CN105315369B - Purification of proteins by cation exchange chromatography - Google Patents
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- CN105315369B CN105315369B CN201410359482.XA CN201410359482A CN105315369B CN 105315369 B CN105315369 B CN 105315369B CN 201410359482 A CN201410359482 A CN 201410359482A CN 105315369 B CN105315369 B CN 105315369B
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Abstract
The invention provides a method for purifying an antibody containing pollutants by cation exchange chromatography, which obtains better removal effect of the pollutants such as HCP (human serum phosphate), DNA (deoxyribonucleic acid), excellent aggregate removal effect and higher yield of target protein by selecting and optimizing the composition, concentration, pH (potential of hydrogen) and conductivity of buffers such as loading, washing and elution. The method has the advantages of simple operation steps, simple and convenient operation, reduction of production cost and suitability for large-scale industrialized antibody separation and purification.
Description
Technical Field
The invention belongs to the field of protein purification, and particularly relates to a method for purifying an antibody containing a pollutant by using a cation exchange chromatography method.
Background
With the continuous development of bioengineering technology, more and more proteins including antibodies are prepared on a large scale through a bioreactor (such as engineering bacteria or engineering cell strains) or other methods, the separation and purification of the obtained culture containing the target protein is an essential step in the downstream technology of the production process, and how to improve the purification conditions of the protein, further increase the efficiency of removing pollutants, improve the purity and yield of the protein and the like through the improvement of a protein purification method is a problem which exists in the industrial production of the protein.
In general, a method for expressing a target protein using an engineered bacterium or an engineered cell line is to introduce a recombinant vector containing a target gene into a host cell and culture the cell under appropriate conditions to express the target protein. The obtained culture contains Host Cell Protein (HCP), Host cell DNA, RNA, culture medium components, target protein variants and/or aggregates, target protein fragments, and other contaminants including toxins, viruses, microorganisms, and the like, which may be present, in addition to the target protein; therefore, a series of purification processes is necessary to obtain a relatively pure protein suitable for the intended purpose.
Ion Exchange Chromatography (Ion Exchange Chromatography abbreviated as IEC) is a purification method commonly used in the field of biochemistry at present. The ion exchange chromatography is a chromatography method which takes an ion exchanger as a stationary phase and achieves the separation purpose by reversibly exchanging component ions in a mobile phase with counter ions on the exchanger. By ion exchange is meant a process in which one type of ion in solution is reversibly exchanged with another type of ion bound to a support, i.e. the ions in solution are bound to the support and the ions on the support are replaced. If the carrier is combined with active groups with positive charges, the carrier can exchange anions and is an anion exchanger; if a negatively charged active group is bound to the support, the cation can be exchanged, which is a cation exchanger. Under certain conditions, different proteins in the mixture have different charges, some can be combined with a specific ion exchanger, and some cannot be combined; the proteins capable of binding to the ion exchanger do not always have the same binding force, and they can be efficiently separated by using an appropriate elution condition. Ion exchange chromatography is widely used for separating and purifying various biochemical substances such as amino acid, protein, saccharide, nucleotide and the like.
Due to the complexity of biological samples and other factors, the binding condition of biological macromolecules and ion exchangers is difficult to estimate, especially the complexity of protein structure, and the binding force of the biological macromolecules and the ion exchangers is related to the number of charges carried by protein molecules and also has a certain relation with other properties such as the size and charge arrangement of the protein molecules. Therefore, it is often necessary to search through a large number of experiments to obtain an antibody with higher purity by optimizing and selecting a suitable ion exchange chromatography purification process for different proteins, particularly for antibodies used as drugs, to reduce the production cost as much as possible and to increase the antibody yield.
Patent 200880119331.X discloses a method for purification of antibodies by cation exchange chromatography wherein a high pH wash step is used to remove contaminants before elution of the desired antibody using an elution buffer with increased conductivity; by including at least two washing steps in a cation exchange purification scheme, at least the first of which is performed at a high pH (about pH 6.8 or greater), the purification efficacy can be significantly improved, achieving higher step yields. This patent removes contaminants by adding a high pH wash step prior to elution of the antibody, but the addition of process steps increases process time and increases production costs.
Disclosure of Invention
In order to reduce production costs and obtain antibodies of higher purity by performing purification with as few ion exchange chromatography steps as possible, the present invention provides a method for purifying antibodies containing contaminants by cation exchange chromatography, comprising the steps of:
a) loading: loading the solution containing the antibody and the contaminant onto a cation exchange column,
b) cleaning: the cation exchange material is washed with a washing buffer,
c) and (3) elution: the desired antibody is eluted from the cation exchange material with an elution buffer.
The invention optimizes the composition, concentration, pH, conductivity and the like of various buffers through a large number of tests, thereby achieving better pollutant removal effect and higher yield.
In a preferred embodiment of the invention, the conductivity of the wash buffer is 5 to 9ms/cm, preferably 6 to 8 ms/cm.
In a preferred embodiment of the invention, the pH of the wash buffer is 5.5-5.9, preferably 5.7-5.9;
the buffer substance in the washing buffer solution is MES, citric acid, phosphoric acid, citrate, phosphate or a mixture of two or more of the MES, the citric acid, the phosphoric acid, the citrate and the phosphate; the buffer solution contains a salt selected from potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or a mixture of two or more of these components.
In a preferred embodiment of the invention, the wash buffer is a solution containing 20-25mM MES, 40-50mM NaCl;
in a preferred embodiment of the invention, the conductivity of the elution buffer is 9-14ms/cm, preferably 9-10ms/cm, more preferably 9.0-9.6 ms/cm.
In another preferred embodiment of the invention, the pH of the elution buffer is between 5.5 and 5.9, preferably between 5.7 and 5.9.
The buffer substance in the elution buffer solution is MES, citric acid, phosphoric acid, citrate, phosphate or a mixture of two or more of the MES, the citric acid, the phosphoric acid, the citrate and the phosphate; the buffer solution contains a salt selected from potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or a mixture of two or more of these components.
In another preferred embodiment of the invention, the elution buffer is a solution containing 20-25mM MES, 75-110mM NaCl, preferably 80-95mM NaCl.
The antibody containing contaminants may be subjected to one or more additional purification steps prior to, during or after cation exchange chromatography using the methods of the invention. For example, a better antibody purification effect can be obtained by performing cation exchange chromatography after primary purification by depth filtration and protein A affinity chromatography.
The antibody purified by the cation exchange chromatography purification process of the present invention is an antibody that binds to human VEGF (human vascular endothelial growth factor) or an antibody that binds to human CD 20.
Antibodies binding to human VEGF that can be used in the purification process of the present invention are meant to include polyclonal antibodies, monoclonal antibodies, antibody fragments with binding specificity, or various modified forms of antibodies, etc. that bind to human VEGF; preferred anti-VEGF antibodies of the invention that can be used for purification are antibodies having the amino acid sequence of SEQ ID NO: 1(CDR H1), SEQ ID NO: 2(CDR H2), SEQ ID NO: 3(CDRH3) and the hypervariable region sequence of the heavy chain shown in SEQ ID NO: 4(CDR L1), SEQ ID NO: 5(CDR L2), SEQ ID NO: 6(CDR L3), more preferably Bevacizumab (Bevacizumab).
The anti-VEGF antibody of the present invention can be obtained by the following method: combining the synthesized SEQ ID NO: 1-3 grafted onto a human heavy chain subgroup iii framework, SEQ ID NO: 4-6 light chain CDR region sequences are transplanted to a framework of a human light chain kappa subgroup 1 to obtain anti-VEGF antibody VH and VL sequences, then the VH and VL are respectively cloned to expression vectors containing a human IgG heavy chain constant region and a light chain constant region, the expression vectors containing the anti-VEGF antibody heavy chain and the light chain are co-transfected into CHO cells, the CHO cells are cultured under proper culture conditions, anti-VEGF antibodies expressed by the CHO cells are secreted into an extracellular culture medium, cell cultures containing the anti-VEGF antibodies are collected, and after primary purification of the cell cultures, a mixture containing the anti-VEGF antibodies and pollutants for further ion exchange chromatography is obtained.
The antibody that binds to human CD20 that can be used in the purification process of the present invention is meant to include polyclonal antibodies, monoclonal antibodies, antibody fragments with binding specificity, or various modified forms of antibodies that bind to human CD20, and the like, and the anti-CD 20 antibody that can be preferably used in the present invention is a monoclonal antibody having the amino acid sequence of SEQ ID NO: 7 and the variable region sequence of SEQ ID NO: 8, more preferably Rituximab (Rituximab).
The anti-CD 20 antibody of the present invention can be obtained by the following method: combining the synthesized SEQ ID NO: 7 and the variable heavy chain sequence of SEQ ID NO: 8, the expression vector containing the heavy chain and the light chain of the anti-CD 20 antibody is co-transfected into CHO cells, the CHO cells are cultured under proper culture conditions, the anti-CD 20 antibody expressed by the CHO cells is secreted into an extracellular culture medium, cell cultures containing the anti-CD 20 antibody are collected, and after the cell cultures are subjected to primary purification, a mixture containing the anti-CD 20 antibody and pollutants for further ion exchange chromatography is obtained.
In another embodiment of the invention, the cation exchange column is equilibrated with an equilibration buffer prior to loading, which may be the same buffer as the wash buffer.
Solution loading solution containing antibody and contaminant before loading, the pH of the loading solution is optimized to be within 0.05 of the pH of the equilibration buffer selected for purification.
The conductivity of the sample solution is adjusted according to the properties of the ion exchange material, if the SP-HP and other ion exchange materials which are not resistant to high salt are adopted, the conductivity of the sample solution is required to be less than or equal to 3.5ms/cm, and if the poros and other high salt resistant fillers are adopted, the conductivity of the sample solution does not need to be adjusted.
In another embodiment of the present invention, the exchange material used is an ion exchange material having a particle size diameter of not more than about 50 μm. In some preferred embodiments, the cation exchange column uses SP-HP (SP-Sepharose HP, High Performance GE), Capto SP ImpRes, poros HS and poros XS (polystyrene resin; i.e., a copolymer of divinylbenzene and styrene), fractogel (polymethacrylate resin), Nuvia HR S and other High resolution strong cation exchange resin resins, and the like.
The invention optimizes the composition, concentration, pH, conductivity and the like of various buffers through a large number of tests, thereby achieving better pollutant removal effect and higher target protein yield. In the selection optimization of the conductivity of the washing buffer solution, the required antibody is still bonded on the exchange material while the pollutants can be removed by improving the conductivity, and the required antibody and the balance buffer solution can be the same buffer solution, so that the simplicity and convenience of the process operation are improved, and the production cost is saved. By adopting one-step cleaning step, better removal efficiency of HCP and DNA pollutants is obtained, the selection of the pH value and the conductivity of the elution buffer solution is optimized, and by controlling the range of the pH value and the conductivity, excellent aggregate removal effect is obtained, and meanwhile, higher antibody yield can be obtained.
The pH value of the present invention is measured at 24 ℃ with a Mettler Toledo pH meter.
"conductivity" herein refers to the ability of a solution to conduct an electrical current. The conductivity of the solution may be altered by changing the salt concentration in the solution, e.g. the conductivity of the elution buffer may be increased by increasing the salt concentration in the elution buffer. Salts that may be used to increase conductivity include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or a mixture of two or more of these salts.
The conductivity value of the invention is measured by a Mettler Toledo conductivity meter at 24 ℃, and the measuring method comprises the following steps: the probe was placed in the solution and the reading was taken directly.
The contaminant-containing protein used for purification may be a protein obtained by chemical synthesis or other synthetic methods, or a protein derived from a natural source, or a protein obtained by culturing an engineered bacterium or an engineered cell strain.
The antibodies described herein include polyclonal antibodies, monoclonal antibodies, multispecific antibodies or antibody fragments with binding specificity, or various modified forms of antibodies, and the like.
In general, a method for expressing a target protein using an engineered bacterium or an engineered cell line is to introduce a recombinant vector containing a target gene into a host cell and culture the cell under appropriate conditions to express the target protein. Suitable host cells for cloning or expressing the gene of interest are prokaryotes, yeast cells or higher eukaryotic cells. Currently, most of antibody drugs are prepared in a large-scale culture of animal cells. Commonly used animal cell lines include, but are not limited to, chicken embryo, pig kidney, human embryonic kidney, hamster kidney, monkey kidney, hamster kidney (BHK), Chinese Hamster Ovary (CHO), human cervical cancer (HELA), human lung, human liver, mouse breast, human hepatoma, and the like. Among them, CHO cell is the most widely used antibody expression system at present.
By inoculating the cells into a bioreactor for cell culture, the protein of interest is expressed, and the expressed protein can be secreted into the cell culture medium or accumulated inside the cells. For proteins that accumulate within cells, it is necessary to first collect the cells for lysis and then remove the host cells and debris resulting from cell lysis by, for example, centrifugation, filtration or ultrafiltration. For proteins secreted into the culture medium, the supernatant containing the protein of interest can be obtained directly by filtration or centrifugation.
A preferred embodiment of the present invention purifies the antibody produced by animal cell culture. Preferably, the vector containing the target gene is introduced into Chinese Hamster Ovary (CHO), and the culture containing the target antibody is collected and purified by cell culture. A range of contaminants are produced by CHO cell culture, such as Chinese Hamster Ovary Protein (CHOP); cellular DNA, media components, variants and/or aggregates of antibodies, antibody fragments, and other contaminants including toxins, viruses, microorganisms, etc., that may be present, and the like. After obtaining a cell culture fluid containing the antibody of interest and other contaminants, the antibody containing the contaminants can be subjected to one or more additional purification steps before, during, or after being subjected to cation exchange chromatography.
Human VEGF is a human vascular endothelial growth factor (vascular permeability factor, VPF for short) which induces angiogenesis in vivo. The antibody binding to human VEGF that can be used in the purification process of the present invention is intended to include polyclonal antibodies, monoclonal antibodies, antibody fragments having binding specificity, or various modified forms of antibodies, etc., which bind to human VEGF.
The CD20 antigen is a surface membrane protein located in B lymphocytes, and is a B cell differentiation antigen, and CD20 is expressed in almost all normal and malignant B cells, and is expressed in over 95% of B cell lymphomas, but not in hematopoietic stem cells, plasma cells, and other normal tissues. Antibodies that bind to human CD20 that can be used in the purification process of the present invention are meant to include polyclonal antibodies, monoclonal antibodies, antibody fragments with binding specificity, or various modified forms of antibodies, etc., that bind to CD20 on the surface of human B lymphocytes.
Cation exchangers used in cation exchange chromatography refer to stationary phase media having anionic groups bound to the surface of the stationary phase, the anionic groups of the solid phase having positively charged counterions (counterions) bound to them that reversibly exchange with positively charged proteins in solution. The ion exchangers are strongly acidic, strongly basic, weakly acidic and weakly basic, depending on the nature of the ionization of the active groups. Useful ion exchange materials include ion exchange resins, ion exchange celluloses, ion exchange sephadex, sepharose, polyacrylamide gels, silica gels and the like.
A variety of commercially available cation exchange materials are currently available for use in the present invention, including Sulfopropyl (SP) (e.g., SP 650S or SP 650M of Tosoh Biosciences) immobilized on methyl acrylate, Sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE HIGHPERFOMANCE (SP-HP), SP-SEPHAROSEFFLOWTM or SP-SEPHAROSE FAST W XLTM of GE Helthcare), FRACTOGEL-SE HICAPTM, FRACTEL-SO 3TM, and FRACTOPREP (EMDMERCK), sulfonyl immobilized on agarose (e.g., S-SEPHAROSEFFLOW of GE, Capto SP pRFLOS and Tosoh Biosciences), sulfopropyl (e.g., Applied Biosystems), or macroporous bioscience filled hydrogel such as Porphys corporation, immobilized on styrene-divinylbenzene, immobilized on coated styrene-divinylbenzene, (e.g., immobilized Biosystems, immobilized on methacrylic acid), hyaluronic acid immobilized on hydrogel such as immobilized on hyaluronic acid, or immobilized on hydrogel such as Sephacrys 6335, immobilized on silica, immobilized on methacrylate, immobilized on silica, immobilized on styrene-alumina, silica, immobilized on silica, alumina, and sulfo groups immobilized on the polymer (e.g., NuviaHS from Bio-rad). Preferred cation exchange materials of the present invention include SP-HP (SP-Sepharose HP, High Performance GE), Capto SP imprmes, poros HS and poros XS (polystyrene resin; i.e., copolymer of divinylbenzene and styrene), fractogel (polymethacrylate resin), Nuvia HR S, and the like.
After a mixture containing the antibody and the pollutant is purified by the cation exchange chromatography process, the pollutant removal effect is remarkable, wherein host cell protein has higher removal efficiency, and in some embodiments, the content of the host cell protein is even lower than 20ppm, even lower than 10 ppm; the content of DNA is lower than 1pg/mg Mab; leached protein a content <10 ppm; the antibody purified by the process has a very obvious aggregate separation effect, the aggregate content after purification is lower than 5%, in some embodiments, the aggregate content is even lower than 1%, and components such as culture medium, toxin and the like are basically not detected in the antibody solution purified by the purification process.
Drawings
FIG. 1: content of major contaminants after cation exchange chromatography of 5 different batches of anti-VEGF antibody samples
The present invention is illustrated by the following specific examples, which are not intended to limit the invention in any way.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1 preparation of a mixture containing anti-VEGF antibodies and contaminants
Combining the synthesized SEQ ID NO: 1-3 grafted human heavy chain subgroup iii framework with the anti-VEGF antibody heavy chain CDR region sequences of SEQ ID NOs: 4-6 light chain CDR region sequences are transplanted to a framework of a human light chain kappa subgroup 1 to obtain anti-VEGF antibody VH and VL sequences, then the VH and VL are respectively cloned to constructed expression vectors containing human IgG heavy chain constant regions and light chain constant regions, the expression vectors containing the anti-VEGF antibody heavy chain and light chain are co-transfected into CHO cells, the CHO cells are cultured under proper culture conditions, anti-VEGF antibodies expressed by the CHO cells are secreted into an extracellular culture medium, cell cultures containing the anti-VEGF antibodies are collected, and after primary purification of the cell cultures, a mixture containing the anti-VEGF antibodies and pollutants for further ion exchange chromatography is obtained.
Example 2 preparation of a mixture containing anti-CD 20 antibody and contaminants
Combining the synthesized SEQ ID NO: 7 and the variable heavy chain sequence of SEQ ID NO: 8, cloning the light chain variable region sequence to a constructed expression vector containing a human IgG heavy chain constant region and a light chain constant region respectively, co-transfecting CHO cells with the expression vector containing the heavy chain and the light chain of the anti-CD 20 antibody, culturing under a proper culture condition, secreting the anti-CD 20 antibody expressed by the CHO cells into an extracellular culture medium, collecting cell culture containing the anti-CD 20 antibody, and primarily purifying the cell culture to obtain a mixture containing the anti-CD 20 antibody and pollutants for further ion exchange chromatography.
Example 3: purification of anti-VEGF antibodies
Experimental methods and results: filling cation exchange resin SP-HP into a chromatographic column with the height of the chromatographic column being 20 cm; balancing the packed chromatographic column with a balancing buffer solution; after column equilibration, the solution containing the anti-VEGF antibody and contaminants obtained from example 1 was slowly added along the wall of the chromatography column; washing the chromatographic column with a washing buffer; then, the antibody is eluted by using an elution buffer, and the eluate is collected. The process parameters of the steps used in this example are shown in table 1 below.
Table 1: the purification process conditions adopted in this example were:
table 2: main pollutant content of purified antibody monomer
After the cation exchange chromatography purification step, the contents of antibody monomers and main impurities in the obtained antibody solution are shown in table 2, and experimental results show that pollutants in the anti-VEGF antibody mixture are removed to a great extent, the yield of the antibody is high, and components of the culture medium are not detected.
In addition, through the optimization of process parameters, the same buffer solution can be selected for the balance buffer solution and the washing buffer solution in the experiment, and the process cost can be further reduced.
The influence of different concentrations, pH values, electric conductivities and the like of the solution used in each step of the purification process on the pollutant removal effect and the protein yield is respectively considered through experimental design, and the optimal concentration range, the pH value and the electric conductivity (shown in table 3) for the anti-VEGF antibody cation exchange chromatography purification process are screened out, and under the conditions of the optimal concentration range, the pH value and the electric conductivity of the purification process of the invention, the content of the host cell protein in the purified antibody solution is even lower than 20ppm or even lower than 10 ppm; the content of DNA is lower than 1pg/mg Mab; leached protein a content <10 ppm; the aggregate separation effect is very significant, with an aggregate content after purification of less than 5%, in some embodiments even less than 1%,
TABLE 3 preferred concentration ranges, pH values and conductivities for anti-VEGF antibody cation exchange chromatography purification Process
Example 4: purification of anti-CD 20 antibodies
Experimental methods and results: filling cation exchange resin (POROS HS 50) into a chromatographic column with a bed height of 10 cm; balancing the packed chromatographic column with a balancing buffer solution; after equilibration of the column, the solution containing the anti-CD 20 antibody and contaminants obtained from example 2 was added slowly along the wall of the chromatography column; washing the chromatographic column with a washing buffer; then, the antibody is eluted by using an elution buffer, and the eluate is collected. The process parameters used in this example are shown in Table 4 below.
Table 4: the purification process conditions are as follows:
table 5: purified antibody monomer and main pollutant content
After the cation exchange chromatography purification step, the contents of antibody monomers and various main impurities in the obtained antibody solution are shown in table 5, and experimental results show that pollutants in the anti-CD 20 antibody mixture are removed to a great extent, the yield of the antibody is high, and the components of the culture medium are not detected.
The influence of different concentrations, pH values, electric conductivities and the like of the solution used in each step of the purification process on the pollutant removal effect and the protein yield is respectively considered through experimental design, and the optimal concentration range, pH value and electric conductivity for the anti-CD 20 antibody cation exchange chromatography purification process are screened out (see Table 6). Under the conditions of concentration range, pH value and conductivity which are optimized in the purification process of the invention, the content of host cell protein in the purified antibody solution is even lower than 20ppm, even lower than 10 ppm; the content of DNA is lower than 1pg/mg Mab; leached protein a content <10 ppm; the aggregate separation effect is very significant, with an aggregate content after purification of less than 5%, in some embodiments even less than 1%,
TABLE 6 preferred molar concentration ranges, pH values and conductivities for cation exchange chromatography purification of anti-CD 20 antibodies
Example 5: stability of the Process of the invention
The experimental method comprises the following steps: filling cation exchange resin SP-HP into a chromatographic column with a bed height of 20 cm; balancing the packed chromatographic column with a balancing buffer solution; after column equilibration, the solution containing the anti-VEGF antibody and contaminants obtained from example 1 was slowly added along the wall of the chromatography column; washing the chromatographic column with a washing buffer; then, the antibody is eluted by using an elution buffer, and the eluate is collected. The parameters of the process steps by which 5 batches of experimental samples were purified are given in this example in Table 7 below.
Table 7: purification process parameters
The experimental results (see figure 1) show that 5 batches of samples are respectively effectively removed after being subjected to the cation exchange chromatography purification step, the process disclosed by the invention is good in removal effect, simple and convenient to operate, consistent in removal effect among different batches, and good in stability.
Example 6: compared with the purification process of patent 200880119331.X, the purification process of the invention
The experimental method comprises the following steps: filling cation exchange resin SP-HP into a chromatographic column with a bed height of 20 cm; purifying the anti-VEGF antibody and pollutant solution by respectively adopting the purification process conditions of the table 8, repeating the purification process for 5 times respectively to perform a purification experiment, respectively collecting eluent, measuring the content of the antibody solution obtained by the purification process of the patent 200880119331.X and the purification method of the invention, and comparing the removal effect and the antibody yield of the process of the patent 200880119331.X and the purification method of the invention.
Table 8: comparison of antibody purification Processes
Table 9: comparison of antibody monomer and major contaminant content
Note: the numerical values in table 9 are represented as: mean value. + -. standard deviation: (
)
Experimental results (see table 9), through statistical analysis of T-test, the contents of HCP, DNA and low molecular substance in the obtained antibody solution after purification by the process of the present invention are all greater than 0.05 in P value compared with the purification results of the process of the patent 200880119331.X, so the purification process of the present invention has the same better removal effect of HCP, DNA and low molecular substance; the content of antibody monomer and aggregate is different from that of the purification result of the patent process 200880119331.X in T test, the P value is less than 0.01, and the difference is very significant; therefore, the purification process has higher aggregate pollutant removal effect, thereby obtaining higher content of target antibody monomers.
The experimental results show that compared with the process of the patent 200880119331.X, the process of the invention reduces the washing step by using the high pH value washing buffer solution before eluting the expected antibody, and also achieves better removal effect of the pollutants such as HCP, DNA and the like, and has more excellent removal efficiency of the aggregates. The method has simple operation steps and simple and convenient operation, reduces the production cost, can further reduce the cost by adopting the same buffer solution for the balance buffer solution and the washing buffer solution, and is suitable for large-scale industrialized antibody separation and purification.
Claims (6)
1. A method for purifying an antibody using cation exchange chromatography comprising the steps of:
a) balancing: equilibrating the cation exchange material with an equilibration buffer, wherein the equilibration buffer has a pH between 5.5 and 5.9 and a conductivity between 6 and 8mS/cm,
b) loading: loading a solution containing the antibody and the contaminant onto a cation exchange column, wherein the solution containing the antibody and the contaminant has a pH of 5.5 to 5.9 and has a conductivity of not more than 3.5ms/cm,
c) cleaning: washing the cation exchange material with a wash buffer, wherein the wash buffer has a pH of from 5.5 to 5.9 and a conductivity of from 6 to 8mS/cm,
d) and (3) elution: eluting the target antibody from the cation exchange material with an elution buffer, wherein the elution buffer has a pH of from 5.5 to 5.9 and a conductivity of from 9 to 14mS/cm,
wherein the antibody is bevacizumab.
2. The method of claim 1, wherein the equilibration buffer and wash buffer are the same buffer.
3. The method according to claim 2, wherein the buffer material in the washing buffer is MES, citric acid, phosphoric acid, citrate, phosphate or a mixture of two or more thereof, and the buffer contains a salt selected from the group consisting of potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate or a mixture of two or more thereof.
4. The method according to claim 1, wherein the buffer material in the elution buffer is MES, citric acid, phosphoric acid, citrate, phosphate or a mixture of two or more thereof, and the buffer contains a salt selected from the group consisting of potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate or a mixture of two or more thereof.
5. The method of claim 1, wherein the exchange material used in the cation exchange column is an ion exchange material having a particle size diameter of no greater than 50 μm.
6. The method of claim 5, wherein the cation exchange column uses a strong cation exchange material with high resolution of SP-HP, Capto SP, ImpRes, poros HS, poros XS, fractogel, Nuvia HR S.
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| CN1299370A (en) * | 1998-05-06 | 2001-06-13 | 基因技术股份有限公司 | Protein purification by ion exchange chromatography |
| CN103554215A (en) * | 2007-10-30 | 2014-02-05 | 健泰科生物技术公司 | Antibody purification by cation exchange chromatography |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5110913A (en) * | 1990-05-25 | 1992-05-05 | Miles Inc. | Antibody purification method |
| CN1299370A (en) * | 1998-05-06 | 2001-06-13 | 基因技术股份有限公司 | Protein purification by ion exchange chromatography |
| CN103554215A (en) * | 2007-10-30 | 2014-02-05 | 健泰科生物技术公司 | Antibody purification by cation exchange chromatography |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023187826A1 (en) * | 2022-03-30 | 2023-10-05 | Dr. Reddy’S Laboratories Limited | Method to purify an antibody composition |
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