CN110128538B - Method for purifying anti-CD 20 human-mouse chimeric monoclonal antibody - Google Patents
Method for purifying anti-CD 20 human-mouse chimeric monoclonal antibody Download PDFInfo
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- CN110128538B CN110128538B CN201810135774.3A CN201810135774A CN110128538B CN 110128538 B CN110128538 B CN 110128538B CN 201810135774 A CN201810135774 A CN 201810135774A CN 110128538 B CN110128538 B CN 110128538B
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- C07—ORGANIC CHEMISTRY
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- 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
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- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
Abstract
The invention belongs to the field of medicines, relates to a monoclonal antibody purification technology, and particularly discloses a method for purifying an anti-CD 20 human-mouse chimeric monoclonal antibody by ion exchange chromatography. The invention adds additive in the ion exchange chromatography process and adjusts the conductance by salt, thus obviously improving the chromatography performance, improving the protein quality and reducing the protein turbidity. Through the technical scheme of the invention, the purity of the target protein can reach more than 99.7%, and meanwhile, acid-base isomers, other impurity components and contents are well controlled within the quality requirements of the antibody. The method is simple to operate, low in cost and high in safety, and can be used for industrial large-scale production.
Description
Technical Field
The invention belongs to the field of medicines, relates to a purification technology of a monoclonal antibody, and particularly relates to a method for purifying an anti-CD 20 human-mouse chimeric monoclonal antibody by ion exchange chromatography.
Background
CD20 is a non-glycosylated transmembrane phosphoprotein expressed on the surface of most mature B cells and CD20 disappears after differentiation into plasma cells. Research shows that it can play the role of calcium ion channel and participate in the regulation of B cell differentiation and development. Whereas the expression level in B-cell lymphoma and non-Hodgkin's lymphoma (NHL) was abnormally increased, CD20 was not internalized or shed from the cell membrane after antibody binding. The above-mentioned features of CD20 make it an ideal target antigen for the treatment of B-cell lymphomas and non-hodgkin lymphomas (NHLs) using monoclonal antibodies. In view of this advantage, an anti-CD 20 human murine chimeric monoclonal antibody was developed which, upon binding to antigens on the surface of B cell lymphomas, can destroy and eliminate tumor cells by activating complement-mediated cytolysis and cytotoxicity.
By adopting a recombination technology and applying a CHO cell expression system, a large-scale anti-CD 20 human-mouse chimeric monoclonal antibody can be obtained, but the diversity of cell environments and the addition of culture cell feeding materials lead to the diversity of impurities in the obtained anti-CD 20 human-mouse chimeric monoclonal antibody, mainly comprising host residual protein (HCP) nucleic acid, acid-base isomer and the like, and trace impurities can generate immune reaction in a human body, so that the impurity components and the concentration in the antibody are required to be controlled within the quality requirement of the antibody. The removal of impurities is suitable for the purification stage, the purification process is optimized, and the improvement of the purification efficiency is of great importance in the production of antibody medicines.
The manufacture of antibody drugs is most complicated by the downstream process. The downstream process comprises a plurality of steps, and liquid chromatography separation methods of affinity chromatography, hydrophobic chromatography, cation exchange chromatography, and anion exchange chromatography are currently commonly used. Although these several chromatographic methods are used, the specific parameters and the choice of the filler will produce completely different results.
In the chromatography method, the crude pure affinity chromatography column can adsorb the target antibody through the specificity of the adsorption of the antibody and the Fc fusion protein so as to remove more than 90% of impurities in cell supernatant, so that the process optimization of multidimensional purification is required after protein A affinity chromatography. Aiming at the problems of more residual isomers, DNA and the like of the antibody CD20 human-mouse chimeric monoclonal antibody obtained by affinity chromatography, the subsequent cation anion chromatography is optimized in process so as to obtain a high-quality antibody and ensure the medication safety.
CN105017418A discloses a purification method of a monoclonal antibody, (1) affinity chromatography, (2) adjusting the pH of an eluent of the affinity chromatography to 3.3-3.8 for virus inactivation, (3) adjusting the pH to neutral deep filtration, (4) anion exchange chromatography, and (5) cation exchange chromatography. The method can ensure that the purity of the SEC of the target protein reaches 99.6 percent. CN105263947A discloses a process for purifying monoclonal antibody, which comprises centrifugation and depth filtration followed by readjustment for cell separation and purification of culture supernatant, affinity chromatography, low pH incubation, neutralization readjustment, hydrophobic chromatography, ultrafiltration-diafiltration, anion exchange column chromatography, nanofiltration, ultrafiltration, diafiltration, microfiltration and other processes; the antibody includes an anti-CD 20 antibody. The hydrophobic chromatography used in the method needs high-concentration salt loading, the structural activity of the protein is easily influenced by too high salt concentration, the target protein is denatured or precipitated, and the removal of acid and base isomers cannot be considered. Aiming at the problems that the anti-CD 20 human mouse chimeric monoclonal antibody obtained by affinity chromatography in the prior art contains various impurities, and the drug effect and the clinical medication safety of the anti-CD 20 human mouse chimeric monoclonal antibody are influenced, a purification method of the anti-CD 20 human mouse chimeric monoclonal antibody, which has simple steps and better protein purification effect, is very necessary to be provided.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for purifying an anti-CD 20 human-mouse chimeric monoclonal antibody by an ion exchange method, and the inventor finds that the combination of cation chromatography and anion chromatography is adopted, the chromatography condition is optimized, and trace impurities in the anti-CD 20 human-mouse chimeric monoclonal antibody can be effectively removed, so that the quality and the stability of the anti-CD 20 human-mouse chimeric monoclonal antibody are improved. The technical scheme of the invention is as follows:
a method for purifying an anti-CD 20 human-mouse chimeric monoclonal antibody by ion exchange chromatography, which comprises the following steps:
(1) adding an additive into a sample obtained by affinity chromatography, and adjusting the pH value to 5.0-6.0;
(2) performing cation chromatography on the sample obtained in the step (1);
(3) and (3) carrying out anion chromatography on the sample obtained in the step (2).
Preferably, the additive in the step (1) is one or more of arginine, glycine or mannitol; the addition amount of the additive is 0.02-0.05 mol per liter of sample.
Preferably, the cation chromatography in step (2) utilizes a sample adsorption elution mode, including an equilibration, loading, washing and elution process; the buffer solution used for cation chromatography is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution containing additives, and the conductance is 5.0-8.5mS/cm, wherein the salt used for adjusting the conductance is selected from one of sodium chloride, potassium chloride and ammonium chloride.
The balancing process is to balance 5CV to the cation chromatographic column by using a balancing buffer solution, wherein CV represents the column volume; wherein the balance buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH5.0-6.0, conductivity 5.0-7.0mS/cm) containing additive, and the concentration of the additive is 0.02-0.05 mol/L.
The washing process comprises the steps of firstly carrying out rebalance on the cation column by using the balance buffer solution for 3CV, then washing by using the washing buffer solution for 3-8 CV, and finally rebalancing by using the balance buffer solution for 3 CV; wherein the washing buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH is 5.0-6.0, electric conductivity is 6.0-8.5mS/cm) containing additives, and the concentration of the additives is 0.02-0.05 mol/L.
The elution process is to elute the cation chromatographic column by using elution buffer solution, and collect effluent liquid after peak emergence; wherein the elution buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.0-6.5, conductivity 6.5-8.5mS/cm) containing additive, and the concentration of the additive is 0.02-0.05 mol/L.
Preferably, anion chromatography in step (3) utilizes a sample flow-through mode, including equilibration, loading, washing processes.
The buffer solution used in the equilibration process is the same as the elution buffer solution used in the cation chromatography; the sample loading process is to load a sample collected by cation chromatography, and collect effluent liquid after peak emergence; the washing process is the same as the cation chromatography elution process, the effluent liquid is continuously collected until the baseline is stable, the collection is stopped, and the collected sample is the stock solution of the anti-CD 20 human-mouse chimeric monoclonal antibody.
Preferably, a method for purifying an anti-CD 20 human murine chimeric monoclonal antibody by ion exchange chromatography comprising the steps of:
(1) adding 0.02-0.05 mmol of additive into 100mL of sample obtained by affinity chromatography, and adjusting the pH value to 5.0-6.0 for later use;
cation chromatography:
(2) balancing: balancing the cation chromatographic column by using a balance buffer solution with the pH of 5.0-6.0, the conductivity of 5.0-7.0mS/cm and the additive concentration of 0.02-0.05 mol/L, wherein the flow rate is 200 cm/h;
(3) loading: loading the sample at a flow rate of 200 cm/h;
(4) washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; washing the cation chromatographic column by using a washing buffer solution with the pH of 5.0-6.0, the conductivity of 6.0-7.0 mS/cm and the additive concentration of 0.02-0.05 mol/L for 6CV at the flow rate of 200 cm/h; then re-balancing 3CV by using a balancing buffer solution at the flow speed of 200 cm/h;
(5) and (3) elution: eluting the cation chromatographic column by using an elution buffer solution with the pH value of 6.0-6.5, the electric conductivity of 6.5-8.5mS/cm and the additive concentration of 0.02-0.05 mol/L at the flow speed of 200cm/h, and recording the effluent liquid after peak emergence as CEX Pro;
anion chromatography:
(6) balancing: balancing 5CV of the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow speed is 300 cm/h;
(7) loading: sampling by using the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent liquid after peak emergence;
(8) washing: continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro; and analyzing and detecting the samples obtained in the experiment.
The cation exchange chromatography packing used in the invention is Capto S Impact or Sepharose High Performance, but is not limited to the packing, and other cation exchange chromatography packing with High resolution is also applicable. The anion exchange chromatography packing used in the invention is Q Sepharose Fast Flow or Capto Q, but is not limited to the packing, and other anion exchange chromatography packing is also applicable to the invention. These fillers are commercially available.
The anti-CD 20 human-mouse chimeric monoclonal antibody is obtained by in vitro mammalian cell fermentation expression, wherein the animal cell is Chinese Hamster Ovary (CHO) cell, and the monoclonal antibody is IgG1 type.
The sample for ion exchange chromatography of the present invention is obtained after affinity chromatography, and the present invention is not limited to the affinity chromatography packing, and may be, for example, the packing Mabselect core, or other packing having affinity for Fc region, such as Mabselect core lx.
The invention has the advantages of simple operation, low cost, high safety and capability of ensuring the quality of the target protein, is convenient and easy to operate, and can be used for industrial large-scale production. The buffer solution used in the invention is a common buffer system, and has low price and simple operation. Arginine, glycine or mannitol and the like are added as additives in the sample loading process, so that the formation of protein aggregates is reduced. Whereas the addition of salts and additives to the mobile phase can reduce protein turbidity. By the technical scheme, the protein purity of the anti-CD 20 human-mouse chimeric monoclonal antibody can reach more than 99.7%, acid-base isomers, other impurity components and contents are well controlled within the quality requirement of the antibody, and the total yield reaches more than 72%.
Detailed Description
In order to better understand the content of the present invention, the following examples are provided to further illustrate the technical solution of the present invention, but the present invention is not limited by these examples.
The raw materials and consumables used in the examples can be prepared by commercial methods or known methods. The chromatography system, packing and column used in the examples were purchased from GE, the chromatography system being AKTAprime plus and the column being XK16 column.
The sample used in the embodiment of the invention is a CHO-expressed anti-CD 20 human-mouse chimeric monoclonal antibody fermentation broth, and the sample is obtained by performing protein A affinity chromatography and virus inactivation after deep filtration, wherein the sample contains 8.0g/L of protein, 91.01% of SEC purity, CEX (protein main peak 65.39%, acid peak 22.01%, alkaline peak 12.60%), 967ppm of HCP residue and 69pg/mL of DNA residue. The column heights used in the examples were all 20cm, the protein load was 800mg, and the protein load was 40 mg/mL.
Example 1
(1) Adding 3mmol arginine into 100mL of the sample obtained by affinity chromatography, and adjusting the pH value to 5.5 for standby.
Cation chromatography:
filling: sepharose High Performance
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH5.5, conductivity adjusted to 5.0mS/cm with sodium chloride) containing 0.02mol/L arginine at a flow rate of 200 cm/h.
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; washing the cation chromatographic column with 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH5.5, conductivity adjusted to 6.0mS/cm by sodium chloride) containing 0.02mol/L arginine for 6CV at flow rate of 200 cm/h; then, 3CV of the solution was re-equilibrated with the equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 6.5mS/cm with sodium chloride) containing 0.02mol/L arginine at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 1.
TABLE 1 data table of the results of sample analysis and detection after cation chromatography and anion chromatography in example 1
Example 2
(1) 100mL of the sample obtained by affinity chromatography is added with 5mmol of glycine, and the pH value is adjusted to 6.0 for standby.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated by 5CV at a flow rate of 200cm/h with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductance adjusted to 6.0mS/cm by potassium chloride) containing 0.05mol/L glycine.
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; then washing the cation chromatographic column with 7CV of 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.0, potassium chloride is used for adjusting the conductance to 7.1mS/cm) containing 0.05mol/L glycine at the flow rate of 200 cm/h; then, 3CV of the solution was re-equilibrated with the equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with potassium chloride) containing 0.05mol/L glycine at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: capto Q
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent liquid after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and marking the collected effluent as AEX Pro. The results of analysis and detection of the samples obtained in the experiment are shown in Table 2.
TABLE 2 data table of sample analysis and detection results after cation chromatography and anion chromatography in example 2
Example 3
(1) A sample obtained by affinity chromatography (100 mL) was added with 2mmol of mannitol, and the pH was adjusted to 5.0 for further use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated by 5CV at a flow rate of 200cm/h with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH5.0, conductivity adjusted to 5.5mS/cm with ammonium chloride) containing 0.03mol/L mannitol.
(3) Sampling: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; then washing the cation chromatographic column for 3CV with 0.03mol/L mannitol-containing 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH5.0, conductivity adjusted to 7.0mS/cm by ammonium chloride) at the flow rate of 200 cm/h; then, 3CV of the solution was re-equilibrated with the equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.5mS/cm with ammonium chloride) containing 0.03mol/L mannitol at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 3.
TABLE 3 data sheet of the results of sample analysis and detection after cation chromatography and anion chromatography in example 3
Example 4
(1) 100mL of the sample obtained by affinity chromatography is added with 5mmol of glycine, and the pH value is adjusted to 6.0 for standby.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 5.5mS/cm with sodium chloride) containing 0.03mol/L glycine at a flow rate of 200 cm/h. .
(3) Sampling: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; then, a 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.0 and conductivity adjusted to 6.5mS/cm by sodium chloride) containing 0.03mol/L glycine is used for balancing the cation chromatographic column for 5CV, and the flow rate is 200 cm/h; then, 3CV of the solution was re-equilibrated with the equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.03mol/L glycine in 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.5mS/cm with sodium chloride) at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: capto Q
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 4.
TABLE 4 data table of the results of sample analysis and detection after cation chromatography and anion chromatography in example 4
Example 5
(1) 100mL of the sample obtained by affinity chromatography is added with 5mmol of arginine, and the pH value is adjusted to 6.0 for standby.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.05mol/L arginine at a flow rate of 200 cm/h. .
(3) Loading: the sample is loaded at a flow rate of 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) containing 0.05mol/L arginine and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.05mol/L arginine at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 5.
TABLE 5 data sheet of the results of sample analysis and detection after cation chromatography and anion chromatography in example 5
Example 6
(1) A100 mL sample obtained by affinity chromatography was added with 5mmol of mannitol, and adjusted to pH6.0 for further use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.05mol/L mannitol at a flow rate of 200 cm/h. .
(3) Loading: the sample is loaded at a flow rate of 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) containing 0.05mol/L mannitol, and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.05mol/L mannitol at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing 5CV of the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow speed is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 6.
TABLE 6 data table of the results of sample analysis and detection after cation chromatography and anion chromatography in example 6
Comparative example 1
(1) 100mL of the sample obtained by affinity chromatography was adjusted to pH6.0 for use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) at a flow rate of 200 cm/h. .
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: eluting the cation chromatographic column with 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH6.5, conductivity adjusted to 8.5mS/cm by sodium chloride) at flow rate of 200cm/h, and collecting eluate after peak emergence as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Sampling: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and marking the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 7.
TABLE 7 data table of sample analysis and detection results after cation chromatography and anion chromatography in comparative example 1
Comparative example 2
(1) 100mL of the sample obtained by affinity chromatography was added with 5mmol of histidine, and the pH was adjusted to 6.0 for further use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation column was equilibrated by 5CV at a flow rate of 200cm/h with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.05mol/L histidine. .
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) containing 0.05mol/L histidine, and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.05mol/L histidine at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 8.
TABLE 8 data table of sample analysis and detection results after cation chromatography and anion chromatography in comparative example 2
Comparative example 3
(1) A100 mL sample obtained by affinity chromatography was added with 1mmol of mannitol, and adjusted to pH6.0 for further use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.01mol/L mannitol at a flow rate of 200 cm/h. .
(3) Sampling: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation chromatography column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) containing 0.01mol/L mannitol, and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.01mol/L mannitol at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of analyzing and detecting the samples obtained in the experiment are shown in Table 9.
TABLE 9 data table of sample analysis and detection results after cation chromatography and anion chromatography in comparative example 3
Comparative example 4
(1) A sample obtained by affinity chromatography (100 mL) was added with 10mmol of mannitol, and the pH was adjusted to 6.0 for further use.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.1mol/L mannitol at a flow rate of 200 cm/h. .
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation chromatography column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 8.0mS/cm with sodium chloride) containing 0.1mol/L mannitol, and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.1mol/L mannitol at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing 5CV of the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow speed is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 10.
TABLE 10 data table of sample analysis and detection results after cation chromatography and anion chromatography in comparative example 4
Comparative example 5
(1) 100mL of the sample obtained by affinity chromatography is added with 5mmol of arginine, and the pH value is adjusted to 6.0 for standby.
Cation chromatography:
filling: capto S Impact
(2) Balancing: the cation chromatography column was equilibrated with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.0, conductivity adjusted to 7.0mS/cm with sodium chloride) containing 0.05mol/L arginine at a flow rate of 200 cm/h. .
(3) Loading: the sample is loaded, and the flow rate is 200 cm/h.
(4) Washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; the cation column was washed with 5CV of 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH7.2) containing 0.05mol/L arginine, and then with 3CV of equilibration buffer at a flow rate of 200 cm/h.
(5) And (3) elution: the cation column was eluted with 0.02mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH6.5, conductivity adjusted to 8.5mS/cm with sodium chloride) containing 0.05mol/L arginine at a flow rate of 200cm/h, and the effluent was collected as CEX Pro.
Anion chromatography:
filling: q Sepharose Fast Flow
(6) Balancing: and (4) balancing the anion chromatographic column by using the elution buffer solution in the step (5) for 5CV, wherein the flow rate is 300 cm/h.
(7) Loading: and (5) loading the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent after peak emergence.
(8) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of the analysis and detection of the samples obtained in the experiment are shown in Table 11.
TABLE 11 data table of sample analysis and detection results after cation chromatography and anion chromatography in comparative example 5
Comparative example 6
(1) 100mL of the sample obtained by affinity chromatography was adjusted to pH6.5 for use.
Hydrophobic chromatography:
filling: capto phenyl Jmpres
(2) Balancing: equilibrating the chromatographic column with 5CV of 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH6.5, conductivity adjusted by sodium chloride is greater than 90mS/cm) at a flow rate of 200 cm/h;
(3) loading: adjusting the conductance of the sample to 90mS/cm by using sodium chloride, and loading the sample at the flow rate of 200 cm/h;
(4) and (3) elution: eluting with 0.02mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH6.5, conductivity adjusted with sodium chloride of 8.5mS/cm), collecting the effluent as: HIC Pro
Anion chromatography:
filling: q Sepharose Fast Flow
(5) Balancing: and (5) balancing the anion chromatographic column by using the elution buffer solution in the step (4) for 5CV, wherein the flow rate is 300 cm/h.
(6) Loading: and (4) loading the sample obtained in the step (4), wherein the flow speed is 300cm/h, and collecting effluent liquid after peak emergence.
(7) Washing: and (4) continuously washing the anion chromatographic column by using the elution buffer solution in the step (4), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro. The results of analyzing and testing the samples obtained in the experiment are shown in Table 12.
TABLE 12 data table of the results of sample analysis and detection after cation chromatography and anion chromatography in comparative example 6
Claims (2)
1. A method for purifying a human-murine chimeric monoclonal antibody against CD20 by ion exchange chromatography, comprising the steps of:
(1) adding an additive into 100mL of a sample obtained by affinity chromatography, and adjusting the pH value to 5.0-6.0 for later use;
cation chromatography:
(2) balancing: balancing the cation chromatographic column by using a disodium hydrogen phosphate-sodium dihydrogen phosphate balance buffer solution with the pH value of 5.0-6.0, the electric conductivity of 5.0-7.0mS/cm and the additive concentration of 0.02-0.05 mol/L for 5CV at the flow rate of 200 cm/h;
(3) loading: loading the sample at the flow speed of 200 cm/h;
(4) washing: re-balancing 3CV with balancing buffer solution at flow rate of 200 cm/h; washing the cation chromatographic column by using a disodium hydrogen phosphate-sodium dihydrogen phosphate washing buffer solution with the pH of 5.0-6.0, the conductivity of 6.0-7.0 mS/cm and the additive concentration of 0.02-0.05 mol/L for 6CV at the flow rate of 200 cm/h; then, re-balancing 3CV by using a balance buffer solution at the flow speed of 200 cm/h;
(5) and (3) elution: eluting the cation chromatographic column by using a disodium hydrogen phosphate-sodium dihydrogen phosphate elution buffer solution with the pH value of 6.0-6.5, the electric conductivity of 6.5-8.5mS/cm and the additive concentration of 0.02-0.05 mol/L at the flow rate of 200cm/h, and collecting effluent liquid after peak emergence and marking as CEX Pro;
anion chromatography:
(6) balancing: balancing 5CV of the anion chromatographic column by using the elution buffer solution in the step (5), wherein the flow speed is 300 cm/h;
(7) loading: sampling by using the sample obtained in the step (5), wherein the flow speed is 300cm/h, and collecting effluent liquid after peak emergence;
(8) washing: continuously washing the anion chromatographic column by using the elution buffer solution obtained in the step (5), wherein the flow rate is 300cm/h, continuously collecting the effluent until the baseline is stable, and recording the collected effluent as AEX Pro;
the additive is one or more of arginine, glycine or mannitol; the addition amount of the additive is 0.02-0.05 mol of additive added into each liter of sample.
2. The method of claim 1, wherein the conductivity is adjusted by using a salt selected from the group consisting of sodium chloride, potassium chloride, and ammonium chloride.
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| CN105263947A (en) * | 2013-06-25 | 2016-01-20 | 卡迪拉保健有限公司 | Purification process for monoclonal antibodies |
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| CA2478925A1 (en) * | 2002-04-26 | 2003-12-11 | Robert Lee Fahrner | Non-affinity purification of proteins |
| CN101535335A (en) * | 2006-08-28 | 2009-09-16 | 阿雷斯贸易股份有限公司 | Process for the purification of Fc-containing proteins |
| CN105263947A (en) * | 2013-06-25 | 2016-01-20 | 卡迪拉保健有限公司 | Purification process for monoclonal antibodies |
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