CN116675759A - Novel purification method for recombinant human nerve growth factor expressed by CHO cells - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 48
- 108010025020 Nerve Growth Factor Proteins 0.000 title claims abstract description 42
- 102000015336 Nerve Growth Factor Human genes 0.000 title claims abstract description 42
- 229940053128 nerve growth factor Drugs 0.000 title claims abstract description 39
- 210000004978 chinese hamster ovary cell Anatomy 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title claims abstract description 17
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 30
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 21
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 21
- 238000010828 elution Methods 0.000 claims abstract description 19
- 238000012856 packing Methods 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 230000008569 process Effects 0.000 claims abstract description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 7
- 239000003480 eluent Substances 0.000 claims abstract description 6
- 239000000945 filler Substances 0.000 claims abstract description 6
- 238000004191 hydrophobic interaction chromatography Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 14
- 238000011068 loading method Methods 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 9
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 150000001450 anions Chemical class 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 239000003446 ligand Substances 0.000 claims description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 2
- 239000006143 cell culture medium Substances 0.000 claims 2
- 239000012634 fragment Substances 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 239000002609 medium Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 7
- 238000009776 industrial production Methods 0.000 abstract description 5
- 239000011347 resin Substances 0.000 description 16
- 229920005989 resin Polymers 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
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- 239000000523 sample Substances 0.000 description 4
- 125000000129 anionic group Chemical group 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000531 effect on virus Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000005709 nerve cell growth Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000002856 peripheral neuron Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention discloses a novel purification method for recombinant human nerve growth factor expressed by CHO cells, which comprises the following steps: in the anion exchange chromatography process, the supernatant of the CHO culture is filtered by sterilization and then is loaded into an anion chromatography filler, and anion chromatography flow-through products are collected to realize anion exchange chromatography; and in the hydrophobic interaction chromatography process, the anion chromatography flow-through material is loaded into a hydrophobic chromatography packing material, and the recombinant human nerve growth factor is obtained after elution by eluent. The method can obtain the product with the purity of 99 percent and the recovery rate of more than 95 percent after two-step chromatography, and has less steps, short period and wide industrial production prospect.
Description
Technical Field
The invention relates to the field of biological medicine purification, in particular to a novel purification method for recombinant human nerve growth factor expressed by CHO cells.
Background
Human nerve growth factor (hNGF), which is a nerve cell growth regulating factor with the dual biological functions of nourishing neurons and promoting protrusion growth, has important regulation and control functions on development, differentiation, growth, regeneration and expression of functional characteristics of central and peripheral neurons. Because of the importance of nerve growth factor and the trace amount of synthesis in humans, synthesis of hNGF by artificial means has been an irreplaceable trend. Expression of recombinant human growth factors in chinese hamster ovary Cells (CHO) by genetic engineering techniques is an important means of synthesizing hNGF. However, CHO expression supernatants contain many impurities, such as HCP, HCD, NGF variants, which must be purified strictly before use.
The current purification method for CHO expressed recombinant human nerve growth factor is mainly disclosed as follows:
1. chinese patent CN113024655a discloses a purification method for efficiently removing rhNGF precursor, which adopts hydroxyapatite chromatography to separate precursor substances, but the patent does not mention the recovery rate and purity of the product, and one or more chromatography means such as ion exchange chromatography, hydrophobic chromatography, etc. are reserved before the hydroxyapatite chromatography, so that the process steps are increased and the production period is prolonged.
2. Chinese patent CN102702341A discloses a recombinant human nerve growth factor purification method based on a CHO cell expression system, which adopts a cation exchange and molecular sieve two-step chromatography process to prepare the recombinant human nerve growth factor with purity of more than 98% (Tricine-SDS-PAGE electrophoresis method), but a molecular sieve sample needs ultrafiltration concentration, and the loading capacity needs less than or equal to 4% of column volume, which is not suitable for industrial production.
3. Chinese patent CN106478801A discloses a method for separating recombinant human nerve growth factor from mammalian cell culture, which adopts a cation exchange and hydrophobic interaction two-step chromatography method to prepare the recombinant human nerve growth factor with purity of more than 99% and recovery rate of more than 71.4%, but adopts linear gradient elution when hydrophobic chromatography elution is carried out, thereby having higher requirements on equipment and being unfavorable for large-scale industrial production.
4. Chinese patent CN108239146a discloses a preparation method of high purity rhNGF, which adds a washing step before cation elution and hydrophobic elution to avoid the problem of linear elution, but the cation must undergo one or more steps of chromatography before loading, which increases the production cycle.
Since CHO cell expression systems are relatively expensive, if the conventional purification process is used, there are great disadvantages in terms of protein yield, activity and time loss, and further, the product cost is increased, which is also a major reason for the industrial production of recombinant human nerve growth factor which has been affected by CHO cells.
Based on the above, the existing purification technology of recombinant human nerve growth factor has the disadvantages of complicated steps, low yield, long period and the like, and is not suitable for large-scale production of recombinant human nerve growth factor. Therefore, development of a purification process which is applicable to the CHO cell system expression recombinant human nerve growth factor and has high purity, high recovery rate, short period and low cost is not easy.
Disclosure of Invention
The invention aims to provide a novel purification method for expressing recombinant human nerve growth factor by CHO cells, so as to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a novel purification method for recombinant human nerve growth factor expressed by CHO cells comprises
S1: in the anion exchange chromatography process, the supernatant of the CHO culture is filtered by sterilization and then is loaded into an anion chromatography filler, and anion chromatography flow-through products are collected to realize anion exchange chromatography;
s2: and in the hydrophobic interaction chromatography process, the anion chromatography flow-through material is loaded into a hydrophobic chromatography packing material, and the recombinant human nerve growth factor is obtained after elution by eluent.
Preferably, the CHO culture in S1 is a CHO cell culture expressing recombinant human nerve growth factor, and the cell culture is centrifuged at 4000rpm for 10min to remove cells and cell debris, and the supernatant is collected.
Preferably, the pore size of the sterilization filtration membrane used in the sterilization filtration in S1 is 0.22. Mu.m, and the material is PES.
Preferably, the pH of the cell supernatant loaded in S1 into the anionic chromatographic packing is in the range of 6.5-8.5, the conductance < 5.000mS/cm.
Preferably, the loading of recombinant human nerve growth factor loaded into the anionic chromatographic packing in S1 is 1.0-2.0mg/ml, and the anionic chromatographic packing in S1 is a strong quaternary ammonium (Q) anion exchange medium.
Preferably, the loading of S1 pre-anion chromatography column is equilibrated with 25mM Tris-HCl for 10CV, wherein 25mM Tris-HCl, pH6.5-8.5, conductance < 5.000mS/cm.
Preferably, the pH of the anion flow-through solution loaded in S2 into the hydrophobic chromatography packing is 6.0-8.0, and the salt concentration is 0.8-1.5M, wherein the salt is NaCl, KCl or (NH) 4 ) 2 SO 4 Any one or two of the compositions.
Preferably, the hydrophobic chromatography packing in S2 has butyl or phenyl ligand, and the recombinant human nerve growth factor loading amount loaded into the hydrophobic chromatography packing in S2 is 3.0-5.0mg/ml.
Preferably, the pre-loading hydrophobic chromatography packing in S2 is equilibrated with 20mM PB for 10CV, wherein 20mM PB, pH6.0-8.0, salt concentration 0.8-1.5mol/L, wherein salt is NaCl, KCl or (NH) 4 ) 2 SO 4 Any one or two of the compositions.
Preferably, the elution condition of the hydrophobic chromatography in S2 is linear elution, the linear condition is a reduced salt concentration gradient, wherein the salt concentration is 0.5-1.5mol/L, the elution volume is 10-30CV, wherein the salt is NaCl, KCl or (NH) 4 ) 2 SO 4 Any one or two of the compositions.
Preferably, the eluent in the linear elution contains 5% -25% of alcohol, and the alcohol is any one of methanol, ethanol or isopropanol.
Compared with the prior art, the invention has the beneficial effects that:
1. the method has fewer process steps, shortens the production period and reduces the sample pollution probability. Meanwhile, the anion chromatography in the flow-through mode has remarkable removal effect on HCP and HCD, and has a certain removal effect on viruses in the sample. Under the hydrophobic chromatography condition, NGF variant is effectively removed, and the purity of NGF is greatly improved.
2. The purification method provided by the invention is simple and efficient, has few steps and short period, and is suitable for large-scale production. After two-step chromatography, the product with purity more than 99% and recovery rate more than 95% can be obtained.
Drawings
FIG. 1 is an anion exchange chromatography chart in example 1;
FIG. 2 is a hydrophobic chromatography chart in example 2;
FIG. 3 is a chart showing the results of SEC-HPLC analysis of the hydrophobic product of example 2.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: anion exchange chromatography, as shown in fig. 1:
the CHO cell culture expressing the recombinant human nerve growth factor was centrifuged at 5000rpm for 10min to remove cells and cell debris, and the supernatant was collected. Then, the supernatant was sterilized and filtered by using a sterilizing filter having a pore size of 0.22. Mu.m, and the filtrate was collected.
UsingAnion chromatography with Avant150 chromatography system (Cytiva) as HiScreen Capto TM Q (4.7 ml, cytiva). The instrument was operated according to the instructions and the A1 pump was Buffer A (25 mM Tris-HCl Buffer, pH 8.0).
The specific chromatography steps are as follows:
(1) Cleaning: capto was rinsed with 1M NaOH at a flow rate of 0.94ml/min TM Q resin column 2 column volumes, pause for 15min, and continue rinsing Capto with 1M NaOH, flow rate 0.94ml/min TM Q resin column 1 column volume;
(2) Pre-balancing: capto was rinsed with 1M NaCl, flow rate 0.94ml/min TM Q resin column 3 column volumes;
(3) Balance: capto was rinsed with 25mM Tris-HCl buffer, flow rate 0.94ml/min TM Q resin column 10 column volumes to UV baseline;
(4) Loading: regulating the pH value of a cell supernatant sample to 8.0, wherein the conductivity is less than 5.000mS/cm, loading at a flow rate of 0.94ml/min, loading load of 1mg/ml, detecting the wavelength of 280nm, and collecting the flow-through liquid with UV (ultraviolet) more than 15 mAU;
(5) Balance: capto was rinsed with 25mM Tris-HCl buffer, flow rate 0.94ml/min TM Q resin column 5 column volumes;
(6) Regeneration: capto was rinsed with 1M NaCl, flow rate 0.94ml/min TM Q resin column 3 Column Volumes (CVs);
(7) Cleaning: capto was rinsed with 1M NaOH at a flow rate of 0.94ml/min TM Q resin column 2 Column Volumes (CV), pause for 15min, and continue rinsing Capto with 1M NaOH at a flow rate of 0.94ml/min TM Q resin column 1 column volume, remove impurity residue on resin;
and (3) preserving: rinsing Capto with 20% ethanol at a flow rate of 0.94ml/min TM Q resin column 3 column volumes.
Example 2: hydrophobic interaction chromatography, as shown in figure 2:
usingAvant150 (Cytiva) chromatography system was used for hydrophobic chromatography, and the column was HiTrap Phenyl HP (5 ml). The instrument operation was performed according to the instructions, with the A1 pump being Buffer A (20mM PB+1MNaCl,pH7.0) and the B1 pump being Buffer B (20 mM PB+20% ethanol, pH 7.0).
The specific chromatography steps are as follows:
(1) Cleaning: flushing the HiTrap Phenyl HP resin column with ultrapure water at a flow rate of 1.00ml/min for 3 column volumes;
(2) Balance: washing the HiTrap Phenyl HP resin column with Buffer A (20mM PB+1M NaCl,pH7.0) at a flow rate of 1.00ml/min for 10 column volumes to a baseline plateau;
(3) Loading: the anion flow-through sample of example 1 was adjusted to pH7.0, conductance 89.00mS/cm, and loaded at a flow rate of 1.00 ml/min;
(4) Balance: washing the HiTrap Phenyl HP resin column with Buffer A (20mM PB+1M NaCl,pH7.0) at a flow rate of 1.00ml/min for 5 column volumes until the column volumes are stable;
(5) Eluting: linear elution was performed with Buffer A (20mM PB+1M NaCl,pH7.0), buffer B (20 mM PB+20% ethanol, pH 7.0) at a flow rate of 1.00ml/min, with a linear elution gradient of 100% -0% Buffer A, 0% -100% Buffer B, and an elution volume of 20CV. Collecting eluent with the linear range of 58% -87% of Buffer B and the UV of more than 10 mAu;
(6) Regeneration: 3 column volumes of the HiTrap Phenyl HP resin column were washed with 0.5M NaCl, flow rate 1.00 ml/min;
(7) Cleaning: the washing was continued with 0.01M NaOH at a flow rate of 0.2ml/min for 15min after 2CV washing and with a continued washing of 1CV to remove very tightly bound, precipitated or denatured impurities;
(8) Flushing: flushing the HiTrap Phenyl HP resin column for 3 column volumes with ultrapure water at a flow rate of 0.2 ml/min;
(9) And (3) preserving: the HiTrap Phenyl HP resin column was flushed with 20% ethanol at a flow rate of 1.00ml/min for 3 column volumes.
The purity of recombinant human nerve growth factor obtained after one-step sterilization and two-step chromatography was analyzed by SEC-HPLC method, which was used in combination with example 1 and example 2. As shown in the detection result of FIG. 3, the product with the purity of more than 99 percent can be obtained through the purification process of the invention, and the recovery rate of the product is more than 95 percent.
In conclusion, the recombinant human nerve growth factor product with high purity is prepared by the method and the process through two-step chromatography, the recovery rate of the product is high, the purification steps are fewer, the production period is short, the industrial production can be realized, and the method and the process have good market application prospects.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A novel purification method for recombinant human nerve growth factor expressed by CHO cells, which is characterized by comprising the following steps:
s1: in the anion exchange chromatography process, the supernatant of the CHO culture is filtered by sterilization and then is loaded into an anion chromatography filler, and anion chromatography flow-through products are collected to realize anion exchange chromatography;
s2: and in the hydrophobic interaction chromatography process, the anion chromatography flow-through material is loaded into a hydrophobic chromatography packing material, and the recombinant human nerve growth factor is obtained after elution by eluent.
2. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the CHO culture medium in S1 is a CHO cell culture medium expressing recombinant human nerve growth factor, and cells and cell fragments are removed after the cell culture medium is centrifuged at 4000rpm for 10min, and supernatant is collected.
3. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the pore diameter of the sterilization filter membrane used in the sterilization filter in the step S1 is 0.22 mu m, and the sterilization filter membrane is made of PES.
4. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the pH range of the cell supernatant loaded into the anion chromatography packing in the S1 is 6.5-8.5, and the electric conductivity is less than 5.000mS/cm.
5. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the recombinant human nerve growth factor loading amount loaded into the anion chromatographic packing in S1 is 1.0-2.0mg/ml, and the anion chromatographic packing in S1 is a strong quaternary ammonium (Q) anion exchange medium.
6. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the loading pre-anion chromatographic column in S1 was equilibrated with 25mM Tris-HCl for 10CV, wherein 25mM Tris-HCl, pH6.5-8.5, conductance < 5.000mS/cm.
7. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the pH of the anion flow-through solution loaded in the S2 into the hydrophobic chromatography packing is 6.0-8.0, and the salt concentration is 0.8-1.5M, wherein the salt is NaCl, KCl or (NH) 4 ) 2 SO 4 Any one or two of the compositions.
8. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the ligand of the hydrophobic chromatography filler in S2 is butyl or phenyl, and the load of the recombinant human nerve growth factor loaded in the S2 into the hydrophobic chromatography filler is 3.0-5.0mg/ml.
9. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the hydrophobic chromatography filler before loading in S2 is balanced by 20mM PB for 10CV, wherein the pH is 6.0-8.0, and the salt concentration is 0.8-1.5mol/L, and the salt is NaCl, KCl or (NH) 4 ) 2 SO 4 Any one or two of the compositions.
10. The method for purifying recombinant human nerve growth factor expressed by a novel CHO cell according to claim 1, wherein the method comprises the steps of: the elution condition of the hydrophobic chromatography in the S2 is linear elution, the linear condition is a reduced salt concentration gradient, wherein the salt concentration is 0.5-1.5mol/L, the elution volume is 10-30CV, and the salt is NaCl, KCl or (NH) 4 ) 2 SO 4 The eluent contains 5% -25% of alcohol in the linear elution, and the alcohol is any one of methanol, ethanol or isopropanol.
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