CN116655726B - Antibody purification method based on ion exchange chromatography - Google Patents
Antibody purification method based on ion exchange chromatography Download PDFInfo
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- CN116655726B CN116655726B CN202310945170.6A CN202310945170A CN116655726B CN 116655726 B CN116655726 B CN 116655726B CN 202310945170 A CN202310945170 A CN 202310945170A CN 116655726 B CN116655726 B CN 116655726B
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- 238000000034 method Methods 0.000 title claims abstract description 67
- 238000004255 ion exchange chromatography Methods 0.000 title claims abstract description 37
- 238000011091 antibody purification Methods 0.000 title abstract description 13
- 239000003480 eluent Substances 0.000 claims abstract description 72
- 238000002386 leaching Methods 0.000 claims abstract description 63
- 238000011068 loading method Methods 0.000 claims abstract description 19
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims abstract description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000018417 cysteine Nutrition 0.000 claims abstract description 12
- 229960003067 cystine Drugs 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 77
- 230000014759 maintenance of location Effects 0.000 claims description 46
- 239000007974 sodium acetate buffer Substances 0.000 claims description 34
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 238000011067 equilibration Methods 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 6
- 230000008929 regeneration Effects 0.000 claims description 6
- 238000011069 regeneration method Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 238000005342 ion exchange Methods 0.000 claims 2
- 238000000746 purification Methods 0.000 abstract description 7
- 235000018102 proteins Nutrition 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- 238000005406 washing Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000012607 strong cation exchange resin Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- 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
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Abstract
The application relates to an antibody purification method based on ion exchange chromatography, which comprises the steps of loading an antibody sample to be purified to an ion exchange chromatography column, eluting and eluting in sequence; the leaching comprises a first leaching, a second leaching, a third leaching and a fourth leaching, wherein the leaching solution adopted by the third leaching comprises cysteine with the concentration of 1.5 mM-3 mM and cystine with the concentration of 0.4 mM-0.8 mM, and the pH value is 7-7.8. According to the antibody purification method based on ion exchange chromatography, cysteine and cystine with specific concentration are adopted on the basis of ion exchange chromatography, and eluting is carried out by using the eluent with pH of 7-7.8, so that antibody purification can be realized, antibody restoration can be realized, the content of the antibody with complete molecular structure obtained after purification is improved, and the protein yield is high.
Description
Technical Field
The application relates to the technical field of antibody purification, in particular to an antibody purification method based on ion exchange chromatography.
Background
An Antibody (anti) is a globular protein produced by the immune system of the body against foreign substances such as bacterial, viral proteins, and is therefore also called an immunoglobulin (Ig). In recent years, as antibody drugs are increasingly used in more therapeutic fields, the structures of antibody drugs are also becoming more diversified. In particular, with the development of high titer cell culture processes and the appearance of complex molecules such as bispecific antibodies and trispecific antibodies, the problem of molecular heterogeneity caused by incomplete disulfide bond pairing or mismatch is frequently and frequently encountered, resulting in a greatly reduced proportion of antibodies with complete molecular structures.
In addition, antibody molecules are usually purified by column chromatography, which inevitably results in further loss of the antibody with complete molecular structure during purification, thus keeping the production costs of the antibody high. There are methods of increasing the proportion of antibodies with intact molecular structure by removing more components of the antibody fragments by peak cutting during the elution phase of column chromatography, but this method will lose a large amount of protein, resulting in a greatly reduced protein yield.
Disclosure of Invention
Based on this, the present application provides a method for purifying an antibody based on ion exchange chromatography. The antibody purification method can purify and improve the content of the antibody with complete molecular structure, and the protein yield is high.
The specific technical scheme is as follows:
an antibody purification method based on ion exchange chromatography comprises the steps of loading an antibody-containing sample to an ion exchange chromatography column, eluting and eluting in sequence;
wherein the leaching solution adopted by leaching comprises cysteine with the concentration of 1.5 mM-3 mM and cystine with the concentration of 0.4 mM-0.8 mM, and the pH value is 7-7.8; the leaching comprises the following steps:
under the condition of room temperature, performing first leaching by adopting a first leaching solution, wherein the first leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6;
under the condition of room temperature, performing second leaching by adopting a second leaching solution, wherein the second leaching solution comprises 15 mM-25 mM phosphate buffer solution, and the pH value is 7.5-8.5;
performing third leaching by adopting a third leaching solution at the temperature of 35-37 ℃, wherein the third leaching solution comprises cysteine with the concentration of 1.5 mM-3 mM and cystine with the concentration of 0.4 mM-0.8 mM, and the pH value is 7-7.8; the retention time of the third eluent is 5-10 minutes;
and under the room temperature condition, leaching for the fourth time by adopting a fourth leaching solution, wherein the fourth leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6.
In some embodiments, the first eluent is used in an amount equal to or greater than 3 column volumes and a retention time equal to or greater than 5 minutes; and/or the number of the groups of groups,
the usage amount of the second eluent is more than or equal to 3 times of the volume of the column, and the retention time is more than or equal to 5 minutes.
In some embodiments, the fourth eluent is used in an amount equal to or greater than 3 column volumes and has a retention time equal to or greater than 5 minutes.
In some of these embodiments, the packing of the ion exchange chromatography column is a POROS XS resin.
In some embodiments, the loading capacity is 20-40 g/L.
In some of these embodiments, the eluting comprises the step of isocratic eluting with eluent a and eluent B;
the eluent A comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and the pH value of 5-6;
the eluent B comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and NaCl with the concentration of 0.5M-1.5M, and the pH value is 5-6.
In some embodiments, the volume percentage of the eluent A and the eluent B is 40% -60% 40%.
In some of these embodiments, the total amount of eluent A and eluent B is greater than or equal to 5 column volumes and the retention time is greater than or equal to 5 minutes.
In some embodiments, the average molecular weight of the antibody is 90kDa to 180kDa.
In some embodiments, the loading further comprises one or more of sterilizing, pre-equilibrating, and equilibrating the ion exchange chromatography column, and the eluting further comprises one or more of regenerating, sterilizing, and preserving the ion exchange chromatography column.
According to the antibody purification method based on ion exchange chromatography, cysteine and cystine with specific concentration are adopted on the basis of ion exchange chromatography, and eluting is carried out by using the eluent with pH of 7-7.8, so that antibody purification can be realized, antibody restoration can be realized, the content of the antibody with complete molecular structure obtained after purification is improved, and the protein yield is high.
Detailed Description
The method for purifying an antibody according to the present application based on ion exchange chromatography is described in further detail below with reference to specific examples. The present application may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other.
Herein, "one or more" refers to any one, any two, or any two or more of the listed items.
In the present application, "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of technical features indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present application, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The percentage content referred to in the present application refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified.
The percentage concentrations referred to in the present application refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.
The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
The room temperature in the present application is generally 4 ℃ to 30 ℃, preferably 20+ -5 ℃.
Unless otherwise specified, the solvents of the solutions according to the present application are all water for injection.
Unless otherwise specified, the steps involved in the present application are all performed at room temperature.
Some examples of the present application provide a method for purifying an antibody based on ion exchange chromatography, comprising the steps of sequentially loading a sample containing the antibody onto an ion exchange chromatography column, eluting, and eluting;
wherein the leaching solution comprises cysteine with the concentration of 1.5 mM-3 mM and cystine with the concentration of 0.4 mM-0.8 mM, and the pH value is 7-7.8.
Specifically, the concentration of cysteine includes, but is not limited to: 1.5mM, 1.8mM, 2mM, 2.2mM, 2.5mM, 2.8mM, 3mM or a range therebetween.
Specifically, cystine concentrations include, but are not limited to: 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM or a range therebetween.
Specifically, the pH of the rinse solution includes, but is not limited to: 7. 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or a range therebetween.
In some specific examples, the step of rinsing with a rinsing solution having a pH of 7 to 7.8 and comprising cysteine at a concentration of 1.5mM to 3mM and cystine at a concentration of 0.4mM to 0.8mM is performed at a temperature of 35 ℃ to 37 ℃.
In some specific examples, the rinsing includes the following steps S1 to S4:
s1: and under the condition of room temperature, performing first leaching by adopting a first leaching solution, wherein the first leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6.
Specifically, the concentration of the acetic acid-sodium acetate buffer includes, but is not limited to: 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM or a range therebetween.
Specifically, the pH of the first eluent includes, but is not limited to: 5. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or a range therebetween.
S2: and (3) carrying out second leaching by adopting a second leaching solution under the room temperature condition, wherein the second leaching solution comprises 15-25 mM phosphate buffer solution, and the pH value is 7.5-8.5.
In particular, the concentration of phosphate buffer includes, but is not limited to: 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM or a range therebetween.
Specifically, the pH of the second eluent includes, but is not limited to: 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or a range therebetween.
S3: and (3) carrying out third leaching by adopting a third leaching solution at the temperature of 35-37 ℃, wherein the third leaching solution is the leaching solution.
S4: and under the room temperature condition, leaching for the fourth time by adopting a fourth leaching solution, wherein the fourth leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6.
Specifically, the concentration of the acetic acid-sodium acetate buffer includes, but is not limited to: 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM or a range therebetween.
Specifically, the pH of the fourth eluent includes, but is not limited to: 5. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or a range therebetween.
In some specific examples, the first eluent is used in an amount equal to or greater than 3 column volumes. Further, the dosage is 3-5 times of the column volume.
In some specific examples, the retention time of the first rinse is greater than or equal to 5 minutes. Further, the retention time is 5 to 10 minutes.
In some specific examples, the second eluent is used in an amount of 3 times or more the volume of the column. Further, the dosage is 3-5 times of the column volume.
In some specific examples, the retention time of the second rinse is greater than or equal to 5 minutes. Further, the retention time is 5 to 10 minutes.
In some specific examples, the third eluent is used in an amount of 5 times or more the volume of the column. Further, the dosage is 5-8 times of column volume.
In some specific examples, the retention time of the third rinse is greater than or equal to 5 minutes. Further, the retention time is 5 to 10 minutes.
In some specific examples, the fourth eluent is used in an amount of 3 times or more the volume of the column. Further, the dosage is 3-5 times of the column volume.
In some specific examples, the retention time of the fourth rinse is greater than or equal to 5 minutes. Further, the retention time is 5 to 10 minutes.
In some specific examples thereof, the packing of the ion exchange chromatography column is a POROS XS resin. It will be appreciated that the POROS XS resin is a strong cation exchange resin.
In some specific examples, the loading capacity is 20-40 g/L. Specifically, the loading capacity includes, but is not limited to: 20g/L, 25g/L, 30g/L, 35g/L, 40g/L or a range therebetween.
In some specific examples, the eluting includes a step of isocratic eluting with eluent a and eluent B;
the eluent A comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and the pH value of 5-6.
Specifically, the concentration of the acetic acid-sodium acetate buffer includes, but is not limited to: 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM or a range therebetween.
Specifically, the pH of the eluent a includes, but is not limited to: 5. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or a range therebetween.
The eluent B comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and NaCl with the concentration of 0.5M-1.5M, and the pH value is 5-6.
Specifically, the concentration of the acetic acid-sodium acetate buffer includes, but is not limited to: 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM or a range therebetween.
Specifically, the pH of the eluent B includes, but is not limited to: 5. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or a range therebetween.
Specifically, the concentration of NaCl includes, but is not limited to: 0.5M, 0.8M, 1M, 1.2M, 1.5M, or a range therebetween.
Further, the volume percentage of the eluent A and the eluent B is 40% -60% 40%. Specifically, the volume percentages of the eluent a and the eluent B include, but are not limited to: 40% 60%, 45% 55%, 50%, 55% 45%, 60% 40% or a range therebetween.
In some specific examples, the total amount of eluent A and eluent B is greater than or equal to 5 column volumes and the retention time is greater than or equal to 5 minutes. Further, the total dosage is 5-8 times of the column volume; further, the retention time is 5 to 10 minutes.
In addition, in some specific examples, the average molecular weight of the antibody is 90kda to 180kda. In particular, the average molecular weight of the antibody includes, but is not limited to: 90kDa, 95kDa, 100kDa, 105kDa, 110kDa, 115kDa, 120kDa, 125kDa, 130kDa, 135kDa, 140kDa, 145kDa, 150kDa, 155kDa, 160kDa, 165kDa, 170kDa, 174kDa, 175kDa, 180kDa or a range therebetween.
In some specific examples thereof, the antibodies in the antibody sample to be purified are monoclonal antibodies.
It will be further appreciated that the antibody purification method may further comprise one or more of sterilization, pre-equilibration and equilibration of the ion exchange chromatography column prior to loading, and one or more of regeneration, sterilization and preservation of the ion exchange chromatography column after elution.
Specifically, in some specific examples thereof, the ion exchange chromatography-based antibody purification method comprises the steps of:
(1) Front disinfection: washing the chromatographic column with 0.3-0.7M NaOH solution for at least 3 times of column volume, wherein the process retention time is not less than 5 minutes;
(2) Pre-balancing: flushing the chromatographic column with a pre-equilibration solution (45 mM-55 mM acetic acid-sodium acetate buffer, 0.5M~1.5M NaCl,pH 5~6) for at least 3 column volumes, wherein the process retention time is not less than 5 minutes;
(3) Balance: washing the chromatographic column with a balancing solution (45 mM-55 mM acetic acid-sodium acetate buffer solution, pH 5-6) for at least 5 times of column volume, wherein the process retention time is not less than 5 minutes;
(4) Loading: sampling an antibody sample, wherein the loading capacity is 20-40 g/L, and the process retention time is not less than 5 minutes;
(5) Leaching for the first time: eluting (45 mM-55 mM acetic acid-sodium acetate buffer solution, pH 5-6) the chromatographic column by using a first eluting solution, wherein the column volume is at least 3 times, and the process retention time is not less than 5 minutes;
(6) And leaching for the second time: eluting (15 mM-25 mM phosphate buffer solution, pH 7.5-8.5) the chromatographic column by using a second eluting solution, wherein the column volume is at least 3 times, and the process retention time is not less than 5 minutes;
(7) And leaching for the third time: rinsing the chromatographic column with a third eluent (1.5 mM-3 mM cysteine, 0.4 mM-0.8 mM cystine, pH 7.0-7.8) at 35-37 ℃ for at least 5 times of column volume, wherein the process retention time is not less than 5 minutes;
(8) Leaching for the fourth time: eluting (45 mM-55 mM acetic acid-sodium acetate buffer solution, pH 5-6) the chromatographic column by using a fourth eluting solution, wherein the column volume is at least 3 times, and the process retention time is not less than 5 minutes;
(9) Eluting: isocratic elution is carried out by using an eluent A and an eluent B (the eluent A is 45-55 mM acetic acid-sodium acetate buffer solution, the pH value is 5-6, the eluent B is 45-55 mM acetic acid-sodium acetate buffer solution, 0.5M~1.5M NaCl,pH 5~6, the volume percentage of the eluent A and the eluent B is 40-60-40%, the chromatographic column is flushed at least 5 times of column volume, and the process retention time is not less than 5 minutes;
(10) Regeneration: flushing the chromatographic column with a pre-equilibration solution (45 mM-55 mM acetic acid-sodium acetate buffer, 0.5M~1.5MNaCl,pH 5~6) for at least 3 column volumes, wherein the process retention time is not less than 5 minutes;
(11) And (3) disinfection: washing the chromatographic column with 0.3-0.7M NaOH solution for at least 3 times of column volume, wherein the process retention time is not less than 5 minutes;
(12) And (3) preserving: the column was rinsed with 0.005~0.02M NaOH solution at least 3 column volumes and the process retention time was not less than 5 minutes.
The following examples are given with the reagents used in the examples being commercially available unless otherwise specified.
Example 1
The application relates to an antibody purifying method based on ion exchange chromatography, which adopts ion exchange chromatography filler POROS XS (product number: 4404336; manufacturer: thermo) as ion exchange chromatography column, and target molecule as monoclonal antibody molecule with molecular weight of 174KD.
The steps are as follows (except for the third leaching, the rest steps are all carried out under the room temperature condition):
(1) Front disinfection: rinsing the chromatographic column with 0.5M NaOH solution for 3 times of column volume, and keeping the process for 5 minutes;
(2) Pre-balancing: the column was washed 3 column volumes with pre-equilibration solution (50 mM acetic acid-sodium acetate buffer, 1M NaCl, pH 5.5) for a process hold time of 5 minutes;
(3) Balance: washing the chromatographic column with equilibration solution (50 mM acetic acid-sodium acetate buffer, pH 5.5) for 5 column volumes and process retention time of 5 minutes;
(4) Loading: sampling an antibody sample, wherein the loading capacity is 30g/L, and the process retention time is 5 minutes;
(5) Leaching for the first time: eluting (50 mM acetic acid-sodium acetate buffer, pH 5.5) the chromatographic column with the first eluting solution for 3 times of column volume, and keeping the process for 5 minutes;
(6) And leaching for the second time: rinsing the chromatographic column with a second eluent (20 mM sodium hydrogen phosphate-disodium hydrogen phosphate buffer, pH 8) 3 times the column volume, with a process retention time of 5 minutes;
(7) And leaching for the third time: rinsing the chromatographic column by using a third eluent (2.5 mM cysteine, 0.3mM cystine, pH 7) at 35-37 ℃ for 5 times of column volume, wherein the process retention time is 5 minutes;
(8) Leaching for the fourth time: rinsing the chromatographic column with a fourth eluent (50 mM acetic acid-sodium acetate buffer, pH 5.5) for 3 times of column volume, wherein the process retention time is 5 minutes;
(9) Eluting: isocratic elution is carried out by using eluent A and eluent B (eluent A:50mM acetic acid-sodium acetate buffer, pH 5.5; eluent B:50mM acetic acid-sodium acetate buffer, 1M NaCl, pH 5.5; volume percentage of eluent A and eluent B is 50% -50%) to wash the chromatographic column for 5 times of column volume, and process retention time is 5 minutes; collecting the eluent, wherein the ultraviolet peak-collecting range is 50mAU/mm before peak to 50mAU/mm after peak;
(10) Regeneration: the column was washed 3 column volumes with pre-equilibration solution (50 mM acetic acid-sodium acetate buffer, 1M NaCl, pH 5.5) for a process hold time of 5 minutes;
(11) And (3) disinfection: rinsing the chromatographic column with 0.5M NaOH solution for 3 times of column volume, and keeping the process for 5 minutes;
(12) And (3) preserving: the column was washed 3 column volumes with 0.01M NaOH solution and the process retention time was 5 minutes.
Samples before and after purification were tested and the results are shown in table 1 below:
TABLE 1
Comparative example 1
This comparative example provides a method for purifying antibodies based on ion exchange chromatography, the antibody sample lot, target molecule and procedure are the same as example 1, the main differences being that: step (7) is not performed.
Samples before and after purification were tested and the results are shown in table 2 below:
TABLE 2
Comparative example 2
This comparative example provides a method for purifying antibodies based on ion exchange chromatography, the target molecules and steps are the same as in example 1, the process for obtaining antibody samples is the same as in example 1, only the obtained batches are different, the main difference is that: step (7) is not performed and step (9) is replaced as follows:
eluent a:50mM acetic acid-sodium acetate buffer, pH 5.5; eluent B:20mM sodium phosphate monobasic-disodium phosphate buffer, 0.1M NaCl, pH 7.0; with gradient elution, eluent B was varied from 40% to 90%,10CV. I.e., peak cutting is performed during the elution phase to remove antibody fragments.
Samples before and after purification were tested and the results are shown in table 3 below:
TABLE 3 Table 3
Comparative example 3
This comparative example provides a method for purifying antibodies based on ion exchange chromatography, the target molecules and steps are the same as in example 1, the process for obtaining antibody samples is the same as in example 1, only the obtained batches are different, the main difference is that: the filler is replaced by an affinity chromatography filler MabSelect SuRe LX (product number: 17547401; manufacturer: cytiva), and meanwhile, the principle of capturing antibodies by the affinity chromatography filler is specific binding, and the balance and the leaching solution are not needed to be balanced under low pH conditions, so that the balance solution and the leaching solution are slightly different, and in addition, the affinity chromatography filler is needed to be eluted under low pH, so that the eluting solution is slightly different.
The method comprises the following specific steps:
(1) Front disinfection: rinsing the chromatographic column with 0.5M NaOH solution for 3 times of column volume, and keeping the process for 5 minutes;
(2) Balance: washing the chromatographic column by using a balancing solution (50mM Tris,0.15M NaCl,pH7.2) for 6 times of column volume, wherein the process retention time is 10 minutes;
(3) Loading: sampling an antibody sample, wherein the loading capacity is 30g/L, and the process retention time is 5 minutes;
(4) Leaching for the first time: eluting (50mM Tris,0.15M NaCl,pH7.2) the chromatographic column by using the first eluting solution, wherein the volume of the chromatographic column is 3 times that of the chromatographic column, and the process retention time is 5 minutes;
(5) And leaching for the second time: rinsing the chromatographic column with a second eluent (20 mM sodium hydrogen phosphate-disodium hydrogen phosphate buffer, pH 8) 3 times the column volume, with a process retention time of 5 minutes;
(6) And leaching for the third time: rinsing the chromatographic column by using a third eluent (2.5 mM cysteine, 0.3mM cystine, pH 7) at 35-37 ℃ for 5 times of column volume, wherein the process retention time is 5 minutes;
(7) Eluting: washing the chromatographic column with 50mM acetic acid-sodium acetate buffer solution, pH 3.5 for 5 times of column volume, and keeping for 5 minutes; collecting the eluent, wherein the ultraviolet peak-collecting range is 50mAU/mm before peak to 50mAU/mm after peak;
(8) Regeneration: flushing the chromatographic column with a regeneration solution (1M acetic acid solution) for 3 times of column volume, wherein the process retention time is 5 minutes;
(8) Post-equilibration: equilibration (50mM Tris,0.15M NaCl,pH7.2) was performed using equilibration liquid to flush the column 3 column volumes with a process hold-up time of 5 minutes;
(9) And (3) disinfection: washing the chromatographic column with 0.1M NaOH solution for 3 times of column volume, and keeping the process for 5 minutes;
(10) And (3) preserving: the column was rinsed 3 times the column volume with 20% ethanol solution and the process retention time was 5 minutes.
Samples before and after purification were tested and the results are shown in table 4 below:
TABLE 4 Table 4
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely illustrate a few embodiments of the present application, which are convenient for a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. It should be understood that, based on the technical solutions provided by the present application, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent of the application should therefore be determined with reference to the appended claims, which are to be construed as in accordance with the doctrines of claim interpretation.
Claims (9)
1. The method is characterized by comprising the steps of sequentially loading a sample containing an antibody into an ion exchange chromatographic column, eluting and eluting, wherein the antibody is a monoclonal antibody, the average molecular weight is 90 KDa-180 KDa, the filler of the ion exchange chromatographic column is POROS XS resin, and the loading capacity of the loading sample is 20-40 g/L;
the leaching comprises the following steps:
under the condition of room temperature, performing first leaching by adopting a first leaching solution, wherein the first leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6;
under the condition of room temperature, performing second leaching by adopting a second leaching solution, wherein the second leaching solution comprises 15 mM-25 mM phosphate buffer solution, and the pH value is 7.5-8.5;
performing third leaching by adopting a third leaching solution at the temperature of 35-37 ℃, wherein the third leaching solution comprises cysteine with the concentration of 1.5 mM-3 mM and cystine with the concentration of 0.4 mM-0.8 mM, and the pH value is 7-7.8; the retention time of the third eluent is 5-10 minutes;
under the room temperature condition, leaching for the fourth time by adopting a fourth leaching solution, wherein the fourth leaching solution comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM, and the pH value is 5-6;
the elution comprises the step of isocratic elution by adopting eluent A and eluent B;
the eluent A comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and the pH value of 5-6;
the eluent B comprises acetic acid-sodium acetate buffer solution with the concentration of 45 mM-55 mM and NaCl with the concentration of 0.5M-1.5M, and the pH value is 5-6;
the volume percentage of the eluent A and the eluent B is 40% -60%, 40%.
2. The method for purifying an antibody based on ion exchange chromatography according to claim 1, wherein the amount of the first eluent is not less than 3 times the column volume and the retention time is not less than 5 minutes.
3. The method for purifying an antibody based on ion exchange chromatography according to claim 1, wherein the amount of the second eluent is not less than 3 times the column volume and the retention time is not less than 5 minutes.
4. The method for purifying an antibody based on ion exchange chromatography according to claim 1, wherein the fourth eluent is used in an amount of 3 times or more of the column volume and the retention time is 5 minutes or more.
5. The method for purifying an antibody based on ion exchange chromatography according to any one of claims 1 to 4, wherein the loading amount is 30g/L.
6. The method for purifying an antibody based on ion exchange chromatography according to any one of claims 1 to 4, wherein the eluent a contains acetic acid-sodium acetate buffer with a concentration of 50mM and a pH of 5.5;
the eluent B contained acetic acid-sodium acetate buffer at a concentration of 50mM and NaCl at a concentration of 1M, at a pH of 5.5.
7. The method for purifying an antibody based on ion exchange chromatography according to any one of claims 1 to 4, wherein the volume percentage of the eluent a to the eluent B is 50% -50%.
8. The method for purifying an antibody based on ion exchange chromatography according to any one of claims 1 to 4, wherein the total amount of eluent a and eluent B is not less than 5 column volumes and the retention time is not less than 5 minutes.
9. The method according to any one of claims 1 to 4, wherein the step of subjecting the ion exchange chromatography column to one or more of sterilization, pre-equilibration and equilibration is further included before the sample loading, and the step of subjecting the ion exchange chromatography column to one or more of regeneration, sterilization and preservation is further included after the elution.
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