CN114409765B - Method for purifying antibody - Google Patents
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- CN114409765B CN114409765B CN202111592194.5A CN202111592194A CN114409765B CN 114409765 B CN114409765 B CN 114409765B CN 202111592194 A CN202111592194 A CN 202111592194A CN 114409765 B CN114409765 B CN 114409765B
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 60
- 239000007853 buffer solution Substances 0.000 claims abstract description 35
- 238000011118 depth filtration Methods 0.000 claims abstract description 32
- 238000011068 loading method Methods 0.000 claims abstract description 24
- 238000000746 purification Methods 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims abstract description 20
- 238000001914 filtration Methods 0.000 claims abstract description 18
- 238000011010 flushing procedure Methods 0.000 claims abstract description 15
- 230000000415 inactivating effect Effects 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 44
- 229910020820 NaAc-HAc Inorganic materials 0.000 claims description 38
- 239000000872 buffer Substances 0.000 claims description 34
- 239000003480 eluent Substances 0.000 claims description 26
- 238000010828 elution Methods 0.000 claims description 25
- 239000011780 sodium chloride Substances 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 15
- 230000002779 inactivation Effects 0.000 claims description 13
- 238000010829 isocratic elution Methods 0.000 claims description 10
- 239000006167 equilibration buffer Substances 0.000 claims description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 8
- 239000004202 carbamide Substances 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000012516 mab select resin Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 18
- 238000011091 antibody purification Methods 0.000 abstract description 9
- 239000012535 impurity Substances 0.000 abstract description 7
- 229920000642 polymer Polymers 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 5
- 238000005349 anion exchange Methods 0.000 abstract description 3
- 238000005341 cation exchange Methods 0.000 abstract description 3
- 238000004140 cleaning Methods 0.000 abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 238000005406 washing Methods 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 238000005571 anion exchange chromatography Methods 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000005277 cation exchange chromatography Methods 0.000 description 6
- 238000012856 packing Methods 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 238000011072 cell harvest Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 108700015808 potyvirus HC-Pro Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of antibody purification, in particular to a purification method of an antibody. A method of purifying an antibody comprising the steps of: purifying the antibody sample to be treated by affinity chromatography, inactivating and deeply filtering; affinity chromatography purification includes: and (3) balancing the affinity chromatography column by using a balancing buffer solution, loading, respectively flushing by using the balancing buffer solution, an intermediate flushing solution and a second balancing buffer solution, and then eluting the target antibody by using an eluting buffer solution and collecting. According to the invention, by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with a certain depth filtration process condition, impurities such as host proteins, DNA, polymers and the like can be obviously removed, and by matching with the affinity chromatography and the depth filtration, the purification effect equivalent to that of the affinity chromatography, the depth filtration, the anion exchange and the cation exchange in the prior art is achieved, meanwhile, the process steps are simplified, the cost is reduced, and the product yield is improved.
Description
Technical Field
The invention relates to the technical field of antibody purification, in particular to a purification method of an antibody.
Background
Monoclonal antibody drugs are typically expressed using genetically engineered mammalian cells, such as CHO, BHK cells, etc., that produce the protein of interest. The addition of a medium containing various components during mammalian cell culture, host proteins and nucleic acids produced by the host cells themselves, etc., results in complex cell culture product components.
The downstream preparation process of antibody medicine mainly includes antibody capturing and refining purification, after the antibody is captured, it still contains various impurities of DNA, aggregate and host protein, etc., and usually it must be undergone the processes of anion exchange chromatography and cation exchange chromatography. That is, in the current antibody purification, at least three steps of chromatography are included to perform protein purification: (1) Performing monoclonal antibody capturing by using affinity chromatography, and removing most of host proteins, DNA and other impurities; (2) Performing anion exchange chromatography after deep filtration to further remove host protein, DNA, virus and other impurities; (3) Cation exchange chromatography is used to remove impurities such as multimers, charge isomers, and the like.
However, in the existing purification process, the steps are more, the cost is higher, the process time is longer, and the product yield is lower due to the more steps.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a purification method of an antibody, which aims to solve the technical problems of more purification process steps, high cost, low yield and the like in the prior art.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
a method of purifying an antibody comprising the steps of:
purifying the antibody sample to be treated by affinity chromatography, inactivating and deeply filtering;
The affinity chromatography purification comprises the following steps: using a balancing buffer to balance the affinity chromatography column, loading the sample, respectively flushing the affinity chromatography column by using the balancing buffer, an intermediate flushing liquid and a second balancing buffer, and then eluting a target antibody by using an eluting buffer and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc, 150-160mM NaCl,pH 7.4 +/-0.2; the second balance buffer solution is 50-55 mM NaAc-HAc, and the pH value is 5.5+/-0.2; the intermediate flushing liquid is at least one of the following buffers:
buffer a: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
Buffer B: 50-55 mM NaAc-HAc, 0.5-0.6%Triton,pH 5.5-8.0;
The eluting buffer is 50-55 mM NaAc-HAc, and the pH is 3.5-5.5.
The antibody purification method of the invention can obviously remove host protein, DNA, multimer and other impurities by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with a certain depth filtration process condition, and the anion exchange chromatography and the cation exchange chromatography are removed in the steps of affinity chromatography and depth filtration, and the purification effect equivalent to the affinity chromatography, depth filtration, anion exchange and cation exchange in the prior art is achieved by matching with the affinity chromatography and the depth filtration, meanwhile, the process steps are simplified, the cost is reduced, and the product yield is improved.
In a specific embodiment of the present invention, the elution is performed by gradient elution or isocratic elution. Further, the gradient elution procedure is: gradient elution was used. Wherein mobile phase A can be 50mM NaAc-HAc pH 5.5, mobile phase B can be 50mM NaAc-HAc pH 3.6, and linear gradient elution is performed with 0% to 100% mobile phase B.
In a specific embodiment of the invention, the collecting comprises: when isocratic elution is adopted, starting to collect eluent with the ultraviolet value of 50-100 mAU/2mm@280nm until the ultraviolet value is 50-100 mAU/2mm@280nm, and ending the collection; when gradient elution is adopted, the eluent with the ultraviolet value of 50-100 mAU/2mm@280nm starts to be collected until the ultraviolet value of 500-1000 mAU/2mm@280nm ends to be collected.
In a specific embodiment of the present invention, the affinity chromatography packing is MabSelect PrismA.
In a specific embodiment of the invention, the affinity chromatography column is further sterilized prior to equilibration of the affinity chromatography column. Further, the disinfection solution adopted by the disinfection is 0.5-0.6M NaOH aqueous solution.
In a specific embodiment of the present invention, the loading conditions of the depth filtration are: the pH value of the sample is 5.0-8.5, and the conductivity is 5-15 mS/cm.
In a specific embodiment of the invention, the depth filtration loading is 2000g/m 2 or less.
The deep filtration process is matched with the specific affinity chromatography purification treatment, so that the residual host cells of the protein can be greatly reduced, the concentration of the SEC can be reduced to about 1-2 ppm, the purity of the SEC can be improved to more than 99%, and the final quality requirement is met.
In a specific embodiment of the present invention, the depth filtration comprises: the depth filter was equilibrated with equilibration buffer and then loaded. Further, the equilibration buffer is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl, 0-120 mM NaCl, pH 5-8, conductivity 5-15 mS/cm, for example, 50-55 mM Tris-HAc, 95-100mM NaCl,pH 8.1 + -0.2.
In practical operation, in depth filtration, after the sample filtration is finished, the feed liquid in the depth filter is replaced by adopting a flushing buffer solution. Further, the flush buffer is the same as the depth filtration equilibration buffer.
In a specific embodiment of the present invention, the depth filtration uses an X0HC depth filtration membrane (MILLISTAK +HC Pod DEPTH FILTER, X0HC MEDIA SERIES) or X0SP deep filtration membrane (MILLISTAK +HC Pro Pod Depth Filter,X0SP media series)。
In a specific embodiment of the invention, the inactivation is a low pH inactivation. Further, the low pH inactivation comprises: adjusting the pH value of the eluent collected by affinity chromatography to 3.5-3.7 by adopting acetic acid buffer solution, and incubating for 60-90 min at 18-26 ℃; and then adjusting the pH to 5.0-8.5 by adopting Tris buffer solution.
In specific embodiments of the invention, the antibody comprises any one of a monoclonal antibody, a diabody, and a fusion protein.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the antibody purification method, a certain affinity chromatography cleaning, eluting and collecting mode is adopted, and a certain depth filtration process condition is matched, so that impurities such as host proteins, DNA and polymers can be obviously removed, the anion exchange chromatography and the cation exchange chromatography are removed in the affinity chromatography and depth filtration steps, and the affinity chromatography and the depth filtration are matched, so that the purification effect equivalent to the affinity chromatography, the depth filtration, the anion exchange and the cation exchange in the prior art is achieved, meanwhile, the process steps are simplified, the cost is reduced, and the product yield is improved;
(2) By adopting the antibody purification method, the SEC purity can reach more than 99 percent, the residual host cell of the protein is reduced to about 1 to 2ppm, and the final quality requirement is met.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of antibody purification according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of affinity chromatography purification provided in an embodiment of the present invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. 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. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The antibody purification method, specifically referring to the schematic flow chart of antibody purification shown in fig. 1, comprises the following steps:
purifying the antibody sample to be treated by affinity chromatography, inactivating and deeply filtering;
The affinity chromatography purification comprises the following steps: using a balancing buffer to balance the affinity chromatography column, loading the sample, respectively flushing the affinity chromatography column by using the balancing buffer, an intermediate flushing liquid and a second balancing buffer, and then eluting a target antibody by using an eluting buffer and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc, 150-160mM NaCl,pH 7.4 +/-0.2; the second balance buffer solution is 50-55 mM NaAc-HAc, and the pH value is 5.5+/-0.2; the intermediate flushing liquid is at least one of the following buffers:
buffer a: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
Buffer B: 50-55 mM NaAc-HAc, 0.5-0.6%Triton,pH 5.5-8.0;
The eluting buffer is 50-55 mM NaAc-HAc, and the pH is 3.5-5.5.
The antibody purification method of the invention can obviously remove host protein, DNA, multimer and other impurities by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with a certain depth filtration process condition, and the anion exchange chromatography and the cation exchange chromatography are removed in the steps of affinity chromatography and depth filtration, and the purification effect equivalent to the affinity chromatography, depth filtration, anion exchange and cation exchange in the prior art is achieved by matching with the affinity chromatography and the depth filtration, meanwhile, the process steps are simplified, the cost is reduced, and the product yield is improved.
The antibody purification method of the present invention does not include anion exchange chromatography and cation exchange chromatography after depth filtration.
In actual operation, adjusting the intermediate flushing liquid according to the residual amount of host protein in the sample; the pH of the elution buffer is adjusted to remove multimers according to the amount of protein. Specifically, in removing the host protein residue, when using the system of buffer A, the pH of the intermediate rinse solution may be adjusted to a lower pH in the range of 5.0 to 5.5 (e.g., 5.0 to 5.2), or a higher urea concentration in the range of 1 to 3M (e.g., 2.5 to 3M) may be used; when using the buffer B system, the intermediate rinse solution is adjusted to a higher pH (e.g., 7-8) in the range of 5.5-8 to remove more host protein residues. In the elution stage, the higher the elution pH value (such as 3.8-4.1) is used for isocratic elution, more polymer is removed; or using gradient elution (mobile phase A is 50mM NaAc-HAc pH 5.5, mobile phase B is 50mM NaAc-HAc pH 3.6, linear gradient elution is performed with 0% to 100% mobile phase B), and more severe collection conditions (500-1000 mAu/2 mM) can be performed on the ultraviolet peak of eluted protein.
As in the different embodiments, the concentration of urea in the buffer a may be 1M, 1.2M, 1.4M, 1.5M, 1.6M, 1.8M, 2M, 2.2M, 2.4M, 2.5M, 2.6M, 2.8M, 3M, etc.; the mass/volume fraction of Triton in the buffer B may be 0.5%, 0.52%, 0.54%, 0.55%, 0.56%, 0.58%, 0.6%, etc.
The washing mode can further improve the removal rate of host proteins and the like in the washing stage of the affinity chromatography; by adopting the elution mode of the invention, the removal rate of the polymer can be further improved.
As in the various embodiments, the equilibration buffer may be 50mM Tris-HAc,150mM NaCl,pH 7.4; the buffer solution A can be 50mM NaAc-HAc, 0.2-1M NaCl, and the pH value is 5.5; the buffer solution B can be 50mM NaAc-HAc, 1-3M urea and has the pH value of 5.0-5.5; the buffer solution C can be 50mM NaAc-HAc,0.5%Triton,pH 5.5-8.0; the eluent can be 50mM NaAc-HAc, pH 3.5-5.5.
In a specific embodiment of the present invention, the elution is performed by gradient elution or isocratic elution. Further, the gradient elution procedure is: gradient elution was used. Wherein mobile phase A can be 50mM NaAc-HAc pH 5.5, mobile phase B can be 50mM NaAc-HAc pH 3.6, and linear gradient elution is performed with 0% to 100% mobile phase B.
In a specific embodiment of the invention, the collecting comprises: when isocratic elution is adopted, starting to collect eluent with the ultraviolet value of 50-100 mAU/2mm@280nm until the ultraviolet value is 50-100 mAU/2mm@280nm, and ending the collection; when gradient elution is adopted, the eluent with the ultraviolet value of 50-100 mAU/2mm@280nm starts to be collected until the ultraviolet value of 500-1000 mAU/2mm@280nm ends to be collected. Wherein @280nm refers to the ultraviolet value of the eluate at a wavelength of 280 nm. And during isocratic elution, when the ultraviolet value of the eluent at 280nm reaches 50-100 mAU/2mm, starting to collect, and when the ultraviolet value of the eluent changes back to 50-100 mAU/2mm along with the elution, stopping to collect.
By adopting the gradient elution and collection mode, the polymer removal can be further optimized, and the product purity can be improved.
In a specific embodiment of the present invention, the affinity chromatography packing is MabSelect PrismA.
In a specific embodiment of the invention, the affinity chromatography column is further sterilized prior to equilibration of the affinity chromatography column. Further, the disinfection solution adopted by the disinfection is 0.5-0.6M NaOH aqueous solution, such as 0.5M NaOH aqueous solution.
The process flow of affinity chromatography purification can refer to fig. 2, wherein the affinity chromatography column is disinfected, balanced, loaded, flushed with the balanced buffer solution 1, flushed with the intermediate buffer solution 2, flushed with the balanced buffer solution 3, and eluted; and after the elution is finished, adopting a sterilizing solution to sterilize the affinity chromatography column, and then flushing and preserving.
In a specific embodiment of the present invention, the loading conditions of the depth filtration are: the pH value of the sample is 5.0-8.5, and the conductivity is 5-15 mS/cm.
As in the various embodiments, the pH of the sample in the loading conditions of the depth filtration may be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, etc.; the conductivity of the sample may be 5mS/cm, 6mS/cm, 7mS/cm, 8mS/cm, 9mS/cm, 10mS/cm, 11mS/cm, 12mS/cm, 13mS/cm, 14mS/cm, 15mS/cm, etc.
In a specific embodiment of the invention, the depth filtration loading is 2000g/m 2 or less. The loading is adjusted according to the amount of protein, the lower the loading, the higher the amount of polymer, host cell residue and nucleic acid residue removed.
The deep filtration process is matched with the specific affinity chromatography purification treatment, so that the residual host cells of the protein can be greatly reduced, the concentration of the SEC can be reduced to about 1-2 ppm, the purity of the SEC can be improved to more than 99%, and the final quality requirement is met.
In a specific embodiment of the present invention, the depth filtration comprises: the depth filter was equilibrated with equilibration buffer and then loaded. Further, the balance buffer is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl, 0-120 mM NaCl, pH 5-8.
As in the various embodiments, the equilibration buffer may be 50mM NaAc-HAc, pH 5.0, 5.5, 5.8, etc.; or may be 50mM His-HCl, pH 5.5, 6.0, 6.5, etc.; or may be 50mM Tris-HAc, pH 7.0, 7.5, 8.0, etc. The NaCl concentration may be 0 to 120mM.
In practical operation, in depth filtration, after the sample filtration is finished, the feed liquid in the depth filter is replaced by adopting a flushing buffer solution. Further, the flush buffer is the same as the depth filtration equilibration buffer.
In a specific embodiment of the present invention, the depth filtration uses an X0HC depth filtration membrane or an X0SP depth filtration membrane.
In a specific embodiment of the invention, the inactivation is a low pH inactivation. Further, the low pH inactivation comprises: adjusting the pH value of the eluent collected by affinity chromatography to 3.5-3.7 by adopting acetic acid buffer solution, and incubating for 60-90 min at 18-26 ℃; and then adjusting the pH to 5.0-8.5 by adopting Tris buffer solution.
In specific embodiments of the invention, the antibody comprises any one of a monoclonal antibody, a diabody, and a fusion protein.
Example 1
(1) 3-4 CV of 0.5M NaOH aqueous solution is adopted to disinfect an affinity chromatography column (packing is MabSelect PrismA, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8 mL), and then 5-6 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 balancing buffer solution is adopted to balance the affinity chromatography column; loading the sample to an affinity chromatographic column, wherein the loading capacity is less than or equal to 57g/L; wherein the sample is a cell harvest supernatant derived from perfusion cultured Chinese Hamster Ovary (CHO) cells.
(2) Washing the affinity chromatographic column with 3-4 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 equilibrium buffer, washing the affinity chromatographic column with 3-4 CV of 50mM NaAc-HAc,3M urea and pH 5.3 intermediate washing liquid, and washing the affinity chromatographic column with 3-4 CV of 50mM NaAc-HAc and pH 5.5 equilibrium buffer; then, a linear gradient elution was performed with 10CV from 0% to 100% mobile phase B at pH 5.5 for mobile phase A of 50mM NaAc-HAc and pH 3.6 for mobile phase B of 50mM NaAc-HAc. When the ultraviolet value of the eluent at the wavelength of 280nm reaches 100mAU/2mm, the eluent starts to collect, and reaches 500mAU/2mm, the eluent stops collecting.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, an acetic acid buffer solution is adopted to adjust the pH value of the eluent collected by affinity chromatography purification to 3.5-3.7, and the eluent is incubated for 60-90 min at 18-26 ℃; the pH was then adjusted to 8.1 with Tris buffer.
(4) Carrying out deep filtration on the sample subjected to the low pH inactivation treatment in the step (3), wherein the specific steps are as follows: after the deep filter X0HC is rinsed with water with the concentration of more than or equal to 100L/m 2, the solution is balanced by 20 to 30L/m 2 with 50 to 55mM Tris-HAc and buffer solution with concentration of 95 to 100mM NaCl,pH 8.1 +/-0.1. When the sample is filtered, the control pressure is less than or equal to 2bar, and the maximum load is set to 2000g/m 2. After the loading was completed, the filtrate was washed with 20 to 25L/m 2 of 50 to 55mM Tris-HAc,95mM NaCl,pH 8.1.+ -. 0.1 buffer and harvested.
Example 2
(1) 3-4 CV of 0.5M NaOH aqueous solution is adopted to disinfect an affinity chromatography column (packing is MabSelect PrismA, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8 mL), and then 5-6 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 balancing buffer solution is adopted to balance the affinity chromatography column; loading the sample to an affinity chromatographic column, wherein the loading capacity is less than or equal to 57g/L; wherein the sample is a cell harvest supernatant derived from perfusion cultured Chinese Hamster Ovary (CHO) cells.
(2) Washing the affinity chromatography column with 3-4 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 equilibrium buffer, washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc,0.5%Triton,pH 8.0 intermediate washing liquid, and washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc equilibrium buffer with pH of 5.5; then, 5CV of 50mM NaAc-HAc, pH 4.1 elution buffer was used for isocratic elution, and collection was started when the UV value of the eluate at 280nm was 100mAU/2mM, until the UV value of the eluate at 280nm was changed back to 100mAU/2mM again, and collection was stopped.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, an acetic acid buffer solution is adopted to adjust the pH value of the eluent collected by affinity chromatography purification to 3.5-3.7, and the eluent is incubated for 60-90 min at 18-26 ℃; the pH was then adjusted to 5.5.+ -. 0.2 with Tris buffer.
(4) Carrying out deep filtration on the sample subjected to the low pH inactivation treatment in the step (3), wherein the specific steps are as follows: after the deep filter X0SP is rinsed with water with the concentration of more than or equal to 100L/m 2, the solution is equilibrated with 50-55 mM NaAc-HAc buffer solution with the pH of 5.5+/-0.1 for 20-30L/m 2. When filtering the sample, controlling the pressure to be less than or equal to 2bar, and setting the maximum load to be 1000-2000 g/m 2. After loading, the filtrate was washed with 20 to 25L/m 2 of 50 to 55mM NaAc-HAc, pH 5.5.+ -. 0.1 buffer and harvested.
Comparative example 1
Comparative example 1 provides a method for purifying a conventional antibody comprising the steps of:
(1) 3-4 CV of 0.5M NaOH aqueous solution is adopted to disinfect an affinity chromatography column (packing is MabSelect PrismA, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8 mL), and then 5-6 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 balancing buffer solution is adopted to balance the affinity chromatography column; loading the sample to an affinity chromatographic column, wherein the loading capacity is less than or equal to 57g/L; wherein the sample is a cell harvest supernatant derived from perfusion cultured Chinese Hamster Ovary (CHO) cells.
(2) Washing the affinity chromatography column with 3-4 CV of 50mM Tris-HAc,150mM NaCl,pH 7.4 equilibrium buffer, washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc,1M NaCl,pH 5.5 intermediate washing liquid, and washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc equilibrium buffer with pH of 5.5; then, 5CV of 50mM NaAc-HAc, pH 3.6 elution buffer was used for isocratic elution, and collection was started when the UV value of the eluate at 280nm was 100mAU/2mM, until the UV value of the eluate at 280nm was changed back to 100mAU/2mM again, and collection was stopped.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, an acetic acid buffer solution is adopted to adjust the pH value of the eluent collected by affinity chromatography purification to 3.5-3.7, and the eluent is incubated for 60-90 min at 18-26 ℃; the pH was then adjusted to 8.1.+ -. 0.2 using Tris buffer.
(4) Carrying out deep filtration on the sample subjected to the low pH inactivation treatment in the step (3), wherein the specific steps are as follows: after the deep filter A1HC is rinsed with water with the concentration of more than or equal to 100L/m 2, the solution is balanced by 20 to 30L/m 2 with 50 to 55mM Tris-HAc and buffer solution with concentration of 95 to 100mM NaCl,pH 8.1 +/-0.1. When the sample is filtered, the control pressure is less than or equal to 2bar, and the maximum load is less than or equal to 2000g/m 2. After the end of the loading, the filtrate was washed with 20 to 25L/m 2 of 50mM Tris-HAc,95mM NaCl,pH 8.1.+ -. 0.1 buffer and harvested.
Comparative example 2
Comparative example 2 reference comparative example 1, differing in that: in step (2), the intermediate washing solution was 50mM NaAc-HAc,3M urea, pH 5.3.
Comparative example 3
Comparative example 3 reference comparative example 1, differing in that: step (4) replaces the deep filter A1HC with X0HC.
Comparative example 4
Comparative example 4 reference is made to the conventional affinity chromatography, virus inactivation and depth filtration steps of comparative example 1.
The filtered sample was purified by conventional flow-through mode anion exchange chromatography, comprising the steps of: the 3CV is balanced by 50mM Tris-HAc buffer solution with the pH value of 7.5 plus or minus 0.1, the loading capacity of 200-400 g/L, the 3CV is washed by 50mM Tris-HAc buffer solution with the pH value of 7.5 plus or minus 0.1, the collection is started when the ultraviolet value of the flow-through liquid at the wavelength of 280nm is 100 in the loading and washing stages, and the collection is stopped until the ultraviolet value of the flow-through liquid at the wavelength of 280nm is changed back to 100mAU/2 mM.
The conventional combination mode cation exchange chromatography purification of the flow-through liquid after anion chromatography comprises the following steps: the solution is equilibrated with 50mM NaAc-HAc, pH 5.5+ -0.1 buffer solution for 3CV, the loading capacity is 30-50 g/L, the solution is washed with 50mM NaAc-HAc, pH 5.5+ -0.1 buffer solution for 3CV, the solution for elution is 50mM NaAc-HAc, pH 5.5+ -0.1, 0.3M NaCl, the collection is started when the ultraviolet value of the eluent is 100mAU/2mM at the wavelength of 280nm, until the ultraviolet value of the eluent is changed back to 100mAU/2mM at the wavelength of 280nm, and the collection is stopped.
Experimental example 1
To comparatively illustrate the differences in the methods of purifying antibodies in the different examples and comparative examples, the products treated in the different examples and comparative examples were tested, and the test results are shown in Table 1.
Table 1 test results for different examples and comparative examples products
According to the test results, the purification method of the antibody can obviously remove impurities such as host proteins, DNA and polymers by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching a certain depth filtration process condition, the removal effect of anion exchange chromatography and cation exchange chromatography is realized in the steps of affinity chromatography and depth filtration, and the purification effect equivalent to the effects of affinity chromatography, depth filtration, anion exchange and cation exchange in the prior art is achieved by matching the affinity chromatography with the depth filtration, meanwhile, the process steps are simplified, the cost is reduced, and the product yield is improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. The method for purifying the antibody is characterized by comprising the following steps:
purifying the antibody sample to be treated by affinity chromatography, inactivating and deeply filtering;
The affinity chromatography purification comprises the following steps: using a balancing buffer to balance the affinity chromatography column, loading the sample, respectively flushing the affinity chromatography column by using the balancing buffer, an intermediate flushing liquid and a second balancing buffer, and then eluting a target antibody by using an eluting buffer and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc,150~160mM NaCl,pH 7.4 +/-0.2; the second balance buffer solution is 50-55 mM NaAc-HAc, and the pH value is 5.5+/-0.2; the intermediate flushing liquid is at least one of the following buffers:
buffer a: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
Buffer B: 50-55 mM NaAc-HAc, 0.5-0.6% Triton, pH 5.5-8.0;
the eluting buffer solution is 50-55 mM NaAc-HAc, and the pH value is 3.5-5.5;
The filler of the affinity chromatography is MabSelect PrismA;
the deep filtration uses an X0HC deep filtration membrane or an X0SP deep filtration membrane;
the loading conditions of the deep filtration are as follows: the pH of the sample is 5.0-8.5, the conductivity is 5-15 mS/cm.
2. The method for purifying an antibody according to claim 1, wherein the elution is performed by gradient elution or isocratic elution.
3. The method of purifying an antibody of claim 1, wherein the collecting comprises: when isocratic elution is adopted, starting to collect eluent with the ultraviolet value of 50-100 mAU/2mm@280nm until the ultraviolet value of 50-100 mAU/2mm@280nm is finished; and when gradient elution is adopted, starting to collect eluent with the ultraviolet value of 50-100 mAU/2mm@280nm until the ultraviolet value of 500-1000 mAU/2mm@280nm is finished.
4. The method of purifying an antibody according to claim 1, wherein the depth filtration load is 2000g/m 2 or less.
5. A method of purifying an antibody according to any one of claims 1 to 3, wherein the depth filtration comprises: the depth filter was equilibrated with equilibration buffer and then loaded.
6. The method according to claim 5, wherein the equilibration buffer is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl,0~120mM NaCl,pH 5~8 in the depth filtration.
7. The method of purifying an antibody according to claim 1, wherein the inactivation is a low pH inactivation.
8. The method of purifying an antibody of claim 7, wherein the low pH inactivation comprises: adjusting the pH of the eluent collected by affinity chromatography purification to 3.5-3.7 by adopting acetic acid buffer solution, and incubating for 60-90 min at 18-26 ℃; and then adopting Tris buffer solution to adjust the pH to 5.0-8.5.
9. The method of purifying an antibody according to claim 1, wherein the antibody comprises any one of a monoclonal antibody and a diabody.
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