CN114409765A - Method for purifying antibody - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 69
- 239000007853 buffer solution Substances 0.000 claims abstract description 56
- 238000001914 filtration Methods 0.000 claims abstract description 33
- 238000005406 washing Methods 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 30
- 238000011068 loading method Methods 0.000 claims abstract description 28
- 238000010828 elution Methods 0.000 claims abstract description 26
- 238000000746 purification Methods 0.000 claims abstract description 26
- 230000002779 inactivation Effects 0.000 claims abstract description 17
- 239000012149 elution buffer Substances 0.000 claims abstract description 11
- 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 42
- 239000003480 eluent Substances 0.000 claims description 26
- 238000011118 depth filtration Methods 0.000 claims description 23
- 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 11
- 238000010829 isocratic elution Methods 0.000 claims description 10
- 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
- 239000000872 buffer Substances 0.000 claims description 6
- 239000000945 filler 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 6
- 108020001507 fusion proteins Proteins 0.000 claims description 3
- 102000037865 fusion proteins Human genes 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 20
- 102000004169 proteins and genes Human genes 0.000 abstract description 20
- 239000012535 impurity Substances 0.000 abstract description 9
- 229920000642 polymer Polymers 0.000 abstract description 9
- 238000011091 antibody purification Methods 0.000 abstract description 8
- 238000004140 cleaning Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- 238000005349 anion exchange Methods 0.000 abstract description 5
- 238000005341 cation exchange Methods 0.000 abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 14
- 238000005571 anion exchange chromatography Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000005277 cation exchange chromatography Methods 0.000 description 8
- 239000006167 equilibration buffer Substances 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000000249 desinfective effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 238000011072 cell harvest Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000011534 incubation Methods 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
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000011010 flushing procedure Methods 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
- 238000006467 substitution reaction Methods 0.000 description 1
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- 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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- 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)
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Abstract
The invention relates to the technical field of antibody purification, in particular to a method for purifying an antibody. A method for purifying an antibody comprising the steps of: carrying out affinity chromatography purification, inactivation and deep filtration on an antibody sample to be treated; the affinity chromatography purification comprises: and (3) balancing the affinity chromatography column by using an equilibrium buffer solution, then loading the column, respectively washing the column by using the equilibrium buffer solution, a middle washing solution and a second equilibrium buffer solution, and then eluting the target antibody by using an elution buffer solution and collecting the target antibody. The invention adopts a certain affinity chromatography cleaning, elution and collection mode, and is matched with a certain deep filtration process condition, so that host protein, DNA, polymer and other impurities can be obviously removed, and through the matching of affinity chromatography and deep filtration, the purification effect equivalent to that of affinity chromatography, deep filtration, anion exchange and 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 method for purifying 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 and host proteins and nucleic acids produced by the host cells themselves during the culture of mammalian cells leads to a complex composition of the cell culture product.
The downstream preparation process of the antibody drug mainly comprises antibody capture and refining purification, wherein after the antibody is captured, the antibody still contains various impurities such as DNA, aggregates, host protein and the like, and generally needs to be treated by anion exchange chromatography and cation exchange chromatography. That is, in the current antibody purification, at least three steps of chromatography are included for protein purification: (1) performing monoclonal antibody capture by using affinity chromatography to remove most of host protein, DNA and other impurities; (2) further removing impurities such as host protein, DNA and virus by anion exchange chromatography after deep filtration; (3) cation exchange chromatography is used to remove impurities such as multimers, charge isomers, etc.
However, the existing purification process has more steps, higher cost, longer process time and lower product yield due to more steps.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for purifying an antibody, which aims to solve the technical problems of multiple purification process steps, high cost, low yield and the like in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for purifying an antibody comprising the steps of:
carrying out affinity chromatography purification, inactivation and deep filtration on an antibody sample to be treated;
the affinity chromatography purification comprises: using an equilibrium buffer solution to balance the affinity chromatography column, then loading the column, respectively washing the column by using the equilibrium buffer solution, a middle washing solution and a second equilibrium buffer solution, and then eluting the target antibody by using an elution buffer solution and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc, 150-160 mM NaCl, and the pH value is 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 washing liquid is at least one of the following buffers:
and (3) buffer solution A: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
and (3) buffer solution B: 50-55 mM NaAc-HAc, 0.5-0.6% Triton and pH 5.5-8.0;
the elution buffer solution is 50-55 mM NaAc-HAc, and the pH value is 3.5-5.5.
The method for purifying the antibody can obviously remove host protein, DNA, polymers and other impurities by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with certain deep filtration process conditions, realizes the removal action of anion exchange chromatography and cation exchange chromatography in the steps of affinity chromatography and deep filtration, achieves the purification effect equivalent to that of affinity chromatography, deep filtration, anion exchange and cation exchange in the prior art by matching of the affinity chromatography and the deep filtration, simplifies the process steps, reduces the cost and improves the product yield.
In a specific embodiment of the invention, the elution is performed by gradient elution or isocratic elution. Further, the procedure of gradient elution is as follows: gradient elution was used. Wherein, the mobile phase A can be 50mM NaAc-HAc pH 5.5, the mobile phase B can be 50mM NaAc-HAc pH 3.6, and the elution is carried out by linear gradient with 0% to 100% of the mobile phase B.
In a specific embodiment of the invention, the collecting comprises: when isocratic elution is adopted, collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280nm from the beginning to the end of collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280 nm; when gradient elution is adopted, the eluent with the ultraviolet value of 50-100 mAU/2mm @280nm is collected from the beginning to the end of the collection with the ultraviolet value of 500-1000 mAU/2mm @280 nm.
In a specific embodiment of the invention, the filler for affinity chromatography is MabSelect prism a.
In a specific embodiment of the invention, the method further comprises sterilizing the affinity chromatography column before equilibrating the affinity chromatography column. Further, the disinfection solution adopted for 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 particular embodiment of the invention, the loading of the depth filtration is 2000g/m or less2。
By adopting the deep filtration process and matching with the specific affinity chromatography purification treatment, the protein residual host cells can be greatly reduced to about 1-2 ppm, the SEC purity can be improved to more than 99%, and the final quality requirement is met.
In a specific embodiment of the invention, the depth filtration comprises: the depth filter was equilibrated with equilibration buffer and then loaded. Further, the balance buffer solution is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl, 0-120 mM NaCl, pH 5-8, and the conductivity is 5-15 mS/cm, such as 50-55 mM Tris-HAc, 95-100 mM NaCl, pH 8.1 + -0.2.
In practice, in depth filtration, after the filtration of the sample is completed, the feed liquid in the depth filter is replaced by a washing buffer. Further, the washing buffer and the equilibration buffer for depth filtration are the same.
In a specific embodiment of the present invention, the depth filtration uses X0HC deep filtration membrane (Millistatk +)
HC Pod Depth filters, X0HC media series) or X0SP deep filtration membrane (Millistatk +
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 eluate collected by affinity chromatography purification to 3.5-3.7 by adopting an acetic acid buffer solution, and incubating for 60-90 min at the temperature of 18-26 ℃; and then regulating the pH value to 5.0-8.5 by adopting a Tris buffer solution.
In a specific embodiment of the present 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, elution and collection mode is adopted, and a certain deep filtration process condition is matched, so that impurities such as host protein, DNA, polymers and the like can be obviously removed, the removal effect of anion exchange chromatography and cation exchange chromatography is realized in the steps of affinity chromatography and deep filtration, the purification effect equivalent to that of affinity chromatography, deep filtration, anion exchange and cation exchange in the prior art is achieved through the matching of affinity chromatography and deep filtration, 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%, and the protein residual host cell is reduced to about 1-2 ppm, thereby meeting the final quality requirement.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a process for purifying an antibody according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of affinity chromatography purification according to an embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The method for purifying the antibody specifically refers to the schematic flow chart of the antibody purification shown in fig. 1, and comprises the following steps:
carrying out affinity chromatography purification, inactivation and deep filtration on an antibody sample to be treated;
the affinity chromatography purification comprises: using an equilibrium buffer solution to balance the affinity chromatography column, then loading the column, respectively washing the column by using the equilibrium buffer solution, a middle washing solution and a second equilibrium buffer solution, and then eluting the target antibody by using an elution buffer solution and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc, 150-160 mM NaCl, and the pH value is 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 washing liquid is at least one of the following buffers:
and (3) buffer solution A: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
and (3) buffer solution B: 50-55 mM NaAc-HAc, 0.5-0.6% Triton and pH 5.5-8.0;
the elution buffer solution is 50-55 mM NaAc-HAc, and the pH value is 3.5-5.5.
The method for purifying the antibody can obviously remove host protein, DNA, polymers and other impurities by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with certain deep filtration process conditions, realizes the removal action of anion exchange chromatography and cation exchange chromatography in the steps of affinity chromatography and deep filtration, achieves the purification effect equivalent to that of affinity chromatography, deep filtration, anion exchange and cation exchange in the prior art by matching of the affinity chromatography and the deep filtration, simplifies the process steps, reduces the cost and improves the product yield.
The antibody purification method of the present invention does not include anion exchange chromatography and cation exchange chromatography after depth filtration.
In actual operation, the intermediate washing liquid is adjusted according to the residual amount of the host protein in the sample; the polymer is removed by adjusting the pH of the elution buffer according to the protein quality. Specifically, in the removal of the host protein residue, when a system of the buffer solution A is used, the pH value of the intermediate washing solution can be adjusted to a lower pH value (e.g., 5.0-5.2) within a range of 5.0-5.5, or a higher urea concentration (e.g., 2.5-3M) within a range of 1-3M; when a system of buffer solution B is used, the pH value of the intermediate flushing liquid is adjusted to be higher (such as 7-8) within the range of 5.5-8, so that more host protein residues can be removed. In the elution stage, the higher the elution pH value (such as 3.8-4.1) is used in isocratic elution, and more aggregates are 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 carried out by using 0% to 100% of mobile phase B), and carrying out severer collection condition (500-1000 mAU/2mM) on the eluted protein ultraviolet peak to remove polymers.
As in various embodiments, the concentration of urea in the buffer a can be 1M, 1.2M, 1.4M, 1.5M, 1.6M, 1.8M, 2M, 2.2M, 2.4M, 2.5M, 2.6M, 2.8M, 3M, and the like; the mass/volume fraction of Triton in buffer B may be 0.5%, 0.52%, 0.54%, 0.55%, 0.56%, 0.58%, 0.6%, etc.
By adopting the washing mode of the invention, the removal rate of host proteins and the like in the washing stage of affinity chromatography can be further improved; by adopting the elution mode of the invention, the removal rate of the polymer can be further improved.
As in 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 pH 5.5; the buffer solution B can be 50mM NaAc-HAc, 1-3M urea and has pH of 5.0-5.5; the buffer solution C can be 50mM NaAc-HAc, 0.5% Triton and has the pH value of 5.5-8.0; the eluent can be 50mM NaAc-HAc, and the pH value is 3.5-5.5.
In a specific embodiment of the invention, the elution is performed by gradient elution or isocratic elution. Further, the procedure of gradient elution is as follows: gradient elution was used. Wherein, the mobile phase A can be 50mM NaAc-HAc pH 5.5, the mobile phase B can be 50mM NaAc-HAc pH 3.6, and the elution is carried out by linear gradient with 0% to 100% of the mobile phase B.
In a specific embodiment of the invention, the collecting comprises: when isocratic elution is adopted, collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280nm from the beginning to the end of collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280 nm; when gradient elution is adopted, the eluent with the ultraviolet value of 50-100 mAU/2mm @280nm is collected from the beginning to the end of the collection with the ultraviolet value of 500-1000 mAU/2mm @280 nm. Wherein @280nm refers to the ultraviolet value of the eluent at a wavelength of 280 nm. During isocratic elution, the eluent is collected when the ultraviolet value of the eluent at 280nm reaches 50-100 mAU/2mm, the ultraviolet value of the eluent is changed along with the elution, and the collection of the eluent is stopped when the ultraviolet value of the eluent at 280nm is changed back to 50-100 mAU/2mm again.
By adopting the gradient elution and collection mode, the polymer can be further optimized to be removed, and the product purity is improved.
In a specific embodiment of the invention, the filler for affinity chromatography is MabSelect prism a.
In a specific embodiment of the invention, the method further comprises sterilizing the affinity chromatography column before equilibrating the affinity chromatography column. Further, the disinfection solution used for disinfection is 0.5-0.6M NaOH aqueous solution, such as 0.5M NaOH aqueous solution.
Referring to fig. 2, the affinity chromatography column is sterilized, then equilibrated, and then loaded, and then washed with the equilibration buffer 1, then washed with the intermediate buffer 2, then washed with the equilibration buffer 3, and then eluted; and after the elution is finished, disinfecting the affinity chromatography column by adopting a disinfection solution, and then washing and storing.
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 various embodiments, the pH of the sample in the depth filtration loading conditions can be 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, etc.; the conductivity of the sample can be 5mS/cm, 6mS/cm, 7mS/cm, 8mS/cm, 9mS/cm, 10mS/cm, 11mS/cm, 12mS/cm, 13mS/cm, 14mS/cm, 15mS/cm, and the like.
In a particular embodiment of the invention, the loading of the depth filtration is 2000g/m or less2. The loading capacity is adjusted according to the protein quality, and the lower the loading capacity, the higher the removed aggregates, the host cell residue and the nucleic acid residue.
By adopting the deep filtration process and matching with the specific affinity chromatography purification treatment, the protein residual host cells can be greatly reduced to about 1-2 ppm, the SEC purity can be improved to more than 99%, and the final quality requirement is met.
In a specific embodiment of the invention, the depth filtration comprises: the depth filter was equilibrated with equilibration buffer and then loaded. Further, the balance buffer solution is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl, 0-120 mM NaCl and pH 5-8.
As in 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.; alternatively, 50mM Tris-HAc, pH 7.0, 7.5, 8.0, etc. may be used. The NaCl concentration may be 0 to 120 mM.
In practice, in depth filtration, after the filtration of the sample is completed, the feed liquid in the depth filter is replaced by a washing buffer. Further, the washing buffer and the equilibration buffer for depth filtration are the same.
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 eluate collected by affinity chromatography purification to 3.5-3.7 by adopting an acetic acid buffer solution, and incubating for 60-90 min at the temperature of 18-26 ℃; and then regulating the pH value to 5.0-8.5 by adopting a Tris buffer solution.
In a specific embodiment of the present invention, the antibody comprises any one of a monoclonal antibody, a diabody and a fusion protein.
Example 1
(1) Disinfecting an affinity chromatography column (the filler is MabSelect prism A, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8mL) by using 3-4 CV of 0.5M NaOH aqueous solution, and then balancing the affinity chromatography column by using 5-6 CV of 50mM Tris-HAc, 150mM NaCl and pH 7.4 balance buffer solution; loading the sample to an affinity chromatography column, wherein the loading capacity of the sample is less than or equal to 57 g/L; wherein the sample is 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 and pH 7.4 balance buffer solution, then washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc, 3M urea and pH 5.3 intermediate washing solution, and then washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc and pH 5.5 balance buffer solution; then, a linear gradient elution was performed with mobile phase A of 50mM NaAc-HAc, pH 5.5, mobile phase B of 50mM NaAc-HAc, pH 3.6, using 10CV from 0% to 100% mobile phase B. When the ultraviolet value of the eluent at the wavelength of 280nm reaches 100mAU/2mm, the collection is started, and when the ultraviolet value reaches 500mAU/2mm, the collection is stopped.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, 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 incubation is carried out for 60-90 min at the temperature of 18-26 ℃; the pH was then adjusted to 8.1 with Tris buffer.
(4) And (3) 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: the depth filter X0HC is prepared by adding water at a ratio of 100L/m or more2After rinsing, the mixture is equilibrated with 50-55 mM Tris-HAc, 95-100 mM NaCl, pH 8.1 + -0.1 buffer solution for 20-30L/m2. When the sample is filtered, the pressure is controlled to be less than or equal to 2bar, and the maximum loading capacity is determined to be 2000g/m2. After the sample loading is finished, using 20-25L/m250-55 mM Tris-HAc, 95mM NaCl, pH 8.1 + -0.1 and collecting the filtrate.
Example 2
(1) Disinfecting an affinity chromatography column (the filler is MabSelect prism A, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8mL) by using 3-4 CV of 0.5M NaOH aqueous solution, and then balancing the affinity chromatography column by using 5-6 CV of 50mM Tris-HAc, 150mM NaCl and pH 7.4 balance buffer solution; loading the sample to an affinity chromatography column, wherein the loading capacity of the sample is less than or equal to 57 g/L; wherein the sample is cell harvest supernatant derived from perfusion cultured Chinese Hamster Ovary (CHO) cells.
(2) Washing the affinity chromatography column with 3-4 CV 50mM Tris-HAc, 150mM NaCl and pH 7.4 balance buffer solution, washing the affinity chromatography column with 3-4 CV 50mM NaAc-HAc, 0.5% Triton and pH 8.0 intermediate washing solution, and washing the affinity chromatography column with 3-4 CV 50mM NaAc-HAc and pH 5.5 balance buffer solution; 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/2 mM.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, 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 incubation is carried out for 60-90 min at the temperature of 18-26 ℃; the pH was then adjusted to 5.5. + -. 0.2 using Tris buffer.
(4) And (3) 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: the depth filter X0SP is prepared by adding water at a ratio of 100L/m or more2After the washing, the mixture is balanced by a buffer solution with pH of 5.5 +/-0.1 and 20-30L/m, wherein the buffer solution is 50-55 mM NaAc-HAc2. When the sample is filtered, the pressure is controlled to be less than or equal to 2bar, and the maximum loading capacity is determined to be 1000-2000 g/m2. After the sample loading is finished, using 20-25L/m250-55 mM NaAc-HAc, pH 5.5 + -0.1 and the filtrate is harvested.
Comparative example 1
Comparative example 1 provides a purification method of a conventional antibody, comprising the steps of:
(1) disinfecting an affinity chromatography column (the filler is MabSelect prism A, the specification of the chromatography column is 0.66 multiplied by 20cm, and the volume is 6.8mL) by using 3-4 CV of 0.5M NaOH aqueous solution, and then balancing the affinity chromatography column by using 5-6 CV of 50mM Tris-HAc, 150mM NaCl and pH 7.4 balance buffer solution; loading the sample to an affinity chromatography column, wherein the loading capacity of the sample is less than or equal to 57 g/L; wherein the sample is 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 and pH 7.4 balance buffer solution, then washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc, 1M NaCl and pH 5.5 intermediate washing solution, and then washing the affinity chromatography column with 3-4 CV of 50mM NaAc-HAc and pH 5.5 balance buffer solution; 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/2 mM.
(3) Inactivating the eluent obtained in the step (2) at low pH; specifically, 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 incubation is carried out for 60-90 min at the temperature of 18-26 ℃; the pH was then adjusted to 8.1. + -. 0.2 using Tris buffer.
(4) And (3) 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: the deep layer filter A1HC is prepared by adding water at a ratio of 100L/m or more2After rinsing, the mixture is equilibrated with 50-55 mM Tris-HAc, 95-100 mM NaCl, pH 8.1 + -0.1 buffer solution for 20-30L/m2. When the sample is filtered, the pressure is controlled to be less than or equal to 2bar, and the maximum loading capacity is determined to be less than or equal to 2000g/m2. After the sample loading is finished, using 20-25L/m250mM Tris-HAc, 95mM NaCl, pH 8.1. + -. 0.1 and the filtrate is harvested.
Comparative example 2
Comparative example 2 reference comparative example 1 with the difference that: in the step (2), the intermediate cleaning solution is 50mM NaAc-HAc, 3M urea and pH 5.3.
Comparative example 3
Comparative example 3 reference comparative example 1 with the difference that: and (4) replacing the deep filter A1HC with X0 HC.
Comparative example 4
Comparative example 4 the conventional affinity chromatography, virus inactivation and depth filtration steps of comparative example 1 were referenced.
The filtered sample was purified by conventional flow-through anion exchange chromatography, comprising the steps of: balancing 3CV by 50mM Tris-HAc and pH 7.5 +/-0.1 buffer solution, loading the sample with 200-400 g/L, washing 3CV by 50mM Tris-HAc and pH 7.5 +/-0.1 buffer solution, and starting to collect the flow-through liquid when the ultraviolet value of the flow-through liquid is 100 at the wavelength of 280nm in the loading and washing stages until the ultraviolet value of the flow-through liquid is changed back to 100mAU/2mM at the wavelength of 280nm to stop collecting.
Purifying the flow-through liquid after anion chromatography by conventional binding mode cation exchange chromatography, and comprises the following steps: balancing 3CV by 50mM NaAc-HAc and buffer solution with pH 5.5 +/-0.1, loading the sample with 30-50 g/L, washing 3CV by 50mM NaAc-HAc and buffer solution with pH 5.5 +/-0.1, wherein the elution buffer solution is 50mM NaAc-HAc and has pH 5.5 +/-0.1 and 0.3M NaCl, starting to collect the eluent when the ultraviolet value of the eluent is 100mAU/2mM at the wavelength of 280nm, and stopping collecting until the ultraviolet value of the eluent is changed back to 100mAU/2mM at the wavelength of 280 nm.
Experimental example 1
In order to compare and illustrate the differences of the antibody purification methods of different examples and comparative examples, the products treated by different examples and comparative examples were tested, and the test results are shown in table 1.
TABLE 1 test results of various examples and comparative examples
From the test results, the method for purifying the antibody can obviously remove host protein, DNA, polymers and other impurities by adopting a certain affinity chromatography cleaning, eluting and collecting mode and matching with certain deep filtration process conditions, realizes the removal action of anion exchange chromatography and cation exchange chromatography in the steps of affinity chromatography and deep filtration, achieves the purification effect equivalent to that of the prior art through matching of affinity chromatography, deep filtration, anion exchange and cation exchange, simplifies the process steps, reduces the cost and improves the product yield.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for purifying an antibody, comprising the steps of:
carrying out affinity chromatography purification, inactivation and deep filtration on an antibody sample to be treated;
the affinity chromatography purification comprises: using an equilibrium buffer solution to balance the affinity chromatography column, then loading the column, respectively washing the column by using the equilibrium buffer solution, a middle washing solution and a second equilibrium buffer solution, and then eluting the target antibody by using an elution buffer solution and collecting the target antibody; wherein the balance buffer solution is 50-55 mM Tris-HAc, 150-160 mM NaCl, and the pH value is 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 washing liquid is at least one of the following buffers:
and (3) buffer solution A: 50-55 mM NaAc-HAc, 1-3M urea, pH 5.0-5.5;
and (3) buffer solution B: 50-55 mM NaAc-HAc, 0.5-0.6% Triton and pH 5.5-8.0;
the elution buffer solution is 50-55 mM NaAc-HAc, and the pH value is 3.5-5.5.
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 for purifying an antibody according to claim 1, wherein the collecting comprises: when isocratic elution is adopted, collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280nm from the beginning to the end of collecting the eluent with an ultraviolet value of 50-100 mAU/2mm @280 nm; when gradient elution is adopted, the eluent with the ultraviolet value of 50-100 mAU/2mm @280nm is collected from the beginning to the end of the collection with the ultraviolet value of 500-1000 mAU/2mm @280 nm.
4. The method for purifying an antibody according to claim 1, wherein the filler for affinity chromatography is MabSelect prism A.
5. The method for purifying an antibody according to any one of claims 1 to 4, wherein the depth filtration is carried out under the following conditions: the pH value of the sample is 5.0-8.5, and the conductivity is 5-15 mS/cm.
6. The method of claim 5, wherein the depth filtration is carried out at a loading of 2000g/m or less2。
7. The method for purifying an antibody according to claim 5, wherein the depth filtration is performed using an X0HC depth filtration membrane or an X0SP depth filtration membrane.
8. The method for purifying an antibody according to any one of claims 1 to 4, wherein the depth filtration comprises: balancing the deep filter by adopting a balance buffer solution, and then loading the sample;
preferably, in the deep filtration, the equilibrium buffer solution is 50-55 mM Tris-HAc or NaAc-HAc or His-HCl, 0-120 mM NaCl and pH 5-8.
9. The method of purifying an antibody according to claim 1, wherein the inactivation is low pH inactivation;
preferably, the low pH inactivation comprises: adjusting the pH value of the eluate collected by affinity chromatography purification to 3.5-3.7 by adopting an acetic acid buffer solution, and incubating for 60-90 min at the temperature of 18-26 ℃; and then regulating the pH value to 5.0-8.5 by adopting a Tris buffer solution.
10. The method for purifying an antibody according to claim 1, wherein the antibody comprises any one of a monoclonal antibody, a diabody and a fusion protein.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115353561A (en) * | 2022-09-29 | 2022-11-18 | 武汉佳惟达生物科技有限公司 | Antibody purification method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130336957A1 (en) * | 2012-05-21 | 2013-12-19 | Abbvie, Inc. | Novel purification of human, humanized, or chimeric antibodies using protein a affinity chromatography |
| JP2014094936A (en) * | 2012-10-10 | 2014-05-22 | Tosoh Corp | Purification method of antibody |
| US20180215785A1 (en) * | 2015-07-22 | 2018-08-02 | Kaneka Corporation | Method for purifying antibody-like protein |
| CN109336969A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | A kind of purification process of antibody |
| CN109336968A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | The method of protein A-sepharose affinity chromatography antibody purification and the purification process of antibody |
| CN109678969A (en) * | 2018-12-29 | 2019-04-26 | 北京东方百泰生物科技有限公司 | A kind of purification process of CTLA4-Ig fusion protein |
| CN112279919A (en) * | 2020-10-02 | 2021-01-29 | 朱吉安 | Preparation method of anti-PD-1 antibody |
-
2021
- 2021-12-23 CN CN202111592194.5A patent/CN114409765A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130336957A1 (en) * | 2012-05-21 | 2013-12-19 | Abbvie, Inc. | Novel purification of human, humanized, or chimeric antibodies using protein a affinity chromatography |
| JP2014094936A (en) * | 2012-10-10 | 2014-05-22 | Tosoh Corp | Purification method of antibody |
| US20180215785A1 (en) * | 2015-07-22 | 2018-08-02 | Kaneka Corporation | Method for purifying antibody-like protein |
| CN109336969A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | A kind of purification process of antibody |
| CN109336968A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | The method of protein A-sepharose affinity chromatography antibody purification and the purification process of antibody |
| CN109678969A (en) * | 2018-12-29 | 2019-04-26 | 北京东方百泰生物科技有限公司 | A kind of purification process of CTLA4-Ig fusion protein |
| CN112279919A (en) * | 2020-10-02 | 2021-01-29 | 朱吉安 | Preparation method of anti-PD-1 antibody |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115353561A (en) * | 2022-09-29 | 2022-11-18 | 武汉佳惟达生物科技有限公司 | Antibody purification method |
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