CN114539416A - Chromatographic purification process of bispecific antibody - Google Patents
Chromatographic purification process of bispecific antibody Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a chromatography purification process of a common light chain bispecific antibody, which comprises the following steps: step 1, processing chromatographic packing and loading; step 2, leaching by using an equilibrium buffer solution; and 3, eluting the product by using an elution buffer solution containing the additive, and collecting the product. The invention achieves the purposes of improving the separation effect of chromatographic purification of the bispecific antibody homodimer and the recovery rate of qualified products by using an additive in the eluent and adjusting the pH value of the eluent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chromatographic purification process of a bispecific antibody.
Background
With the continued development of biopharmaceuticals, therapeutic antibodies, particularly bispecific antibodies, have become the primary drug of choice for patients with cancer, autoimmunity, inflammation, and various other diseases. Bispecific antibodies (biabs) are artificial antibodies containing two specific antigen binding sites, capable of specifically recognizing and binding two different antigens or epitopes, which can bridge between target cells and functional molecules (cells), produce a targeted effector function, redirect specific immune cells to tumor cells to enhance killing of tumors, or block two different mediators/pathways simultaneously to exert unique or overlapping functions.
Bispecific antibodies against GPC3 and anti-CD 3 were developed with two distinct variable regions, one recognizing the binding tumor antigen GPC3 and the other for binding T cells, the bispecific antibody acting as a "linker arm" to recruit T cells to the tumor site. Meanwhile, the antibody adopts a common light chain structure design, and the heavy chains of the antibodies aiming at two target points have the same light chain, so that the mismatching of the light chain and the heavy chain cannot be caused on the expression pairing of the antibodies.
Although the common light chain bispecific antibody has great advantages in terms of drug availability and control of the production process, the two different heavy chains are more complex than the monoclonal antibody in structure, and are more likely to produce different homodimers in the process of making the bispecific antibody. Therefore, the isolation and purification of the target antibody-containing culture is an essential step in the production process thereof. However, in the prior art, there are few reports on methods for purifying bispecific antibodies having a common light chain.
Patent CN101180317B discloses a method for purifying immunoglobulins by ion exchange chromatography. The chromatography method uses a weak ion exchange resin and a single step elution method to purify the immunoglobulin and determines the salt concentration of the immunoglobulin from the ion exchange resin in a single step elution.
Patent CN107922476A discloses a method for purifying bispecific antibodies from a mixture of monospecific antibodies, i.e. bispecific antibodies (consisting of a single heavy chain and two different light chains) will be isolated from a mixture comprising monospecific antibodies with two kappa light chains or parts thereof and monospecific antibodies with two lambda light chains or parts thereof by a multiplex hybrid chromatographic purification method.
Therefore, it is desirable to provide a simple and effective chromatographic purification process for purifying the bispecific antibody homodimer, which can improve the efficiency of removing the homodimer in the preparation process of the bispecific antibody drug and provide a more reliable choice for the purification process of the bispecific antibody drug.
Disclosure of Invention
In order to solve the problems in the prior art, the invention discloses a chromatographic purification process of a bispecific antibody.
The chromatographic purification process of the bispecific antibody comprises the following steps:
step 1, processing chromatographic packing and loading;
step 2, leaching by using an equilibrium buffer solution;
and 3, eluting the product by using an elution buffer solution containing the additive, and collecting the product.
Further, the bispecific antibody is a bispecific antibody against GPC3 and against CD 3.
Further, the specific operation method of step 1 is as follows:
1a, packing a chromatographic packing into a column, and washing the chromatographic column by using an equilibrium buffer solution with the volume of 3-50 times of the column volume;
1b, sterilizing the chromatographic column by using 1M NaOH solution with 2-4 times of column volume;
1c, washing the chromatographic column by using 3-6 times of column volume of equilibrium buffer solution;
and 1d, loading and keeping for 5 +/-1 min.
Further, the chromatography packing is selected from Capto MMC, Diamond SP Mustang or Capto S ImpAct.
Further, the elution buffer comprises elution buffer a and elution buffer B; the elution buffer solution A consists of sodium citrate, sodium dihydrogen phosphate and Tris (hydroxymethyl) aminomethane (Tris); the elution buffer solution B consists of sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane.
Further, the elution buffer produces a linear pH gradient as it passes through the eluted product.
Further, the pH range of the elution buffer A is 4.0-6.0, and the pH range of the elution buffer B is 9.0-11.0.
Further, the pH range of the elution buffer A is 4.5-5.5, and the pH range of the elution buffer B is 9.5-10.5.
Further, the elution buffer also contains additives.
Further, the additive is alcohol, sugar or amino acid.
Further, the amino acid is arginine, glycine, histidine, proline, phenylalanine, or tyrosine.
Further, the additive has a molarity of 1-100 mM; preferably, the additive is present in a molar concentration of 2-50 mM; more preferably, the additive is present in a molar concentration of 5-40 mM; further preferably, the additive is present in a molar concentration of 10-30 mM.
Further, the equilibration buffer of step 2 is 3-6 column volumes.
Further, the equilibration buffer is sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane, ph 5.0.
Further, the elution buffer of step 3 is 10 to 100 times the column volume.
Accordingly, the present invention relates to the following embodiments:
1. a process for chromatographic purification of a bispecific antibody, comprising the steps of:
step 1, processing chromatographic packing and loading;
step 2, leaching by using an equilibrium buffer solution;
and 3, eluting the product by using an elution buffer solution containing the additive, and collecting the product.
2. The chromatographic purification process of claim 1, wherein the bispecific antibody is a bispecific antibody against GPC3 and against CD 3.
3. The chromatographic purification process according to claim 1 or 2, characterized in that the specific operation method of step 1 is:
1a, packing a chromatographic packing into a column, and washing the chromatographic column by using 3-50 times of column volume of an equilibrium buffer solution;
1b, sterilizing the chromatographic column by using 1M NaOH solution with 2-4 times of column volume;
1c, washing the chromatographic column by using 3-6 times of column volume of equilibrium buffer solution;
and 1d, loading and keeping for 5 +/-1 min.
4. The chromatographic purification process according to claims 1-3, wherein the chromatographic packing is selected from Capto MMC, Diamond SP Mustang or Capto S ImpAct.
5. The chromatographic purification process of claims 1-4, wherein the elution buffer comprises elution buffer A and elution buffer B; the elution buffer solution A consists of sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane; the elution buffer solution B consists of sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane.
6. The chromatographic purification process of claim 5, wherein the elution buffer produces a linear pH gradient through the eluted product.
7. The chromatographic purification process according to claim 5 or 6, wherein the pH of elution buffer A is in the range of 4.0-6.0 and the pH of elution buffer B is in the range of 9.0-11.0.
8. The chromatographic purification process according to claim 5 or 6, wherein the pH of elution buffer A is in the range of 4.5-5.5 and the pH of elution buffer B is in the range of 9.5-10.5.
9. The chromatographic purification process according to claims 1-8, wherein the elution buffer further comprises an additive.
10. The chromatographic purification process according to claim 9, wherein the additive is an alcohol, a sugar or an amino acid.
11. The chromatographic purification process according to claim 10, wherein the amino acid is arginine, glycine, histidine, proline, phenylalanine or tyrosine.
12. The chromatographic purification process according to claim 10, wherein the additive is present in a molar concentration of 1-100 mM; preferably, the additive is present in a molar concentration of 2-50 mM; more preferably, the additive is present in a molar concentration of 5-40 mM; further preferably, the additive is present in a molar concentration of 10-30 mM.
13. The chromatographic purification process according to claim 1 or 2, wherein the equilibration buffer of step 2 is 3-6 column volumes.
14. The chromatographic purification process according to claim 1 or 2, wherein the equilibration buffer is sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane, ph 5.0.
15. The chromatographic purification process according to claim 1, wherein the elution buffer of step 3 is 10-100 times the column volume.
Term(s) for
"common light chain" or "common light chain" refers to a polypeptide comprising a light chain variable region that is capable of stably and independently pairing with at least two different heavy chain polypeptides and thereby producing, under each independent condition, an antigen binding domain comprising the heavy chain variable region and the light chain variable region of each heavy chain polypeptide. Thus, the two copies of the common light chain are each capable of stably pairing with the first and second heavy chain polypeptides such that the bispecific antibody is in a native form, such as an IgG isotype form, such as an IgG1 form, an IgG2 form, an IgG3 form, and/or an IgG4 form, with specificity for two different antigens.
"homodimer" refers to a heavy chain comprising two identical heavy chains with one of two antigen specificities, and in certain embodiments, to an antibody specific for GPC3, or to an antibody specific for CD 3.
"amino acids" are designated by the three-letter or one-letter code as follows: alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
A "buffer" is a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
"equilibration buffer" is herein a buffer used to prepare the chromatographic solid phase.
The "elution buffer" is used to elute the protein from the chromatography matrix, i.e., to elute the protein of interest from the solid phase. The conductivity and/or pH of the elution buffer enables the target protein to be eluted from the ion exchange resin.
By "linear elution" is meant a method, for example, in certain embodiments, where the total volume of elution buffer a and elution buffer B is constant throughout the elution process and the volume fraction of elution buffer B is linear with time, i.e., the volume of elution buffer B increases linearly with time.
A "salt" is a compound formed by the interaction of an acid and a base.
The invention has the advantages of
The invention provides a novel, simple and effective chromatographic purification process for removing the homodimers of the bispecific antibody, which can improve the efficiency of removing the homodimers in the preparation process of the bispecific antibody drug and provide more reliable selection for the purification process of the bispecific antibody drug.
Detailed Description
The present invention will be described in detail with reference to specific examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions, and the test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.
EXAMPLE 1 preparation of antibodies
Taking the 105-cell strain from the liquid nitrogen, rapidly melting in water bath at 37 ℃, transferring the cells to a shake flask for culture, wherein the cell inoculation density is 0.4-1.2 × 106Individual cells/ml.
Cells were grown in shake flasksAnd performing amplification culture, and carrying out passage once every 2 days. Changing different specifications of shake flasks according to culture volume, with shaking table parameters of 37 deg.C and 5% CO2115 rpm. When the final cell density reaches 3-6 × 106Each cell/ml, the cell activity is more than 90 percent, and the cells are transferred to a fermentation tank for culture.
The inoculation density of the fermentation tank is 0.8-1.2 multiplied by 106Cell/ml, temperature 37 ℃, rotation speed 180-250 rpm, pH 6.8-7.2, dissolved oxygen (pO)2)50 percent. When the cell density is more than or equal to 10 multiplied by 106At individual cells/ml, the culture temperature was lowered to 32 ℃. In order for the cells to have sufficient nutrients to maintain their high viability and produce the protein of interest, the initial culture volume of 3% feed medium was supplemented on days 4, 6, 8, and 10 of culture. In order to avoid the obvious influence of the excessively low glucose content on the cell viability, 30 percent (m/v) of glucose solution is added into the reactor, so that the glucose content in the culture solution is maintained at 4 g/L. And when the cell viability is reduced to about 80 percent, harvesting cell fermentation liquor.
The harvested fermentation broth is centrifuged AT 8000-12000 rpm for 30 minutes, filtered through a 0.45um filter membrane, and subjected to protein A affinity chromatography (Booglong AT protein A) to obtain the bispecific antibody (about 15% homodimer impurity) against GPC3 and CD 3.
Example 2 addition of salts and different additives to the elution buffer
The purification effect of the salts and additives on homodimers in bispecific antibodies against GPC3 and anti CD3 was investigated by adding the salt different additives in the elution buffer solution.
Chromatography conditions are as follows:
filling: diamond SP Mustang
Column volume: 6.18ml
Flow rate: 1.2ml/min
Sample preparation: bispecific antibodies against GPC3 and CD3 with approximately 15% homodimer impurity
And (3) an equilibrium buffer: 50mM NaAc-HAc, pH5.0
Elution buffer a: 50mM NaAc-HAc, pH5.0
Elution buffer B: 50mM NaAc-HAc, 1M NaCl, pH5.0
The loading was performed with a bispecific antibody against GPC3 and anti CD3 (homodimer impurity of around 15%), and after the loading step the column was washed with 3 column volumes of equilibration buffer at a flow rate of 1.2 ml/min. The homodimers in the bispecific antibody against GPC3 and anti CD3 were eluted with a linear elution method, wherein the pH in the mobile phase was kept constant. The volume fraction of elution buffer B was linearly increased from 5% to 50% in scheme 1 and scheme 3, and from 0% to 50% in scheme 2.
And collecting the elution sample in a branch pipe to determine the purity, taking the product with the purity of more than or equal to 98 percent as a qualified sample, and calculating the recovery rate of the qualified sample, wherein the results are shown in table 1.
Table 1 purification results with different additives
From the results, it was found that when only NaCl was added to the elution buffer without using an additive, the target product and homodimer were purified without separation effect; when the additive uses ethanol and cane sugar, the separation effect is achieved, but the recovery rate is low; when the additive uses L-Arg, the separation effect is better, the purity of the target product is high, and the recovery rate is higher than that when the additive is not used.
Example 3 pH Linear elution
The effect of the additives on the purification effect of homodimers in the bispecific antibody against GPC3 and anti CD3 was examined by adding different additives to the elution buffer solution to which NaCl was added.
Chromatography conditions are as follows:
column volume: 6.18ml
Flow rate: 1.2ml/min
Sample preparation: bispecific antibodies against GPC3 and CD3 with approximately 15% homodimer impurity
And (3) an equilibrium buffer: 20mM sodium citrate, 20mM NaH2PO4，20mM Tris
Elution buffer a: 20mM sodium citrate, 20mM NaH2PO4，20mM Tris
Elution buffer B: 20mM sodium citrate, 20mM NaH2PO4，20mM Tris
The loading was performed with a bispecific antibody against GPC3 and anti CD3 (homodimer impurity of around 15%), and after the loading step the column was washed with 3 column volumes of equilibration buffer at a flow rate of 1.2 ml/min. The homodimers in the bispecific antibody against GPC3 and CD3 were eluted by linear pH elution, with the volume fraction of elution buffer B increasing linearly from 0% to 100%. Protocol 5 no additive was added and the additive used in protocol 6, protocol 7, protocol 8 and protocol 9 was 20mM glycine.
And collecting the elution sample in a branch pipe to determine the purity, taking the product with the purity of more than or equal to 98 percent as a qualified sample, and calculating the recovery rate of the qualified sample, wherein the results are shown in table 2.
TABLE 2 purification results of the linear gradient method using additives
From the results, it was found that when the homodimer in the bispecific antibody against GPC3 and CD3 was eluted by the linear pH elution method, the target product and the homodimer were purified without using an additive, and the separation effect was good, but the recovery rate was low; when 20mM glycine is used, not only the separation effect is good, but also the recovery rate of qualified products is greatly improved. Compared with the solution of example 1 without pH adjustment, the solution of example 2 has better separation effect and higher recovery rate due to the change of pH linear gradient.
It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A process for chromatographic purification of a bispecific antibody, comprising the steps of:
step 1, processing chromatographic packing and loading;
step 2, leaching by using an equilibrium buffer solution;
and 3, eluting the product by using an elution buffer solution containing the additive, and collecting the product.
2. The chromatographic purification process of claim 1, wherein the bispecific antibody is a bispecific antibody against GPC3 and against CD 3.
3. The chromatographic purification process according to claim 1 or 2, characterized in that the specific operation method of step 1 is:
1a, packing a chromatographic packing into a column, and washing the chromatographic column by using an equilibrium buffer solution with the volume of 3-50 times of the column volume;
1b, sterilizing the chromatographic column by using 1M NaOH solution with 2-4 times of column volume;
1c, washing the chromatographic column by using 3-6 times of column volume of equilibrium buffer solution;
and 1d, loading and keeping for 5 +/-1 min.
4. The chromatographic purification process according to claims 1-3, wherein the chromatographic packing is selected from Capto MMC, Diamond SP Mustang or Capto S ImpAct.
5. The chromatographic purification process of claims 1-4, wherein the elution buffer comprises elution buffer A and elution buffer B; the elution buffer solution A consists of sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane; the elution buffer solution B consists of sodium citrate, sodium dihydrogen phosphate and tris (hydroxymethyl) aminomethane.
6. The chromatographic purification process of claim 5, wherein the elution buffer produces a linear pH gradient through the eluted product.
7. The chromatographic purification process according to claim 5 or 6, wherein the pH of elution buffer A is in the range of 4.0-6.0 and the pH of elution buffer B is in the range of 9.0-11.0.
8. The chromatographic purification process according to claim 5 or 6, wherein the pH of elution buffer A is in the range of 4.5-5.5 and the pH of elution buffer B is in the range of 9.5-10.5.
9. The chromatographic purification process according to claims 1-8, wherein the elution buffer further comprises an additive.
10. The chromatographic purification process according to claim 9, wherein the additive is an alcohol, a sugar or an amino acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116655726A (en) * | 2023-07-31 | 2023-08-29 | 上海澳斯康生物制药有限公司 | Antibody purification method based on ion exchange chromatography |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103833852A (en) * | 2012-11-23 | 2014-06-04 | 上海市肿瘤研究所 | Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen |
| CN103833855A (en) * | 2009-06-26 | 2014-06-04 | 瑞泽恩制药公司 | Readily isolated bispecific antibodies with native immunoglobulin format |
| CN107556387A (en) * | 2016-06-30 | 2018-01-09 | 中国科学院深圳先进技术研究院 | Resisting GPC 3 and the double targeting antibodies of CD3 specificity, the minicircle dna containing this pair of targeting antibodies expression cassette and application |
| US20180079797A1 (en) * | 2015-04-10 | 2018-03-22 | Adimab, Llc | Methods for purifying heterodimeric multispecific antibodies from parental homodimeric antibody species |
| CN110066314A (en) * | 2019-04-01 | 2019-07-30 | 上海药明生物技术有限公司 | A kind of efficient affinity purification technique for improving polymer separation resolution ratio |
| US20190256556A1 (en) * | 2016-06-17 | 2019-08-22 | Genentech, Inc. | Purification of multispecific antibodies |
| CN111018969A (en) * | 2019-12-27 | 2020-04-17 | 上海药明生物技术有限公司 | Method for purifying bispecific antibody by light chain select combined chromatography |
| CN111465619A (en) * | 2017-12-21 | 2020-07-28 | 豪夫迈·罗氏有限公司 | Combination therapy of tumor-targeted ICOS agonists with T cell bispecific molecules |
| WO2020177627A1 (en) * | 2019-03-02 | 2020-09-10 | 上海一宸医药科技有限公司 | Bispecific antibody |
-
2020
- 2020-11-26 CN CN202011342663.3A patent/CN114539416A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103833855A (en) * | 2009-06-26 | 2014-06-04 | 瑞泽恩制药公司 | Readily isolated bispecific antibodies with native immunoglobulin format |
| CN103833852A (en) * | 2012-11-23 | 2014-06-04 | 上海市肿瘤研究所 | Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen |
| US20180079797A1 (en) * | 2015-04-10 | 2018-03-22 | Adimab, Llc | Methods for purifying heterodimeric multispecific antibodies from parental homodimeric antibody species |
| US20190256556A1 (en) * | 2016-06-17 | 2019-08-22 | Genentech, Inc. | Purification of multispecific antibodies |
| CN107556387A (en) * | 2016-06-30 | 2018-01-09 | 中国科学院深圳先进技术研究院 | Resisting GPC 3 and the double targeting antibodies of CD3 specificity, the minicircle dna containing this pair of targeting antibodies expression cassette and application |
| CN111465619A (en) * | 2017-12-21 | 2020-07-28 | 豪夫迈·罗氏有限公司 | Combination therapy of tumor-targeted ICOS agonists with T cell bispecific molecules |
| WO2020177627A1 (en) * | 2019-03-02 | 2020-09-10 | 上海一宸医药科技有限公司 | Bispecific antibody |
| CN110066314A (en) * | 2019-04-01 | 2019-07-30 | 上海药明生物技术有限公司 | A kind of efficient affinity purification technique for improving polymer separation resolution ratio |
| CN111018969A (en) * | 2019-12-27 | 2020-04-17 | 上海药明生物技术有限公司 | Method for purifying bispecific antibody by light chain select combined chromatography |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116655726A (en) * | 2023-07-31 | 2023-08-29 | 上海澳斯康生物制药有限公司 | Antibody purification method based on ion exchange chromatography |
| CN116655726B (en) * | 2023-07-31 | 2023-11-14 | 上海澳斯康生物制药有限公司 | Antibody purification method based on ion exchange chromatography |
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Inventor after: Wu Chongbing Inventor after: Jiang Xiaoling Inventor after: Gu Haitao Inventor after: Chang Jiani Inventor after: Zhou Jinhua Inventor after: Zhu Cailin Inventor before: Wu Chongbing Inventor before: Jiang Xiaoling Inventor before: Gu Haitao Inventor before: Chang Jiani Inventor before: Zhou Jinhua Inventor before: Zhu Cailin Inventor before: Gao Chao Inventor before: Li Qiong |