.05～0.5ml/ (hml gel);
Each step of aforesaid method is all carried out in 2～30 ℃ of temperature ranges.
The method of the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast provided by the invention is tested at laboratory scale, 500 person-portion lab scales and 5000 person-portion pilot scales respectively.From cytoclasis, sample passes through anion-exchange chromatography, hydrophobic chromatography, ultrafiltration and gel-filtration successively after centrifugal pre-treatment.In purge process, ion exchange chromatography can be removed host protein and the part lipid material more than 85%.Hydrophobic chromatography can further be removed foreign protein and the part lipid material more than 90%.Gel permeation chromatography can further be removed residual most impurity, makes final sample purity greater than more than 99%, reaches the pure and mild chromatographically pure of electrophoresis.Compare with the prior art of Documentary Records, the present invention has following characteristics:
1) purifying activity recovery height on average can reach (triplicate experiment) more than 30%, and purity can reach more than 99%;
2) the purifying process operation steps is few, is connected compact between step, and the operational cycle is short;
3) avoid using the high equipment of price such as ultracentrifuge, cost is relatively low;
4) production technique is simple, can use the self-control chromatography media, and production cost is low;
5) whole process adopts chromatography and membrane separation technique to combine, and is easy to be amplified to commercial scale production;
In sum, the invention provides a kind of method of hepatitis B surface antigen of easy, quick, the separation and purification expressed by Hansenula yeast that is easy to amplify, have bigger actual application value.
Description of drawings
Fig. 1 is the schema of the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast provided by the invention;
Fig. 2 is embodiment 4 intermediate ion displacement chromatography collection of illustrative plates;
Fig. 3 is a hydrophobic chromatography HIC collection of illustrative plates among the embodiment 4;
Fig. 4 is a gel-filtration GF collection of illustrative plates among the embodiment 4;
Fig. 5 is the SDS-PAGE collection of illustrative plates of the purified product of embodiment 1 and 4;
Fig. 6 is the HPSEC collection of illustrative plates of the purified product of embodiment 1 and 4.
Embodiment
Get the recombination hepatitis B surface antigen culture supernatant of the expressed by Hansenula yeast that 200ml common process fermentation culture obtains, earlier by debaryomyces hansenii cell centrifugal and that washing is used to express hepatitis B surface antigen, place high pressure homogenizer then, add and account for the PMSF that treats broken liquid weight ratio 0.05%Triton X-100 and 1mM, broken 6 times repeatedly, cell crashing ratio reaches 90%.
With the centrifugal 20min under the rotating speed of 20000g of cell after the fragmentation, remove cell debris, supernatant liquor is adjusted pH value to 5.5 with 1.0MNaOH and 1.0M HCl, and specific conductivity is less than 5.0mS/cm, be fed to (GE Healthcare in the Q SepharoseFF ion exchange column then, 30cm * 15cm I.D.), column volume (CV) is 2000ml, and flow velocity is 1.0ml/ (hml gel).The chromatography process is carried out damping fluid I (20mM phosphate buffered saline buffer (PB) successively, pH5.5) steps such as balance, charging, damping fluid I reequilibrate, 0～100% damping fluid II (1.0M NaCl+ damping fluid I) continuous gradient wash-out, damping fluid I regeneration, collect each elution peak component, adopt the ELISA method to measure protein concentration and target protein antigenic activity, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
Merge the active ingredient that above-mentioned anion-exchange chromatography obtains, after adjusting specific conductivity and pH to 50ms/cm and 5.5 respectively with ammonium sulfate and NaOH solution successively, be fed to is PB damping fluid (the damping fluid III of 50ms/cm to specific conductivity with being added with ammonium sulfate in advance, pH5.5) equilibrated Octyl QZT hydrophobic chromatography post (national biochemical engineering center, Beijing, 40cm * 5.5cm I.D.), column volume 1000ml, flow velocity are 1.0ml/ (hml gel).Charging is after carry out gradient elution with 20mM PB (pH5.5) behind the damping fluid III reequilibrate, collects to contain the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
To the active peak of hydrophobic chromatography gained sample, the ultrafiltration and concentration of hollow-fibre membrane ultra-filtration membrane (GEHealthercare) that through molecular weight cut-off is 100KD is to about the 10ml, and protein concn is 0.1mg/ml in the sample; The hollow-fibre membrane flow velocity is 10cm/s during ultrafiltration, and pressure-controlling is below 0.3MPa.
Get sample behind the 10ml ultrafiltration and concentration, be fed to Sepharose 4FF gel-filtration column (GE Healthcare, 100cm * 5.0cm, I.D.), column volume is 2000ml, flow velocity remains 0.5ml/ (hml gel), moving phase is the 20mM PB that contains 0.10M sodium sulfate, pH5.5, detect the protein content and the activity of elution peak component respectively, obtain the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% at last, the active peak of gained component is 32% with respect to the activity recovery of cytoclasis supernatant liquor.
Get the recombination hepatitis B surface antigen culture supernatant of the expressed by Hansenula yeast that 200ml common process fermentation culture obtains, earlier by debaryomyces hansenii cell centrifugal and that washing is used to express hepatitis B surface antigen, place on the ball mill then, add and account for the PMSF that treats broken liquid weight ratio 0.3%Triton X-100 and 0.1mM, broken 2 times repeatedly, cell crashing ratio reaches 50%.
With the centrifugal 30min under the rotating speed of 6000g of cell after the fragmentation, remove cell debris, supernatant liquor is adjusted pH value to 8.5 with 1.0MNaOH and 1.0M HCl, and specific conductivity is less than 20mS/cm, be fed to (GE Healthcare in the Q SepharoseFF ion exchange column then, 30cm * 15cm I.D.), column volume (CV) is 2000ml, and flow velocity is 20ml/ (hml gel).The chromatography process is carried out damping fluid I (20mM Tris-HCl damping fluid successively, pH8.5) steps such as balance, charging, damping fluid I reequilibrate, 0～100% damping fluid II (1.0M sodium sulfate+damping fluid I) continuous gradient wash-out, damping fluid I regeneration, collect each elution peak component, adopt the ELISA method to measure protein concentration and target protein antigenic activity, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
Merge the active ingredient that above-mentioned anion-exchange chromatography obtains, after adjusting specific conductivity and pH to 200ms/cm and 5.5 respectively with NaCl and NaOH solution successively, be fed to is PB damping fluid (damping fluid III) equilibrated Butyl-S Sepharose 6FF hydrophobic chromatography post (the GE Healthcare of 200ms/cm to specific conductivity with adding NaCl in advance, 40cm * 5.5cm I.D.), column volume 1000ml, flow velocity are 20ml/ (hml gel).Charging is after carry out gradient elution with 20mM PB (pH5.5) behind the damping fluid C reequilibrate, collects to contain the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
To the active peak of hydrophobic chromatography gained sample, be that (Millipore USA) divides three ultrafiltration and concentration to about the 20ml, and protein concn is 1.0mg/ml in the sample for the flat ultra-filtration membrane of 300KD through molecular weight cut-off; The hollow-fibre membrane flow velocity is 50cm/s during ultrafiltration, and pressure-controlling is below 0.2MPa.
Get sample behind the 10ml ultrafiltration and concentration, be fed to Sepharose 6FF gel-filtration column (GE Healthcare, 100cm * 5.0cm, I.D.), column volume is 2000ml, flow velocity remains 0.05ml/ (hml gel), moving phase is the 20mM PB that contains 0.2MNaCl, pH7.0, detect the protein content and the activity of elution peak component respectively, obtain the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% at last, the active peak of gained component is 35% with respect to the activity recovery of cytoclasis supernatant liquor.
Get the recombination hepatitis B surface antigen culture supernatant of the expressed by Hansenula yeast that 50ml common process fermentation culture obtains, earlier by debaryomyces hansenii cell centrifugal and that washing is used to express hepatitis B surface antigen, place on the high pressure homogenizer then, add and account for the PMSF that treats broken liquid weight ratio 0.1%Triton X-100 and 10mM, broken 3 times repeatedly, cell crashing ratio reaches 70%.
With the centrifugal 20min under the rotating speed of 10000g of cell after the fragmentation, remove cell debris, supernatant liquor is adjusted pH value to 7.0 with 1.0MNaOH and 1.0M HCl, and specific conductivity is less than 10mS/cm, be fed to (national biochemical engineering center in the DEAE QZTFF ion exchange column then, 30cm * 5.5cm I.D.), column volume (CV) is 500ml, and flow velocity is 10ml/ (hml gel).The chromatography process is carried out damping fluid I (20mM Tris-HCl successively, pH 7.0) balance, charging, buffer A reequilibrate, 0%, 20%, 40%, steps such as 100% damping fluid II (1.0M NaCl+ damping fluid I) step gradient elution, damping fluid I regeneration, collect each elution peak component, adopt the ELISA method to measure protein concentration and target protein antigenic activity, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
Merge the active ingredient that above-mentioned anion-exchange chromatography obtains, after adjusting specific conductivity and pH to 96ms/cm and 7.0 respectively with ammonium sulfate and NaOH solution successively, be fed to is Tris-HCl damping fluid (damping fluid III) equilibrated Butyl-S QZT FF hydrophobic chromatography post (the national biochemical engineering center of 96ms/cm to specific conductivity with adding ammonium sulfate in advance, Beijing, 20cm * 5.5cm I.D.), column volume 200ml, flow velocity are 20ml/ (hml gel).Charging is after carry out gradient elution with 20mM Tris-HCl (pH7.0) behind the damping fluid C reequilibrate, collects to contain the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%.
To the active peak of hydrophobic chromatography gained sample, through molecular weight cut-off be 500KD the hollow-fibre membrane ultra-filtration membrane (Pall company, USA) ultrafiltration and concentration is to about the 10ml, protein concn is 0.5mg/ml in the sample; The hollow-fibre membrane flow velocity is 10cm/s during ultrafiltration, and pressure-controlling is below 0.1MPa.
Get sample behind the 10ml ultrafiltration and concentration, be fed to Sepharose 4FF gel-filtration column (national biochemical engineering center, Beijing, 100cm * 2.6cm, I.D.), column volume is 400ml, flow velocity remains 0.5ml/ (hml gel), moving phase is the 20mM Tris-HCl that contains 0.3M sodium sulfate, pH7.5, detect the protein content and the activity of elution peak component respectively, obtain the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% at last, the active peak of gained component is 44% with respect to the activity recovery of cytoclasis supernatant liquor.
Sample after cytoclasis liquid among the 4000ml embodiment 1 is centrifugal, use 50mM Tris-HCl, pH8.5 damping fluid (buffer A) is diluted to specific conductivity less than 20ms/cm, and with behind 1.0M NaOH and the 1.0M HCl adjusting pH to 8.5, be fed to Q QZT FF ion exchange column (the national biochemical engineering center of crossing with the buffer A balance, 100cm * 30cm I.D, CV=30L), flow velocity 5ml/ (hml gel), after charging is finished successively through the buffer A reequilibrate, 15%, 50% and 100% buffer B (1.0M NaCl+ buffer A) wash-out, the wash-out collection of illustrative plates as shown in Figure 2, adopt the ELISA method to measure protein concentration and target protein antigenic activity, collection contains the elutriant 10000ml of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%, the calculated activity rate of recovery and purifying multiple.
Get the active peak of ion exchange chromatography gained component, add saturated ammonium sulphate solution to its specific conductivity and approach damping fluid C (the 20mM PB that contains 15% ammonium sulfate, specific conductivity is about 120ms/cm), regulate its pH to 8.5 with 0.1M NaOH again, be fed to HIC post (Phenyl QZT FF, the national biochemical engineering center used in advance after the damping fluid C balance, Beijing, 50cm * 15cm I.D, CV=5.0L), flow velocity 10ml/ (hml gel).After charging is finished according to reequilibrate, damping fluid D (10mM Tris-HCl, pH8.5), 1.0M NaOH wash-out.Collect damping fluid D elution peak component, the wash-out collection of illustrative plates is collected and is contained the elutriant 5000ml of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20% as shown in Figure 3.
With the sample at P1 peak (as shown in Figure 3) among the hydrophobic chromatography HIC, be that 300KD ultra-filtration membrane (GE Healthcare) carries out ultrafiltration with molecular weight cut-off, sample volume is concentrated to about 500ml, protein concn is 0.5mg/ml in the sample; The hollow-fibre membrane flow velocity is 100cm/s during ultrafiltration, and pressure-controlling is below 0.3MPa.By active and determination of protein concentration result as can be known, this enrichment step does not have loss of activity.
For ultrafiltration and concentration gained sample, with gel-filtration column (Sepharose 4FF, GE Healthcare, 100cm * 11.3cm, I.D. CV=10.2L) carries out the purifying experiment, moving phase is the 50mM PB solution that contains 0.05M NaCl, the wash-out collection of illustrative plates obtains the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% as shown in Figure 4 at last, and the active peak of gained component is 29% with respect to the activity recovery of cytoclasis supernatant liquor.
In this pilot experiment, the total operating time of technology that comprises ion exchange chromatography, hydrophobic chromatography, gel-filtration and ultrafiltration and concentration was less than 40 hours, and the ult rec of whole technology is 31.5%, greater than 30%.
The recombination hepatitis B surface antigen of the expressed by Hansenula yeast that obtains in embodiment 1 and 4 is carried out the SDS-PAGE electrophoretic analysis.Gel-filtration gained final sample is concentrated, reduced form SDS-PAGE analyzes its purity then, as shown in Figure 5, and wherein, first swimming lane is lower molecular weight Marker, and second and third swimming lane is respectively the sample of embodiment 4 gained of laboratory scale embodiment 1 and pilot experiment.Thus the result as seen, the sample that embodiment 1 and 4 obtains all only contains a master tape, its molecular weight is about 24KD, is the S protein monomer of HBsAg.This presentation of results can obtain electrophoresis pure sample product through the recombination hepatitis B surface antigen of the expressed by Hansenula yeast of method purifying provided by the invention, and purity is greater than 99%.
Embodiment 6, efficient molecular-exclusion chromatography (HPSEC) are analyzed
The recombination hepatitis B surface antigen of the expressed by Hansenula yeast that obtains in embodiment 1 and 4 is carried out efficient molecular-exclusion chromatography (HPSEC) analysis.After gel-filtration gained final sample concentrated 20 and 50 times respectively, with sample preparation liquid (0.1MDTT and 1: 50Tween 80 equal-volumes are than mixed solution) balanced mix, carry out HPSEC and analyze (TSK G5000 post, 300mm * 7.8mm), experimental result as shown in Figure 6, Fig. 6-A and Fig. 6-B are respectively the sample of embodiment 4 gained of laboratory scale embodiment 1 and pilot experiment.As shown in Figure 6,2 kinds of samples all do not contain any foreign protein, and promptly the recombination hepatitis B surface antigen through the expressed by Hansenula yeast of method purifying provided by the invention does not contain foreign protein, and sample reaches chromatographically pure, and purity is greater than more than 99%.
Embodiment 7, sample protein assay
Adopt the Lowry method to carry out the determination of protein concentration experiment to gained GF sample in three batches of pilot plant tests, the result shows as can be known, on average can obtain 42mg target protein antigen HBsAg by every liter of cytoclasis supernatant liquor through above-mentioned separation purifying technique, every batch of test on average can obtain the 168mg target protein.
Claims (9)
1, a kind of method of recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast, it comprises following step:
1) cytoclasis
Get the recombination hepatitis B surface antigen nutrient solution of the expressed by Hansenula yeast that the common process fermentation culture obtains, by debaryomyces hansenii cell centrifugal and that washing is used to express hepatitis B surface antigen, place high pressure homogenizer or ball mill then, it is the enzyme inhibitors of 0.1～10mM that adding accounts for Triton X-100 and the final concentration for the treatment of broken liquid weight ratio 0.05～0.3%, broken 1～6 time repeatedly, reach 50～90% until cell crashing ratio;
2) ion exchange chromatography
With cell after the fragmentation with the centrifugal cell debris of removing of the rotating speed of 5000～20000g, regulate pH value to 5.5～8.5 with 1.0M NaOH and 1.0MHCl, and specific conductivity is less than 5.0～20mS/cm, carry out anion-exchange chromatography then, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%;
3) hydrophobic chromatography
The recombination hepatitis B surface antigen component of the active expressed by Hansenula yeast that the merging anion-exchange chromatography obtains, adding inorganic salt is 50～200ms/cm until its specific conductivity, regulate pH to 5.5～8.5 with 1.0M NaOH and 1.0M HCl then, carry out hydrophobic chromatography then, collection contains the elutriant of the recombination hepatitis B surface antigen activity recovery of expressed by Hansenula yeast greater than the ultraviolet absorption peak more than 20%;
4) ultrafiltration and concentration
To the hydrophobic chromatography obtained component, the employing molecular weight cut-off is that the ultra-filtration membrane of 100～500KD repeats the sample one or many to concentrate, and protein concn is 0.1～1.0mg/ml in sample, and flow velocity is 20～100cm/s during ultrafiltration, and pressure is below 0.3Mpa;
5) gel-filtration
The concentrated solution that step 4) is obtained uses gel-filtration column to carry out gel-filtration, obtains the recombination hepatitis B surface antigen of purity greater than the expressed by Hansenula yeast more than 99% at last;
Each step of aforesaid method is all carried out in 2～30 ℃ of temperature ranges.
2, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: the aglucon of anion-exchange chromatography medium described step 2) is DEAE or quaternary ammonium salt.
3, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: the elutriant of anion-exchange chromatography described step 2) is the 10～50mM damping fluid that contains 0.1～1.0M inorganic salt.
4, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 3 is characterized in that: the inorganic salt in the described elutriant are ammonium sulfate, sodium-chlor or sodium sulfate.
5, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: the inorganic salt of described step 3) are ammonium sulfate, sodium sulfate or sodium-chlor.
6, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: the aglucon of the medium of the hydrophobic chromatography of described step 3) is butyl, phenyl or octyl group.
7, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1, it is characterized in that: during the hydrophobic chromatography of described step 3), use successively and contain 5～20wt% ammonium sulfate, pH5.5～8.5,10～50mM damping fluid balance, charging, drip washing, re-use pH5.5～8.5,10～50mM buffer solution for gradient elution, use the regeneration of 0.5～1.0M NaOH or 20～40wt% Virahol at last.
8, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: in the described step 5), gel-filtration column is Sepharose 4FF or 6FF, and feed volume is 0.5～5% of a gel column volume.
9, the method for the recombination hepatitis B surface antigen of separation and purification expressed by Hansenula yeast as claimed in claim 1 is characterized in that: in the described step 5), moving phase is the damping fluid of pH5.5～7.5 that contain 0.05～0.5M ammonium sulfate, sodium sulfate or sodium-chlor.
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Cited By (9)
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| CN103319572A (en) * | 2012-03-20 | 2013-09-25 | 北京天坛生物制品股份有限公司 | Hepatitis B surface antigen purification method |
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| CN108047316A (en) * | 2018-01-04 | 2018-05-18 | 南京赛威信生物医药有限公司 | The isolation and purification method of recombination hepatitis B core antigen |
| CN110423280A (en) * | 2019-07-31 | 2019-11-08 | 北京泓恩生物科技有限公司 | The preparation method of human papilloma virus and heat shock protein recombinant protein |
| CN111166873A (en) * | 2019-12-27 | 2020-05-19 | 深圳康泰生物制品股份有限公司 | Crude purification process of hand-foot-and-mouth disease vaccine antigen expressed by recombinant hansenula polymorpha, vaccine stock solution and preparation method of vaccine stock solution |
| CN113388609A (en) * | 2021-06-21 | 2021-09-14 | 上海碧博生物医药科技有限公司 | Purification method of large plasmid DNA |
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| US5156968A (en) * | 1988-06-24 | 1992-10-20 | Genentech, Inc. | Purified yeast ubiquitin hydrolase |
| CN1345927A (en) * | 2000-09-30 | 2002-04-24 | 暨南大学 | Recombination basic fibroblast growth factor (bFGF) gene engineering microzyme and production method thereof |
| CN1384205A (en) * | 2002-05-22 | 2002-12-11 | 四川汉龙高新技术开发有限公司 | Large-scale protein process of recombinant human liver cell growth factor rhHGF |
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| CN103319572A (en) * | 2012-03-20 | 2013-09-25 | 北京天坛生物制品股份有限公司 | Hepatitis B surface antigen purification method |
| CN103319572B (en) * | 2012-03-20 | 2015-06-24 | 北京天坛生物制品股份有限公司 | Hepatitis B surface antigen purification method |
| WO2015043160A1 (en) * | 2013-09-29 | 2015-04-02 | 深圳康泰生物制品股份有限公司 | Method for promoting formation of hepatitis b surface antigen covalently crosslinked particles expressed by saccharomyces cerevisiae |
| CN104745492B (en) * | 2015-04-21 | 2018-10-30 | 南京赛威信生物医药有限公司 | A kind of application of Hansenula yeast mutant and its expression vector in terms of recombinant protein expression |
| CN104745492A (en) * | 2015-04-21 | 2015-07-01 | 江苏赛锘威生物医药有限公司 | Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression |
| CN105693826A (en) * | 2016-03-11 | 2016-06-22 | 安徽智飞龙科马生物制药有限公司 | Method for separating and purifying hansenula polymorpha expressed recombinant HBsAgs (hepatitis B surface antigens) and application of recombinant HBsAgs |
| CN108047316A (en) * | 2018-01-04 | 2018-05-18 | 南京赛威信生物医药有限公司 | The isolation and purification method of recombination hepatitis B core antigen |
| CN110423280A (en) * | 2019-07-31 | 2019-11-08 | 北京泓恩生物科技有限公司 | The preparation method of human papilloma virus and heat shock protein recombinant protein |
| CN110423280B (en) * | 2019-07-31 | 2021-02-02 | 北京泓恩生物科技有限公司 | Preparation method of human papilloma virus and heat shock protein recombinant protein |
| CN111166873A (en) * | 2019-12-27 | 2020-05-19 | 深圳康泰生物制品股份有限公司 | Crude purification process of hand-foot-and-mouth disease vaccine antigen expressed by recombinant hansenula polymorpha, vaccine stock solution and preparation method of vaccine stock solution |
| CN111166873B (en) * | 2019-12-27 | 2023-12-15 | 深圳康泰生物制品股份有限公司 | Crude and pure process for recombinant hansenula polymorpha expressed hand-foot-and-mouth disease vaccine antigen, vaccine stock solution and preparation method thereof |
| CN113388609A (en) * | 2021-06-21 | 2021-09-14 | 上海碧博生物医药科技有限公司 | Purification method of large plasmid DNA |
| CN113388609B (en) * | 2021-06-21 | 2022-09-13 | 上海碧博生物医药科技有限公司 | Purification method of large plasmid DNA |
| CN114478745A (en) * | 2022-03-30 | 2022-05-13 | 南京京达生物技术有限公司 | Separation and purification method of recombinant procalcitonin PCT |
| CN114478745B (en) * | 2022-03-30 | 2023-12-15 | 南京京达生物技术有限公司 | Separation and purification method of recombinant procalcitonin PCT |
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Assignee: Hualan Bio-Engineering Co., Ltd. Assignor: Institute of Process Engineering, Chinese Academy of Sciences Contract record no.: 2011410000116 Denomination of invention: Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast Granted publication date: 20090923 License type: Exclusive License Open date: 20070725 Record date: 20110809 |
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