CN106222221A - Prepare the purification process of recombined human granulocyte stimulating factors stock solution - Google Patents
Prepare the purification process of recombined human granulocyte stimulating factors stock solution Download PDFInfo
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- CN106222221A CN106222221A CN201610634431.2A CN201610634431A CN106222221A CN 106222221 A CN106222221 A CN 106222221A CN 201610634431 A CN201610634431 A CN 201610634431A CN 106222221 A CN106222221 A CN 106222221A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
Abstract
The present invention relates to biological technical field, particularly to preparing the purification process of recombined human granulocyte stimulating factors stock solution, step is as follows: 1) selects engineering bacteria e. coli jm109 to do bacterial strain and carries out seed liquor cultivation；2) collect seed liquor to cultivate, ferment and thalline；3) the purest；4) consummate；5) preservation of stock solution；Present invention employs ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, molecular sieve chromatography, obtain recombined human granulocyte stimulating factors stock solution, improve purification efficiency and purification quality, after testing, the stock solution of gained presses Chinese Pharmacopoeia and European Pharmacopoeia detection, purity respectively reaches 95% and more than 98%, and specific activity has respectively reached 6 × 107U/mg and 1.0 × 108More than U/mg, respectively reaches national standard and Europe touchstone.
Description
Technical field
The present invention relates to biological technical field, particularly to the purification side preparing recombined human granulocyte stimulating factors stock solution
Method.
Background technology
Recombined human granulocyte stimulating factors, G-CSF, match Gree, match is strong, southeast Jilin (Filgrastim, Recombinant
Human Granulocyte-Colony Stimulating Factor, rhG-CSF) it is that specific action is in grain system CFU-GM, rush
Enter it to ripe neutrophilic granulocyte propagation, the hemopoietic growth factor of differentiation；Its Main Function is as follows: after (1) promotes bone marrow transplantation
The recovery of neutrophilic granulocyte；(2) neutrophilic granulocytopenia after treatment chemotherapy of tumors；(3) treatment is combined with myelodysplastisches
The neutrophilic granulocytopenia of simulator sickness；(4) treatment is with the Neutrophilic granulocytopenia of hypoplastic anemia；(5) treat congenital
Property, idopathic neutropenia.
The purification process of a patent No. CN1030141000 B recombined human granulocyte stimulating factors, the method includes following step
Rapid: a, selection engineering bacteria do bacterial strain and carry out fermentation culture；B, fermentation culture gained thalline is carried out separation and the washing of inclusion body,
Obtain refined inclusion body；C, refined inclusion body is carried out renaturation, obtain renaturation product；D, renaturation product is carried out anion column layer
Analysis processes, and obtains Anionic column chromatography product；E, Anionic column chromatography product is carried out Cationic column chromatography, obtain cation
Column chromatography product；F, Cationic column chromatography product is carried out fine separation, use reverse phase filler column chromatography to process, obtain recombined human
Granulocyte colony-stimulating factor；Using the reverse phase filler fine separation of this patent, rhG-CSF electrophoresis purity is brought up to by 90%
99%, HPLC purity is brought up to 99% by 90%, and its ratio is lived by 0.8 × 107U/mg brings up to 3 × 108U/mg；Visible, use
The purifying process of the recombinant human granulocyte colony stimulating factor of the present invention, can be greatly improved the purity of rhG-CSF, ratio
Live, and stability.
Summary of the invention
The invention provides a kind of new purification process preparing recombined human granulocyte stimulating factors stock solution, the product of its purification
Product can meet national standard can meet again Europe touchstone, and the method specifically includes:
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C；
2) collect seed liquor to cultivate, ferment and thalline；
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution；
4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography, it is thus achieved that weight
Group human granulocyte stimulating factors stock solution；
5) preservation of stock solution.
Described step 2) collect seed liquor cultivation, fermentation and thalline, being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
Described step 3) the purest: inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution are prepared, specifically
Implementing step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 10-100mmol/L Tris-HCl solution are fully stirred in the ratio of 1:10
Even, centrifugal collecting precipitation；Again in the precipitation collected, add 10-100mmol/L Tris-HCl solution in the ratio of 1:10
The most fully stirring evenly, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, and centrifugal collecting precipitation obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 10-20mmol/L Tris-HCl, 2-4mol/L urea, 1-
The buffer solution of 5mmol/L EDTA is fully stirred evenly in the ratio of 1:15, centrifugal, collects precipitation；
Up walk in the ratio of 1:15 and fully stir evenly after the precipitation obtained adds 10-60mmol/L Tris-HCl solution, centrifugal
Collect precipitation, inclusion body after must washing for such twice；
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 10-100mmol/L Tris-HCl, 5-9mol/L urea, 1-5mmol/L
EDTA(pH8.5), in buffer, in the above-mentioned buffer solution having added inclusion body, add beta-mercaptoethanol, sealing, continue to stir
Mix, obtain inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 10-100mmol/L Tris-HCl buffer of 6-10 times of volume；
Solubilising is completed the solution 10-100mmol/L Tris-HCl buffer ultrafiltration obtained, and it is multiple to promote to stir oxygen supplement
Property；Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 5-50mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
Described step 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, molecule
Sieve chromatography, it is thus achieved that recombined human granulocyte stimulating factors stock solution, being embodied as step is:
A) ion-exchange chromatography
Filler CM Sepharose FF, with 5-50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid
PH is identical；Ion is exchanged sample solution loading, balances to baseline with 5-50mmol/L NaAc-HAc buffer after end of the sample,
Carry out pre-eluting with 5-50mmol/L NaAc-HAc buffer again, after eluting, delay with 5-50mmol/L NaAc-HAc
Rush liquid balance identical with balance liquid pH to effluent pH, then with 5-50mmol/L NaAc-HAc 0.2-0.8mol/L NaCl
Buffer solution elution, and collect eluting peak；
B) hydrophobic chromatography
With 5-50mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
10-100mmol/L Tris-HCl, 1.0-3.0mol/L (NH4) 2SO4 solution, makes hydrophobic sample solution, and (NH4) 2SO4 is eventually
Concentration is 0.3-0.7mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
10-100mmol/L Tris-HCl solution rinses consistent, finally with 10-100mmol/L Tris-HCl solution to effluent conductance
With 10-100mmol/L Tris-HCl 0.3-0.7mol/L (NH4) 2SO4 solution equilibria, to effluent conductance and balance liquid phase
With；
By hydrophobic chromatography sample solution loading, with 10-100mmol/L Tris-HCl, 0.3-0.7mol/L (NH4) after end of the sample
2SO4 solution is balanced, and balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M (NH4) 2SO4 solution is consistent, enters with 10-100mmol/L Tris-HCl, 0.01-0.15mol/L (NH4) 2SO4 solution
Row eluting, collects eluting peak；
C) ultrafiltration desalination
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 5-50mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ
S/cm, after diluting with 5-50mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II
Filler CM Sepharose FF, with 5-50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid
PH is identical；Ion is exchanged II sample solution loading, balances to base with 5-50mmol/L NaAc-HAc buffer after end of the sample
Line, then 5-50mmol/L NaAc-HAc 0.2-0.8mol/L NaCl eluant solution, collect eluting peak；
E) sieve chromatography
Filler Sephacryl S-100, with 5-50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid
PH is identical；By ion-exchanging eluent loading, afterwards by 5-50mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；
Eluting peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
Described step 5) preservation of stock solution, being embodied as step is:
The preservation of stock solution:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
The method have the advantages that
Present invention employs ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, molecular sieve chromatography, obtain
Obtaining recombined human granulocyte stimulating factors stock solution, improve purification efficiency and purification quality, after testing, the stock solution of gained presses middle traditional Chinese medicines
Allusion quotation and European Pharmacopoeia detection, purity respectively reaches 95% and more than 98%, and specific activity has respectively reached 6 × 107U/mg and 1.0 ×
108More than U/mg, respectively reaches national standard and Europe touchstone.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, but the present invention is not appointed by specific embodiment
What limits.
Embodiment 1
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C.
2) collect seed liquor to cultivate, ferment and thalline.
Being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution.
Being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 10mmol/L Tris-HCl solution are fully stirred evenly in the ratio of 1:10, from
The heart collects precipitation；Fully stir after again adding 10mmol/L Tris-HCl solution in the ratio of 1:10 in the precipitation collected
Even, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, centrifugal, collects precipitation, obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 10mmol/L Tris-HCl, 4mol/L urea, 5mmol/L
The buffer solution of EDTA is fully stirred evenly in the ratio of 1:15, centrifugal collecting precipitation；
Up walk in the ratio of 1:15 and fully stir evenly after the precipitation obtained adds 10mmol/L Tris-HCl solution, centrifugal, receive
Collection precipitation, inclusion body after must washing for such twice；
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 10mmol/L Tris-HCl, 5mol/L urea, 5mmol/L EDTA
In buffer solution (pH8.5), in the above-mentioned buffer having added inclusion body, add beta-mercaptoethanol, sealing, continue stirring,
To inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 10mmol/L Tris-HCl buffer of 6 times of volumes；
Solubilising is completed the solution 10mmol/L Tris-HCl buffer ultrafiltration obtained, and stirs oxygen supplement to promote renaturation；
Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 5mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography, to obtain
Obtain recombined human granulocyte stimulating factors stock solution.
Being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, with 5mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged sample solution loading, balances to baseline with 5mmol/L NaAc-HAc buffer after end of the sample, then use
5mmol/L NaAc-HAc buffer carries out pre-eluting, after eluting, by 5mmol/L NaAc-HAc buffer balance to stream
Going out liquid pH identical with balance liquid pH, then by 5mmol/L NaAc-HAc 0.2mol/L NaCl buffer solution elution, and collection is washed
De-peak.
B) hydrophobic chromatography:
With 5mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
10mmol/L Tris-HCl、 1.0mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4Final concentration of
0.3mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
10mmol/L Tris-HCl solution rinses consistent with 10mmol/L Tris-HCl solution to effluent conductance, finally uses
10mmol/L Tris-HCl 0.3mol/L(NH4)2SO4Solution equilibria is identical with balance liquid to effluent conductance；
By hydrophobic chromatography sample solution loading, with 10mmol/L Tris-HCl, 0.3mol/L (NH after end of the sample4)2SO4Solution
It is balanced, balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M(NH4)2SO4Solution is consistent, with 10mmol/L Tris-HCl, 0.01mol/L (NH4)2SO4Solution carries out eluting, collects
Eluting peak.
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 5mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ s/
Cm, after diluting with 5mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 5mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged II sample solution loading, balances to baseline, then with 5mmol/L NaAc-HAc buffer after end of the sample
5mmol/L NaAc-HAc 0.2-0.8mol/L NaCl eluant solution, collects eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 5mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；By ion-exchanging eluent loading, afterwards by 5mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；Eluting
Peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
5) preservation of stock solution.
Being embodied as step is:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
Embodiment 2
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C.
2) collect seed liquor to cultivate, ferment and thalline.
Being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution.
Being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 25mmol/L Tris-HCl solution are fully stirred evenly in the ratio of 1:10, from
The heart collects precipitation；Fully stir after again adding 25mmol/L Tris-HCl solution in the ratio of 1:10 in the precipitation collected
Even, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, centrifugal, collects precipitation, obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 12mmol/L Tris-HCl, 3mol/L urea, 4mmol/L
The buffer solution of EDTA is fully stirred evenly in the ratio of 1:15, centrifugal, collects precipitation；The precipitation obtained up is walked in the ratio of 1:15
Fully stir evenly after middle addition 30mmol/L Tris-HCl solution, centrifugal, collect precipitation, inclusion body after must washing for such twice.
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 25mmol/L Tris-HCl, 7mol/L urea, 3mmol/L EDTA
In buffer solution (pH8.5), in the above-mentioned buffer having added inclusion body, add beta-mercaptoethanol, sealing, continue stirring,
To inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 25mmol/L Tris-HCl buffer of 7 times of volumes；
Solubilising is completed the solution 25mmol/L Tris-HCl buffer ultrafiltration obtained, and stirs oxygen supplement to promote renaturation；
Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 20mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
It is 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography,
Obtain recombined human granulocyte stimulating factors stock solution.
Being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, with 25mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged sample solution loading, balances to baseline with 25mmol/L NaAc-HAc buffer after end of the sample, then use
Carry out pre-eluting with 25mmol/L NaAc-HAc buffer, after eluting, balance with 25mmol/L NaAc-HAc buffer
Identical with balance liquid pH to effluent pH, then by 25mmol/L NaAc-HAc 0.4mol/L NaCl buffer solution elution, and receive
Collection eluting peak；
B) hydrophobic chromatography:
With 25mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
25mmol/L Tris-HCl、 2mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4Final concentration of 0.5mol/
L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
25mmol/L Tris-HCl solution rinses consistent with 25mmol/L Tris-HCl solution to effluent conductance, finally uses
25mmol/L Tris-HCl 0.5mol/L(NH4)2SO4Solution equilibria is identical with balance liquid to effluent conductance；
By hydrophobic chromatography sample solution loading, with 25mmol/L Tris-HCl, 0.3mol/L (NH after end of the sample4)2SO4Solution
It is balanced, balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M(NH4)2SO4Solution is consistent, with 25mmol/L Tris-HCl, 0.08mol/L (NH4)2SO4Solution carries out eluting, collects
Eluting peak；
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 25mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ s/
Cm, after diluting with 25mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 25mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged II sample solution loading, balances to baseline, so with 25mmol/L NaAc-HAc buffer after end of the sample
Rear 25mmol/L NaAc-HAc 0.5mol/L NaCl eluant solution, collects eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 25mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；By ion-exchanging eluent loading, afterwards by 25mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；Eluting
Peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
5) preservation of stock solution.
Being embodied as step is:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
Embodiment 3
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C.
2) collect seed liquor to cultivate, ferment and thalline.
Being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution.
Being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 60mmol/L Tris-HCl solution are fully stirred evenly in the ratio of 1:10, from
The heart collects precipitation；Fully stir after again adding 60mmol/L Tris-HCl solution in the ratio of 1:10 in the precipitation collected
Even, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, centrifugal, collects precipitation, obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 15mmol/L Tris-HCl, 4mol/L urea, 1mmol/L
The buffer solution of EDTA is fully stirred evenly in the ratio of 1:15, centrifugal collecting precipitation；The precipitation obtained up is walked in the ratio of 1:15
Fully stir evenly after middle addition 60mmol/L Tris-HCl solution, centrifugal, collect precipitation, inclusion body after must washing for such twice；
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 60mmol/L Tris-HCl, 9mol/L urea, 3mmol/L EDTA
(pH8.5), in buffer, in the above-mentioned buffer having added inclusion body, add beta-mercaptoethanol, sealing, continue stirring, obtain
Inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 40mmol/L Tris-HCl buffer of 10 times of volumes；
Solubilising is completed the solution 60mmol/L Tris-HCl buffer ultrafiltration obtained, and stirs oxygen supplement to promote renaturation；
Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 40mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
It is 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography,
Obtain recombined human granulocyte stimulating factors stock solution.
Being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, with 40mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged sample solution loading, balances to baseline with 40mmol/L NaAc-HAc buffer after end of the sample, then use
Carry out pre-eluting with 40mmol/L NaAc-HAc buffer, after eluting, balance with 40mmol/L NaAc-HAc buffer
Identical with balance liquid pH to effluent pH, then by 40mmol/L NaAc-HAc 0.6mol/L NaCl buffer solution elution, and receive
Collection eluting peak；
B) hydrophobic chromatography:
With 40mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
60mmol/L Tris-HCl、 3.0mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4Final concentration of
0.6mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
60mmol/L Tris-HCl solution rinses consistent with 60mmol/L Tris-HCl solution to effluent conductance, finally uses
60mmol/L Tris-HCl 0.6mol/L(NH4)2SO4Solution equilibria is identical with balance liquid to effluent conductance；
By hydrophobic chromatography sample solution loading, with 60mmol/L Tris-HCl, 0.6mol/L (NH after end of the sample4)2SO4Solution
It is balanced, balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M(NH4)2SO4Solution is consistent, with 60mmol/L Tris-HCl, 0.08mol/L (NH4)2SO4Solution carries out eluting, collects
Eluting peak；
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 30mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ s/
Cm, after diluting with 30mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 30mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged II sample solution loading, balances to baseline, so with 30mmol/L NaAc-HAc buffer after end of the sample
Rear 30mmol/L NaAc-HAc 0.2-0.8mol/L NaCl eluant solution, collects eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 30mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；By ion-exchanging eluent loading, afterwards by 50mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；Eluting
Peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
5) preservation of stock solution.
Being embodied as step is:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
Embodiment 4
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C.
2) collect seed liquor to cultivate, ferment and thalline.
Being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution.
Being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 80mmol/L Tris-HCl solution are fully stirred evenly in the ratio of 1:10, from
The heart collects precipitation；Fully stir after again adding 80mmol/L Tris-HCl solution in the ratio of 1:10 in the precipitation collected
Even, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, centrifugal, collects precipitation, obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 20mmol/L Tris-HCl, 4mol/L urea, 2mmol/L
The buffer solution of EDTA is fully stirred evenly in the ratio of 1:15, centrifugal, collects precipitation；
Up walk in the ratio of 1:15 and fully stir evenly after the precipitation obtained adds 40mmol/L Tris-HCl solution, centrifugal receipts
Collection precipitation, inclusion body after must washing for such twice.
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 80mmol/L Tris-HCl, 5mol/L urea, 5mmol/L EDTA
(pH8.5), in buffer, in the above-mentioned buffer having added inclusion body, add beta-mercaptoethanol, sealing, continue stirring, obtain
Inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 100mmol/L Tris-HCl buffer of 7 times of volumes；
Solubilising is completed the solution 80mmol/L Tris-HCl buffer ultrafiltration obtained, and stirs oxygen supplement to promote renaturation；
Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 50mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
It is 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography,
Obtain recombined human granulocyte stimulating factors stock solution.
Being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged sample solution loading, balances to baseline with 5-50mmol/L NaAc-HAc buffer after end of the sample, then
Carry out pre-eluting with 50mmol/L NaAc-HAc buffer, after eluting, put down with 50mmol/L NaAc-HAc buffer
Weigh identical with balance liquid pH to effluent pH, then by 50mmol/L NaAc-HAc, 0.8mol/L NaCl buffer solution elution,
And collect eluting peak.
B) hydrophobic chromatography:
With 50mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
80mmol/L Tris-HCl、 3.0mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4Final concentration of
0.7mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
80mmol/L Tris-HCl solution rinses consistent with 80mmol/L Tris-HCl solution to effluent conductance, finally uses
80mmol/L Tris-HCl 、0.7mol/L(NH4)2SO4Solution equilibria is identical with balance liquid to effluent conductance；
By hydrophobic chromatography sample solution loading, with 80mmol/L Tris-HCl, 0.7mol/L (NH after end of the sample4)2SO4Solution
It is balanced, balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M(NH4)2SO4Solution is consistent, with 80mmol/L Tris-HCl, 0.15mol/L (NH4)2SO4Solution carries out eluting, collects
Eluting peak；
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 50mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ s/
Cm, after diluting with 50mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged II sample solution loading, balances to baseline, so with 50mmol/L NaAc-HAc buffer after end of the sample
Rear 50mmol/L NaAc-HAc, 0.8mol/L NaCl eluant solution, collect eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；By ion-exchanging eluent loading, afterwards by 50mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；Eluting
Peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
5) preservation of stock solution.
Being embodied as step is:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
Embodiment 5
Preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C.
2) collect seed liquor to cultivate, ferment and thalline.
Being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution.
Being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 100mmol/L Tris-HCl solution are fully stirred evenly in the ratio of 1:10, from
The heart collects precipitation；Fully stir after again adding 100mmol/L Tris-HCl solution in the ratio of 1:10 in the precipitation collected
Even, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, centrifugal, collects precipitation, obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 20mmol/L Tris-HCl, 4mol/L urea, 5mmol/L
The buffer solution of EDTA is fully stirred evenly as after the ratio of 1:15, centrifugal, collects precipitation；The precipitation obtained up is walked in the ratio of 1:15
Fully stir evenly after middle addition 20mmol/L Tris-HCl solution, centrifugal, collect precipitation, inclusion body after must washing for such twice.
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 100mmol/L Tris-HCl, 9mol/L urea, 5mmol/L EDTA
(pH8.5), in buffer, in the above-mentioned buffer having added inclusion body, add beta-mercaptoethanol, sealing, continue stirring, obtain
Inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 100mmol/L Tris-HCl buffer of 6 times of volumes；
Solubilising is completed the solution 100mmol/L Tris-HCl buffer ultrafiltration obtained, and stirs oxygen supplement to promote renaturation；
Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 50mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
It is 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography,
Obtain recombined human granulocyte stimulating factors stock solution.
Being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged sample solution loading, balances to baseline with 50mmol/L NaAc-HAc buffer after end of the sample, then use
Carry out pre-eluting with 50mmol/L NaAc-HAc buffer, after eluting, balance with 50mmol/L NaAc-HAc buffer
Identical with balance liquid pH to effluent pH, then by 50mmol/L NaAc-HAc 0.8mol/L NaCl buffer solution elution, and receive
Collection eluting peak；
B) hydrophobic chromatography:
With 50mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
100mmol/L Tris-HCl、 3.0mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4Final concentration of
0.7mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
100mmol/L Tris-HCl solution rinses consistent with 100mmol/L Tris-HCl solution to effluent conductance, finally uses
100mmol/L Tris-HCl 、0.7mol/L(NH4)2SO4Solution equilibria is identical with balance liquid to effluent conductance；
By hydrophobic chromatography sample solution loading, with 100mmol/L Tris-HCl, 0.7mol/L (NH after end of the sample4)2SO4Solution
It is balanced, balances consistent with balance liquid to effluent electrical conductivity；
With 20mM Tris-HCl, 0.2M (NH4)2SO4The pre-eluting of solution, is washed till effluent electrical conductivity and 20mM Tris-in advance
HCl、0.2M(NH4)2SO4Solution is consistent, with 100mmol/L Tris-HCl, 0.15mol/L (NH4)2SO4Solution carries out eluting,
Collect eluting peak；
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 50mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ s/
Cm, after diluting with 50mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；Ion is exchanged II sample solution loading, balances to baseline, so with 50mmol/L NaAc-HAc buffer after end of the sample
Rear 50mmol/L NaAc-HAc 0.8mol/L NaCl eluant solution, collects eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid pH
Identical；By ion-exchanging eluent loading, afterwards by 50mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；Eluting
Peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
5) preservation of stock solution.
Being embodied as step is:
Recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, are configured to preserve stock solution, immediately
Become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization and preserve stock solution.
Present invention employs ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography,
Obtaining recombined human granulocyte stimulating factors stock solution, after testing, the stock solution of gained presses Chinese Pharmacopoeia and European Pharmacopoeia detection, and purity is divided
Not reaching 95 % and more than 98%, specific activity has respectively reached 6 × 107U/mg and 1.0 × 108More than U/mg, respectively reaches state
Family's standard and Europe touchstone.
Above example only in order to technical scheme to be described, is not intended to limit；Although with reference to previous embodiment
The present invention is described in detail, it will be understood by those within the art that: it still can be to aforementioned each enforcement
Technical scheme described in example is modified, or wherein portion of techniques feature is carried out equivalent；And these amendment or
Replace, do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (5)
1. preparing the purification process of recombined human granulocyte stimulating factors stock solution, the method comprises the following steps:
1) selecting engineering bacteria e. coli jm109 to do bacterial strain and carry out seed liquor cultivation, this strain derives from A.T.C.C；
2) collect seed liquor to cultivate, ferment and thalline；
3) the purest: prepared by inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution；
It is 4) consummate: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography, it is thus achieved that
Recombined human granulocyte stimulating factors stock solution；
5) preservation of stock solution.
Prepare the purification process of recombined human granulocyte stimulating factors stock solution, described step 2 the most as claimed in claim 1) receive
Collection seed liquor is cultivated, is fermented and thalline, and being embodied as step is:
A) frozen seed liquor being thawed, inoculate 100 μ l after 50ml LB culture medium, 37 DEG C ± 1 DEG C carries out first order seed training
Support；
B) by 1% inoculum concentration, after primary seed solution is inoculated in LB culture medium, 37 DEG C ± 1 DEG C carries out secondary seed cultivation, by two
Level seed liquor is inoculated in fermentation tank by 1:10 inoculum concentration, ferments；
C) controlling fermentation temperature is 37 DEG C ± 1 DEG C, pH6.8 ± 0.2；OD is surveyed in sampling per hour600 0.5, with thalli growth situation pair
Rotating speed, air mass flow implement regulation, enter logarithmic growth after date, start supplemented medium, add IPTG depending on OD value situation, continue
Cultivate to OD600 0.5Till the most significantly raised；Collecting fermentation liquid, centrifugal collecting precipitation obtains thalline；Sweat is expressed and produces
Recombined human granulocyte stimulating factors albumen is present in thalline with inclusion bodies.
Preparing the purification process of recombined human granulocyte stimulating factors stock solution the most as claimed in claim 1, described step 3) is thick
Pure: preparing inclusion body collection, washing, degeneration, renaturation, ion exchange sample solution, being embodied as step is:
A) inclusion body is collected, is washed
Thick inclusion body obtains washing: wet thallus and 10-100mmol/L Tris-HCl solution are fully stirred in the ratio of 1:10
Even, centrifugal collecting precipitation；Again in the precipitation collected, add 10-100mmol/L Tris-HCl solution in the ratio of 1:10
The most fully stirring evenly, the suspension obtained is carried out brokenly bacterium, percentage of damage reaches 95%, and centrifugal collecting precipitation obtains thick inclusion body；
After washing, inclusion body obtains: by the thick inclusion body obtained and 10-20mmol/L Tris-HCl, 2-4mol/L urea, 1-
The buffer solution of 5mmol/L EDTA is fully stirred evenly in the ratio of 1:15, centrifugal, collects precipitation；
Fully stir evenly after adding 10-60mmol/L Tris-HCl solution in the ratio of 1:15 in the precipitation that upper step obtains, centrifugal
Collect precipitation, inclusion body after must washing for such twice；
B) inclusion body degeneration and renaturation
Under stirring, inclusion body re-suspension liquid is added 10-100mmol/L Tris-HCl, 5-9mol/L urea, 1-5mmol/L
In the buffer solution (pH8.5) of EDTA, in the above-mentioned buffer solution having added inclusion body, add beta-mercaptoethanol, sealing, continue
Continuous stirring, obtains inclusion body lysate；
Collect centrifugal for above inclusion body lysate in supernatant extremely aseptic pyrogen removal container, be slowly added to thereto under stirring
The 10-100mmol/L Tris-HCl buffer of 6-10 times of volume；
Solubilising is completed the solution 10-100mmol/L Tris-HCl buffer ultrafiltration obtained, and it is multiple to promote to stir oxygen supplement
Property；Ensureing the liquid level constant of solution in container in ultra-filtration process, buffer continues ultrafiltration to prescribed volume after adding；
Centrifugal collection supernatant, dilutes by 5-50mmol/L NaAc-HAc equimultiple, obtains ion exchange sample solution.
Prepare the purification process of recombined human granulocyte stimulating factors stock solution, described step 4) essence the most as claimed in claim 1
Pure: through ion-exchange chromatography, hydrophobic chromatography, ultrafiltration desalination, ion-exchange chromatography II, sieve chromatography, it is thus achieved that recombined human grain is thin
Born of the same parents' stimulating factor stock solution, being embodied as step is:
A) ion-exchange chromatography:
Filler CM Sepharose FF, uses 5-50mmol/L N, aAc-HAc buffer balance chromatographic column to effluent and balance
Liquid pH is identical；Ion is exchanged sample solution loading, balances to base with 5-50mmol/L NaAc-HAc buffer after end of the sample
Line, then carry out pre-eluting with 5-50mmol/L NaAc-HAc buffer, after eluting, delay with 5-50mmol/L NaAc-HAc
Rush liquid balance identical with balance liquid pH to effluent pH, then with 5-50mmol/L NaAc-HAc 0.2-0.8mol/L NaCl
Buffer solution elution, and collect eluting peak；
B) hydrophobic chromatography:
With 5-50mmol/L NaAc-HAc buffer, liquid dilution is collected in the ion exchange measured, add under stirring
10-100mmol/L Tris-HCl、 1.0-3.0mol/L (NH4)2SO4Solution, makes hydrophobic sample solution, (NH4)2SO4The denseest
Degree is 0.3-0.7mol/L, waits loading；
Filler Phenyl sepharose High performance, after being disposed by 0.5mol/L NaOH solution, then uses
10-100mmol/L Tris-HCl solution rinses consistent, finally with 10-100mmol/L Tris-HCl solution to effluent conductance
With 10-100mmol/L Tris-HCl 0.3-0.7mol/L (NH4)2SO4Solution equilibria, to effluent conductance and balance liquid phase
With；
By hydrophobic chromatography sample solution loading, with 10-100mmol/L Tris-HCl, 0.3-0.7mol/L (NH after end of the sample4)2SO4Solution is balanced, and balances consistent with balance liquid to effluent electrical conductivity；
With the 20mM Tris-HCl pre-eluting of 0.2M ammonium sulfate, be washed till in advance effluent electrical conductivity and 20mM Tris-HCl,
0.2M(NH4)2SO4Solution is consistent, with 10-100mmol/L Tris-HCl, 0.01-0.15mol/L (NH4)2SO4Solution is carried out
Eluting, collects eluting peak；
C) ultrafiltration desalination:
By hydrophobic chromatography eluting peak, carry out ultrafiltration desalination with 5-50mmol/L NaAc-HAc buffer, to electrical conductivity≤1500 μ
S/cm, after diluting with 5-50mmol/L NaAc-HAc buffer, obtains ion exchange II sample solution；
D) ion-exchange chromatography II:
Filler CM Sepharose FF, with 5-50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid
PH is identical；Ion is exchanged II sample solution loading, balances to base with 5-50mmol/L NaAc-HAc buffer after end of the sample
Line, then 5-50mmol/L NaAc-HAc 0.2-0.8mol/L NaCl eluant solution, collect eluting peak；
E) sieve chromatography:
Filler Sephacryl S-100, with 5-50mmol/L NaAc-HAc buffer balance chromatographic column to effluent and balance liquid
PH is identical；By ion-exchanging eluent loading, afterwards by 5-50mmol/L NaAc-HAc buffer solution elution, collect eluting peak liquid；
Eluting peak liquid with 0.22 μm filter filtration sterilization, obtain recombined human granulocyte stimulating factors stock solution.
Prepare the purification process of recombined human granulocyte stimulating factors stock solution, described step 5) stock solution the most as claimed in claim 1
Preservation, being embodied as step is:
The preservation of stock solution: recombined human granulocyte stimulating factors stock solution, polyoxyethylene sorbitan monoleate mix with mannitol buffer, is configured to protect
Deposit stock solution, become recombined human granulocyte stimulating factors through 0.22 μm filter filtration sterilization immediately and preserve stock solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109879930A (en) * | 2019-03-18 | 2019-06-14 | 浙江优诺金生物工程有限公司 | A kind of purification process of recombinant protein |
| CN110041423A (en) * | 2018-01-16 | 2019-07-23 | 江苏奥赛康药业股份有限公司 | A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor |
| CN111285929A (en) * | 2018-12-10 | 2020-06-16 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human epidermal growth factor from genetically engineered rice seeds |
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| CN110041423A (en) * | 2018-01-16 | 2019-07-23 | 江苏奥赛康药业股份有限公司 | A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor |
| CN111285929A (en) * | 2018-12-10 | 2020-06-16 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human epidermal growth factor from genetically engineered rice seeds |
| CN111285929B (en) * | 2018-12-10 | 2023-09-19 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human epidermal cell growth factor from genetically engineered rice seeds |
| CN109879930A (en) * | 2019-03-18 | 2019-06-14 | 浙江优诺金生物工程有限公司 | A kind of purification process of recombinant protein |
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Application publication date: 20161214 |