CN117720613A - Method for improving aggregate removal and yield of Fc fusion protein in chromatography - Google Patents
Method for improving aggregate removal and yield of Fc fusion protein in chromatography Download PDFInfo
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- CN117720613A CN117720613A CN202311555003.7A CN202311555003A CN117720613A CN 117720613 A CN117720613 A CN 117720613A CN 202311555003 A CN202311555003 A CN 202311555003A CN 117720613 A CN117720613 A CN 117720613A
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 108091006020 Fc-tagged proteins Proteins 0.000 title claims abstract description 15
- 238000004587 chromatography analysis Methods 0.000 title abstract description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 63
- 238000010828 elution Methods 0.000 claims abstract description 32
- BDKZHNJTLHOSDW-UHFFFAOYSA-N [Na].CC(O)=O Chemical compound [Na].CC(O)=O BDKZHNJTLHOSDW-UHFFFAOYSA-N 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
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- 230000002378 acidificating effect Effects 0.000 claims abstract description 7
- 238000000746 purification Methods 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 41
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- 239000000872 buffer Substances 0.000 claims description 25
- 239000011780 sodium chloride Substances 0.000 claims description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000005349 anion exchange Methods 0.000 claims description 15
- 238000011068 loading method Methods 0.000 claims description 15
- 238000005571 anion exchange chromatography Methods 0.000 claims description 11
- 239000008351 acetate buffer Substances 0.000 claims description 8
- 150000001450 anions Chemical group 0.000 claims description 7
- 108020001507 fusion proteins Proteins 0.000 claims description 6
- 102000037865 fusion proteins Human genes 0.000 claims description 6
- 239000012475 sodium chloride buffer Substances 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 150000005846 sugar alcohols Polymers 0.000 claims description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- 125000000129 anionic group Chemical group 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 238000011091 antibody purification Methods 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims description 2
- 125000001165 hydrophobic group Chemical group 0.000 claims description 2
- 125000003010 ionic group Chemical group 0.000 claims description 2
- 238000001742 protein purification Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 238000011097 chromatography purification Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 abstract description 2
- 239000000178 monomer Substances 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 150000003384 small molecules Chemical class 0.000 abstract 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 14
- 230000008929 regeneration Effects 0.000 description 9
- 238000011069 regeneration method Methods 0.000 description 9
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 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 description 4
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 4
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 4
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- KPSSIOMAKSHJJG-UHFFFAOYSA-N neopentyl alcohol Chemical compound CC(C)(C)CO KPSSIOMAKSHJJG-UHFFFAOYSA-N 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical compound CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 2
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- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for improving aggregate removal and yield of Fc fusion protein in chromatography, belonging to the technical field of purification. For the acidic Fc fusion protein with PI (isoelectric point) value of about 5.0-6.0, an acetic acid buffer system and an acetic acid-sodium chloride-alcohol buffer system are used for linear gradient elution, and the target protein is collected. The method can effectively remove aggregates and small molecules, reduce HCP and potential viruses, and compared with the traditional chromatographic elution method, the method has the advantages that the yield is improved by about 60 percent, the purity of the monomer is improved by about 1.2 percent, and the production cost is greatly saved. The method is not only suitable for laboratory scale, but also can be used for process amplification as a production process.
Description
Technical Field
The invention belongs to the technical field of purification, and particularly relates to a method for improving aggregate removal and yield of Fc fusion protein in chromatography.
Background
The Fc fusion protein is a recombinant protein formed by combining an Fc segment of immunoglobulin with a functional protein molecule with biological activity by utilizing a genetic engineering technology, not only retains the biological activity of the functional protein molecule, but also has the properties of antibodies, such as prolonging half-life by combining related Fc receptors, inducing antibody-dependent cell-mediated cytotoxicity effects and the like, and has important significance in diagnosis and treatment of diseases.
The anion exchange chromatography matrix binds proteins with negative charges, so that the proteins are retained on the column, and the proteins adsorbed on the column are eluted by means of increasing the salt concentration in the eluate or lowering the pH of the eluate. The weaker protein is eluted first and the stronger protein is eluted later.
The conventional isoelectric point of the acidic Fc fusion protein is between 5.0 and 7.0. Because of the low isoelectric point nature of acidic Fc fusion proteins, such lower PI protein molecules can be purified using an anion-binding elution profile.
Acidic Fc fusion proteins have poor molecular stability and low pH eluting proteins are prone to precipitation, aggregation or degradation, resulting in limited alternative buffer systems. There is a strong need for a chromatographic method that can effectively purify acidic Fc fusion proteins and remove Fc fusion protein aggregates.
Disclosure of Invention
In order to solve the problems, the invention provides an Fc fusion protein anion exchange chromatography purification method, in which a protein and an anion filler are combined with each other through a charge acting force, an acetic acid buffer solution pH6.0 is used as a balance solution, an acetic acid-sodium chloride-alcohol buffer solution pH6.0 is used as an eluent, and 0-100% of B and 10CV are used in a linear elution mode, so that the aim of separating a target protein combined on the anion filler is fulfilled.
Further, in the elution method, the anion-balancing solution is an acetate buffer having a pH in the range of pH5.5 to pH6.5, more preferably pH5.8 to 6.2, and most preferably pH6.0.
Further, in the elution method, the anion eluent is acetic acid-sodium chloride-alcohol buffer, and the pH of the buffer ranges from pH5.5 to pH6.5, more preferably from pH5.8 to 6.2, and most preferably pH6.0.
Further, the alcohol in the acetic acid-sodium chloride-alcohol buffer solution is one or more of monohydric alcohol or polyhydric alcohol with carbon chain length of 2-6.
Preferably, the alcohol in the acetic acid-sodium chloride-alcohol buffer solution is one or more of isopropanol, n-propanol, isobutanol, tert-butanol, n-pentanol, isopentanol and neopentyl alcohol.
Specifically, the alcohol in the acetic acid-sodium chloride-alcohol buffer is isopropanol or isobutanol.
Preferably, the acetate buffer concentration in the acetate-sodium chloride-alcohol buffer is preferably 40 mM-60 mM, more preferably 45 mM-55 mM, most preferably 50mM.
Preferably, the concentration of sodium chloride in the acetic acid-sodium chloride-alcohol buffer is preferably 1.0M to 1.2M, most preferably 1.1M.
Preferably, the alcohol content in the acetic acid-sodium chloride-alcohol buffer is preferably 8% to 12%, most preferably 10%.
Further, in the elution method, the elution mode is that the equilibrium solution and the eluent are linearly eluted, the elution gradient is 0-100% B, and the elution volume is 10 column volumes.
The invention also provides an anion exchange chromatography method of the fusion protein, which comprises the following steps:
(1) Pre-balancing an anion exchange chromatographic column by using the eluent B;
(2) Balancing the anion exchange chromatographic column by using the balance liquid A;
(3) Filtering the solution containing the fusion protein, and loading the filtered solution on an anion exchange chromatographic column;
(4) Then balancing the chromatographic column by using the balance liquid A;
(5) Eluting the main peak of the antibody by using the balance A and the eluent B and recovering the main peak;
(6) The regenerated peak was eluted using high concentration sodium chloride buffer.
In one embodiment of the present invention, the eluent B in step (1) is acetic acid-sodium chloride-alcohol buffer.
In one embodiment of the invention, the alcohol in the acetic acid-sodium chloride-alcohol buffer is one or more of a monohydric or polyhydric alcohol having a carbon chain length of 2 to 6.
In one embodiment of the present invention, preferably, the alcohol in the acetic acid-sodium chloride-alcohol buffer is one or more of isopropanol, n-propanol, isobutanol, t-butanol, n-pentanol, isopentanol, neopentyl alcohol.
In one embodiment of the present invention, preferably, the alcohol in the acetic acid-sodium chloride-alcohol buffer is specifically selected to be isopropanol or isobutanol.
In one embodiment of the invention, the amount of the B eluent in the step (1) is 2 to 8 column volumes.
In one embodiment of the present invention, the amount of the B eluent in the step (1) may be specifically selected to be 5 column volumes.
In one embodiment of the present invention, the pH of the B eluate ranges from pH5.5 to pH6.5.
In one embodiment of the invention, preferably, the pH of the B eluate is in the range of pH5.8-6.2.
In one embodiment of the present invention, preferably, the pH of the B eluate is specifically selected to be pH6.0.
In one embodiment of the present invention, the acetic acid concentration in the acetic acid-sodium chloride-alcohol buffer is 40 mM-60 mM.
In one embodiment of the present invention, the acetic acid concentration in the acetic acid-sodium chloride-alcohol buffer is preferably 45 mM-55 mM.
In one embodiment of the present invention, it is preferable that the concentration of acetic acid in the acetic acid-sodium chloride-alcohol buffer is specifically selected to be 50mM.
In one embodiment of the present invention, the concentration of sodium chloride in the acetic acid-sodium chloride-alcohol buffer is 1.0M to 1.2M.
In one embodiment of the present invention, it is preferable that the concentration of sodium chloride in the acetic acid-sodium chloride-alcohol buffer is specifically selected to be 1.1M.
In one embodiment of the invention, the alcohol content of the acetic acid-sodium chloride-alcohol buffer is 8% -12%.
In one embodiment of the invention, the alcohol content of the acetic acid-sodium chloride-alcohol buffer is preferably chosen to be 10%.
In one embodiment of the present invention, the balancing solution a in step (2) is an acetate buffer.
In one embodiment of the present invention, the amount of the balancing liquid a in the step (2) is 20 to 30 column volumes.
In one embodiment of the present invention, the amount of the balancing solution a in the step (2) may be specifically selected to be 25 column volumes.
In one embodiment of the present invention, the pH of the a-balance liquid ranges from pH5.5 to pH6.5.
In one embodiment of the present invention, preferably, the pH of the a-equilibria solution ranges from pH5.9 to 6.1.
In one embodiment of the present invention, preferably, the pH of the a-balance solution is specifically selected to be pH6.0.
In one embodiment of the present invention, the acetic acid concentration in the acetic acid buffer is 40 mM-60 mM.
In one embodiment of the present invention, the acetic acid concentration in the acetic acid buffer is preferably 45 mM-55 mM.
In one embodiment of the present invention, it is preferable that the concentration of acetic acid in the acetic acid buffer is specifically selected to be 50mM.
In one embodiment of the invention, the support in step (3) is an anionic filler comprising ionic groups and hydrophobic groups.
In one embodiment of the invention, the support in step (3) is preferably an anionic packing of agarose bed frame.
In one embodiment of the present invention, the carrier in step (3) may be specifically selected to be nano-NM 90-boost TM HAM, boegron EzScreen MIX-a.
In one embodiment of the invention, the protein in step (3) is an acidic Fc fusion protein molecule having a PI value between 5.0 and 6.0.
In one embodiment of the invention, the filtration in step (3) is filtration of the solution containing the Fc fusion protein using a 0.22 μm filter membrane.
In one embodiment of the present invention, the chromatography column loading in step (3) is 10 to 50g/L.
In one embodiment of the present invention, preferably, the chromatography column loading in step (3) is specifically selected to be 30g/L.
In one embodiment of the present invention, the amount of the balancing solution a in the step (4) is 7 to 11 column volumes.
In one embodiment of the present invention, the amount of the balancing solution a in the step (4) may be specifically selected to be 9 column volumes.
In one embodiment of the present invention, the elution in step (5) is performed by linear elution of the equilibrium liquid A and the eluent B, with an elution gradient of 0-100% B, and an elution volume of 10 column volumes.
In one embodiment of the present invention, the high concentration sodium chloride solution in step (6) is acetic acid and sodium chloride solution.
In one embodiment of the present invention, the concentration of sodium chloride in the high concentration sodium chloride solution in step (6) is 0.5 to 2M.
In one embodiment of the present invention, the concentration of sodium chloride in the high concentration sodium chloride solution in step (6) is specifically selected to be 1M.
In one embodiment of the present invention, the concentration of acetic acid in the high concentration sodium chloride solution in step (6) is 40 mM-60 mM.
In one embodiment of the present invention, the concentration of acetic acid in the high concentration sodium chloride solution in step (6) is specifically selected to be 50mM.
In one embodiment of the present invention, the high concentration sodium chloride solution in step (6) has a pH in the range of pH5.5 to pH6.5.
In one embodiment of the present invention, the pH of the high concentration sodium chloride solution in step (6) is specifically selected to be pH6.0.
In one embodiment of the present invention, the high concentration sodium chloride solution in step (6) is used in an amount of 2 to 6 column volumes.
In one embodiment of the present invention, the amount of the high concentration sodium chloride solution used in step (6) may be specifically selected to be 4 column volumes.
The method provided by the invention is applied to recombinant protein purification, antibody purification and vaccine purification.
The beneficial effects are that:
compared with a classical antibody platform, the anion chromatography step is mostly in a flow-through mode, and the anion chromatography step in the invention is in a combined elution mode, so that aggregates, fragments, host protein residues and potential viruses can be removed, and the method has the greatest advantages that impurities and target molecules can be effectively separated, and compared with the traditional chromatography elution method, the yield is improved by about 60%, the monomer purity is improved by about 1.2%, and the production cost is greatly saved.
Drawings
FIG. 1 is a chromatogram of example 1.
FIG. 2 is a SEC-HPLC purity detection profile of example 1.
FIG. 3 is an AEX-HPLC purity detection profile of example 1.
Detailed Description
The following examples are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
The raw material sources are as follows:
CHO (chinese hamster ovary cells) expresses a protein of interest and is purified by the following two steps:
1. affinity chromatography:
chromatography column: nano-micro NMab, column height of 20.9cm, column volume of 210.0mL
Chromatography apparatus: AKTA pure150L
Chromatographic sample: harvesting liquid for CHO cell culture expression
Balancing solution: 20mM Tris-HCl,150mM NaCl,pH7.2
Eluent: 50mM acetic acid pH 3.5
Regeneration liquid: 2% acetic acid
Disinfectant: 0.1M sodium hydroxide
Sample conditioning fluid: 2M Tris
Experimental operation flow: washing the affinity chromatography column with 3 column volumes of balancing solution for balancing, loading the obtained product of CHO cell culture expression onto the affinity chromatography column (the loading capacity is 30 g/L), washing the affinity chromatography column with 3 column volumes of balancing solution for balancing, eluting with 5 column volumes of eluent, taking UV280 as a judgment standard, starting collection when the absorption value of UV280 is increased to 100mAu, stopping collection when the absorption value of UV280 is reduced to 100mAu, named as affinity collection, and regulating pH of the affinity collection solution to 6.0 with 2M Tris. After the elution, the regeneration peak was eluted with 3 column volumes of regeneration solution, the regeneration solution was washed with 3 column volumes of equilibration solution, the chromatographic column was washed with 3 column volumes of antiseptic solution, and then stored.
2. Hydrophobic interaction chromatography:
chromatography column: nami UniHR Butyl-30L, column height 19.2cm, column volume 184.6mL
Chromatography apparatus: AKTA pure150L
Chromatographic sample: affinity collection liquid neutralization liquid
Regulating solution: 20mmol/L Tris-HCl,1mol/L NaCl, pH 7.5
Balancing solution: 20mmol/L Tris-HCl,10mmol/L NaCl, pH 7.5
Eluent: 20mmol/L Tris-HCl, pH 7.5
Regeneration liquid: 2% acetic acid
Disinfectant: 0.5M sodium hydroxide
Experimental operation flow: the hydrophobic interaction chromatography column is washed by 3 column volumes of balance liquid for balancing, then neutralization liquid of the affinity collection liquid is loaded on the hydrophobic interaction chromatography column (the loading capacity is 30 g/L), when the UV280 absorption value is increased to 100mAu, collection is started, 5 column volumes of balance liquid are washed by the hydrophobic interaction chromatography column for balancing, when the UV280 absorption value is reduced to 100mAu, collection is stopped, and the collection liquid is named as the hydrophobic interaction chromatography collection liquid. After the collection, the regeneration peak was eluted with 3 column volumes of regeneration solution, and the column was washed with 3 column volumes of sterilization solution, followed by storage.
The collected liquid of the hydrophobic interaction chromatography is filtered by 0.22 mu m to obtain the sample liquid of the anion chromatography.
The detection equipment used in the invention mainly comprises:
SEC-HPLC liquid phase detection device: a liquid chromatography system (Thermo UltiMate 3000), a chromatographic column (nano BioCore SEC-150), mobile phase 44mM PB+300mM NaCl+20% CAN, pH6.5; the flow rate was 0.5mL/min.
AEX-HPLC liquid phase detection apparatus: liquid chromatography system (Thermo UltiMate 3000), column (Sepax Proteomix SAX-NP 5), mobile phase A:20mM triethanolamine+20 mM 1-methylpiperazine+5 mM NaCl+20mM1, 3-bis [ tris (hydroxymethyl) methylamino ] propane+20 mM piperazine+10% acetonitrile, pH8.0; mobile phase B:20mM triethanolamine+20 mM 1-methylpiperazine+5 mM NaCl+20mM1, 3-bis [ tris (hydroxymethyl) methylamino ] propane+20 mM piperazine+10% acetonitrile, pH4.0; the flow rate was 0.8mL/min.
Example 1
Chromatography column: nami NM90Agarose HAM, column height 10.0cm, column volume 4.65mL
Chromatographic sample: anion chromatography loading solution with loading capacity of 30g/L
Balancing solution: 50mM acetate buffer pH6.0
Eluent: 50mM acetic acid-1.1M sodium chloride-10% isopropyl alcohol pH6.0
Sodium chloride buffer: experimental procedure for 50mM acetic acid-1M sodium chloride pH 6.0: the anion exchange chromatographic column is washed by eluent with 5 column volumes for pre-balancing, then the anion exchange chromatographic column is washed by balance liquid with 25 column volumes for balancing, then the anion chromatographic loading liquid is loaded on the anion exchange chromatographic column, the loading capacity is 30g/L, then the anion exchange chromatographic column is washed by balance liquid with 9 column volumes for balancing, then the elution is started, the elution mode is that the balance liquid and the eluent are linearly eluted, the elution gradient is 0-100% B, the elution volume is 10 column volumes, the UV280 is taken as a judgment standard after the eluent starts to be eluted, the collection is started when the UV absorption value is reduced to 1000mAu, and the collection is named AEX-PM. After the elution was completed, the regeneration peak was eluted with 4 column volumes of sodium chloride buffer, and the column was sterilized and then stored.
TABLE 1 example 1 anion chromatography mass data
Example 2
Chromatography column: bognon EzScreen MIX-A, column height 10.0cm, column volume 4.9mL
Chromatographic sample: anion chromatography loading solution with loading capacity of 30g/L
Balancing solution: 50mM acetate buffer pH6.0
Eluent: 50mM acetic acid-1.1M sodium chloride-10% isopropyl alcohol pH6.0
Sodium chloride buffer: experimental procedure for 50mM acetic acid-1M sodium chloride pH 6.0: the anion exchange chromatographic column is washed by eluent with 5 column volumes for pre-balancing, then the anion exchange chromatographic column is washed by balance liquid with 10 column volumes for balancing, then the anion chromatographic loading liquid is loaded on the anion exchange chromatographic column, the loading capacity is 30g/L, then the anion exchange chromatographic column is washed by balance liquid with 9 column volumes for balancing, then the elution is started, the elution mode is that the balance liquid and the eluent are linearly eluted, the elution gradient is 0-100% B, the elution volume is 10 column volumes, the UV280 is taken as a judgment standard after the eluent starts to be eluted, the collection is started when the UV absorption value is reduced to 1000mAu, and the collection is named AEX-P. After the elution was completed, the regeneration peak was eluted with 4 column volumes of sodium chloride buffer, and the column was sterilized and then stored.
TABLE 2 example 2 anion exchange chromatography
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The anion exchange chromatographic purification process of fusion protein features that acetic acid buffer solution is used as balance liquid and acetic acid-sodium chloride-alcohol buffer solution is used as eluent in the linear elution mode with 0-100% gradient and 10 column volumes to separate target protein bound to anion stuffing.
2. The method of claim 1, wherein the acetate buffer has a pH in the range of about pH5.5 to about pH6.5.
3. The method according to claim 1, wherein the pH of the acetic acid-sodium chloride-alcohol buffer is in the range of pH5.5 to pH6.5.
4. The method according to claim 1, wherein the concentration of acetate buffer in acetate-sodium chloride-alcohol buffer is 40mM to 60mM; the concentration of sodium chloride is 1.0M-1.2M; the alcohol is one or more of monohydric alcohol or polyhydric alcohol with carbon chain length of 2-6; the alcohol content is 8-12%.
5. An anion exchange chromatography method of a fusion protein, comprising the steps of:
(1) Pre-balancing an anion exchange chromatographic column by using the eluent B;
(2) Balancing the anion exchange chromatographic column by using the balance liquid A;
(3) Filtering the solution containing the fusion protein, and loading the filtered solution on an anion exchange chromatographic column;
(4) Then balancing the chromatographic column by using the balance liquid A;
(5) Eluting the main peak of the protein by using the balance A and the eluent B and recovering the main peak;
(6) The regenerated peak was eluted using high concentration sodium chloride buffer.
6. The method of claim 5, wherein the B eluent in step (1) is acetic acid-sodium chloride-alcohol buffer; the pH range of the eluent B is between pH5.5 and pH6.5; acetic acid concentration in the acetic acid-sodium chloride-alcohol buffer solution is 40 mM-60 mM; the concentration of sodium chloride in the acetic acid-sodium chloride-alcohol buffer solution is 1.0M-1.2M; the alcohol in the acetic acid-sodium chloride-alcohol buffer solution is one or more of monohydric alcohol or polyhydric alcohol with carbon chain length of 2-6; the alcohol content in the acetic acid-sodium chloride-alcohol buffer solution is 8% -12%.
7. The method of claim 5, wherein the a-equilibration solution in step (2) is an acetate buffer; the pH range of the balance liquid A is between pH5.5 and pH6.5; the acetic acid concentration in the acetic acid buffer is 40 mM-60 mM.
8. The method according to claim 5, wherein the carrier in step (3) is an anionic filler containing ionic groups and hydrophobic groups; the fusion protein is an acidic Fc fusion protein molecule with the PI value between 5.0 and 6.0.
9. The method of claim 5, wherein the elution in step (5) is performed by linear elution of the equilibrium liquid A with the eluent B, with an elution gradient of 0-100% B, and an elution volume of 10 column volumes.
10. Use of the method of any one of claims 1 to 4 or the method of any one of claims 5 to 9 in recombinant protein purification, antibody purification, vaccine purification.
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