CN109593133B - Method for separating charge isomers of anti-human nerve growth factor antibody - Google Patents
Method for separating charge isomers of anti-human nerve growth factor antibody Download PDFInfo
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Abstract
The invention discloses a charge isomer separation method of an anti-human nerve growth factor antibody. Comprises affinity chromatography, and adjusting pH of the collected elution peak to 5.0-5.50; the conductance is adjusted to 4.90-5.10 ms/cm; and (4) passing through an anion exchange chromatography column, and collecting flow-through liquid when a penetration peak appears and the A280 absorption value reaches 20mAU, wherein the collected flow-through liquid is the antihuman nerve growth factor after the charge isomers are removed. The anti-human nerve growth factor antibody obtained by the method has high yield of charge isomers and good activity.
Description
Technical Field
The invention relates to the field of separation of antibodies, in particular to a method for separating charge isomers of an anti-human nerve growth factor antibody.
Background
The anti-human nerve growth factor antibody can block a nerve growth factor-mediated pain transfer pathway, so that the antibody has a wide treatment effect on painful diseases such as neuropathic pain, fracture pain, osteoarthritis, rheumatoid arthritis, gout joint pain and the like. During production, extraction and storage, antibodies undergo a series of post-translational modification processes, including glycosylation, polymerization, fragmentation, deamidation, etc., resulting in antibodies exhibiting various types of heterogeneity, which in turn leads to different types of variants that differ in their physicochemical properties, such as molecular weight, hydrophobicity, charge, etc. Charge isomers are generally classified as acidic or basic isomers according to their isoelectric points relative to the main peak. In general, acidic isomers have a lower isoelectric point relative to the main peak, while basic isomers have a higher isoelectric point relative to the main peak.
The current preparative method for separating antibody charge isomers generally uses cation exchange chromatography technology, and uses an adsorption elution mode to remove an acidic peak and a basic peak so as to obtain a main peak. The analytical method for separating the antibody charge isomers comprises ion exchange chromatography, reversed phase liquid chromatography, hydrophobic chromatography, size exclusion chromatography, SDS-polyacrylamide gel electrophoresis, capillary isoelectric focusing, capillary zone electrophoresis, mass spectrometry and the like, wherein the capillary isoelectric focusing is used for analyzing the antibody charge isomers and has the advantages of high efficiency, rapidness, high separation degree, economy and high automation.
Disclosure of Invention
The invention aims to provide a method for separating charge isomers of an anti-human nerve growth factor antibody with high yield and good activity.
In order to achieve the above object, the present invention provides a method for separating charge isomers of an anti-human nerve growth factor antibody, comprising the steps of,
affinity chromatography: performing affinity chromatography column chromatography on cell fermentation liquid containing the anti-human nerve growth factor antibody, and collecting A280 to obtain an elution peak; adjusting the pH of the collected elution peak to 5.0-5.50; the conductance is adjusted to 4.90-5.10 ms/cm;
anion exchange chromatography: and (4) passing through an anion exchange chromatography column, and collecting flow-through liquid when a penetration peak appears and the A280 absorption value reaches 20mAU, wherein the collected flow-through liquid is the antihuman nerve growth factor after the charge isomers are removed.
Furthermore, the cell fermentation liquid containing the anti-human nerve growth factor antibody is the fermentation liquid of recombinant anti-human nerve growth factor antibody CHO cells.
Further, the pH adjustment was adjusted with a sodium acetate buffer; preferably, the pH is adjusted to 5.2.
Further, the conductance is adjusted by using a sodium acetate buffer solution of 5.0-5.50.
Further, the anion exchange chromatography step is to balance the anion exchange chromatography column with 25mM sodium acetate buffer solution with pH5.00-5.50 for 15-25 column volumes; preferably 20 column volumes; loading the feed liquid with the adjusted pH and conductivity to an anion exchange chromatographic column until a penetration peak appears and the A280 absorption value reaches 15-25 mAU; preferably 20mAU, the flow through starts to collect; after the sample loading is finished, washing the chromatographic column by using 25mM sodium acetate buffer solution with pH of 5.00-5.50, and when the absorption value of A280 is reduced to 15-25 mAU; and (4) stopping collecting the flow-through liquid when the concentration is preferably 20mAU, wherein the collected flow-through liquid is the anti-human nerve growth factor after the charge isomers are removed.
Taking the anti-human nerve growth factor antibody feed liquid after affinity chromatography, and adjusting the pH value to be within the range of 5.0-5.50 by using 2M Tris-Cl buffer solution (pH9.0). When the pH value is higher than 5.50, all or most of the antihuman nerve growth factor is hung on the anion exchange chromatographic column during loading, and the antihuman nerve growth factor comprises a main peak, a basic peak and an acidic peak, and has few penetrating peaks and no separation effect. The pH is lower than 5.0, and the main peak, the alkaline peak and the acidic peak are all or most penetrated, and the separation effect is not realized. In the pH range of 5.0-5.50 and the conductance range, the main peak penetration is more, the basic peak penetration and the acidic peak penetration are less, the yield of the protein after the Capto Q purification is 75-85%, if the yield of the protein is lower than the range, the purification loses industrial value, and if the yield of the protein is higher than the range, the content ratio of the basic charge isomer and the acidic charge isomer is increased.
The conductance was adjusted to a range of 4.90-5.10ms/cm with 25mM sodium acetate buffer (pH 5.0-5.50).
The anion exchange chromatography column was equilibrated with 25mM sodium acetate buffer (pH5.00-5.50) for 20 column volumes.
And (3) loading the feed liquid with the adjusted pH and conductivity to an anion exchange chromatographic column, and collecting the flow-through liquid when a penetration peak appears and the A280 absorption value reaches 20 mAU.
After the sample loading is finished, washing the chromatographic column by using 25mM sodium acetate buffer solution (pH5.00-5.50), stopping collecting the flow-through solution when the A280 absorption value is reduced to 20mAU, and collecting the obtained flow-through solution, namely the anti-human nerve growth factor after the charge isomers are removed.
The anti-human nerve growth factor after the anion exchange chromatography penetration mode provided by the invention separates the charge isomers is detected by capillary isoelectric focusing (cIEF): the main peak isoelectric point (pI) is 6.81, which is consistent with the isoelectric point after cation exchange chromatography; the main peak area percentage is 65.77 percent, which is improved by 36.85 percent compared with the main peak area percentage (48.06 percent) of cation exchange chromatography. The cell activity test shows that the IC50 is 50.3ng/ml, and the activity is improved by 36.3 percent compared with the cell activity of cation exchange chromatography (the IC50 is 78.9 ng/ml).
The invention discloses an application of anion exchange chromatography penetration mode in separation of anti-human nerve growth factor antibody charge isomers. Compared with the method for separating the antibody charge isomers by cation exchange chromatography through an adsorption-elution mode, the method has the advantages of simplicity and convenience, more thorough removal of alkaline and acidic isomers and higher biological activity of the obtained anti-human nerve growth factor antibody. According to the results of capillary isoelectric focusing, the isoelectric points of main peaks of samples after affinity chromatography, Capto Q and Capto S are all 6.81, and the area percentages of the main peaks are respectively 35.01%, 65.77% and 48.06%. Cell activity assay (inhibition of nerve growth factor activity) results: the IC50 for the post-Capto Q and post-Capto S samples were 102.4ng/mL, 50.3ng/mL, 78.9ng/mL, respectively, meaning that half the amount of nerve growth factor was inhibited, and the post-Capto Q sample was 28.6ng/mL (36.3%) less than the amount required for the post-Capto S sample.
Drawings
FIG. 1 is a liquid phase diagram of the purification of the affinity chromatography of the anti-human nerve growth factor fermentation broth.
FIG. 2 is a liquid phase diagram of the purification of anti-human nerve growth factor by affinity chromatography followed by Capto Q anion exchange chromatography.
FIG. 3 is a liquid phase diagram of the purification of anti-human nerve growth factor by affinity chromatography followed by Capto S cation exchange chromatography.
FIG. 4 is the capillary isoelectric focusing detection diagram after the affinity chromatography of anti-human nerve growth factor fermentation liquor.
FIG. 5 is a capillary isoelectric focusing detection graph of a sample after affinity chromatography of anti-human nerve growth factor fermentation broth and Capto Q anion exchange chromatography.
FIG. 6 is a capillary isoelectric focusing detection graph of a sample after affinity chromatography of anti-human nerve growth factor fermentation broth and then cation exchange chromatography of Capto S.
FIG. 7 is a graph of the cellular biological activity of samples after affinity chromatography of anti-human nerve growth factor antibody and further after chromatography of Capto Q and Capto S.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Recombinant anti-human nerve growth factor antibody CHO cells used in the following examples: the heavy chain sequence of the anti-human nerve growth factor antibody is shown as SEQ ID NO. 1, and the light chain sequence is shown as SEQ ID NO. 2, and the anti-human nerve growth factor antibody can be synthesized artificially; connecting the heavy chain sequence of the anti-human nerve growth factor antibody with a pEE6.4 vector through double enzyme digestion (Hind III/EcoRI) to obtain a heavy chain recombinant vector, connecting the light chain sequence of the anti-human nerve growth factor antibody with a pEE12.4 vector through double enzyme digestion (Hind III/EcoRI) to obtain a light chain recombinant vector, connecting a heavy chain expression frame on the heavy chain recombinant vector with a light chain recombinant vector of the same double enzyme digestion (Not I/Sal I) after double enzyme digestion (Not I/Sal I), and transferring the constructed heavy chain expression frame to a CHO cell to obtain the recombinant anti-human nerve growth factor antibody CHO cell.
Example 1: affinity chromatography of anti-human nerve growth factor antibody
Affinity chromatography column: a5 ml pre-packed Hitrap Mabselect column was purchased from GE Healthcare and the chromatography system was AKTA Purifier.
The recombinant anti-human nerve growth factor antibody CHO cells are cultured in 200mL CD FortiCHO medium at the initial fermentation concentration of 3 multiplied by 106Cell/ml, at 37 ℃ 5% CO2Shaking in incubator at 50rpm for 7 days, centrifuging the culture solution at 4 deg.C and 5000rpm for 10min, collecting supernatant of fermentation broth, and filtering with 0.45 μm membrane; equilibrating affinity chromatography column Hitrap Mabselect 5ml pre-packed column with 30ml phosphate buffer (pH7.0), and then loading the supernatant 35ml of the above fermentation liquid to the column; washing the column with 30ml phosphate buffer (pH7.0), eluting the affinity column with acetate buffer (pH3.2), collecting the elution peak when the absorption value of A280 reaches 20mAU, and stopping collecting when the absorption value of A280 decreases to 20 mAU. The results are shown in FIG. 1 (FIG. 1 is a purified liquid phase diagram of affinity chromatography of anti-human nerve growth factor fermentation broth). As can be seen from FIG. 1, the first peak is the broth penetration peak, containing CHO host proteins, pigments, etc., and this fraction is discarded. The second peak is the peak of eluting anti-human nerve growth factor, and the part with A280 absorption value higher than 20mAU is collected.
The collected solution was adjusted to the desired pH with 2M Tris-Cl buffer (pH9.0) (adjusted to pH5.2 by Capto Q chromatography and 5.0 by Capto S chromatography) and frozen at-20 ℃ until use.
Example 2: capto Q anion exchange chromatography of anti-human nerve growth factor antibody
Anion exchange chromatography column: a Hitrap Capto Q5 ml pre-column was purchased from GE Healthcare and the chromatography system was AKTA Purifier.
The anti-human nerve growth factor antibody feed liquid after the affinity chromatography with the pH adjusted to 5.2 obtained in example 1 is adjusted to the conductivity of 5.2ms/cm by 25mM sodium acetate buffer solution (pH 5.2); equilibrating Capto Q5 ml pre-column with 100ml of 25mM sodium acetate buffer (pH 5.2); loading 5ml of the feed liquid with the adjusted pH and conductivity to a Capto Q chromatographic column, and collecting the flow-through liquid when a penetration peak appears and the absorption value of A280 reaches 20 mAU; after the sample loading is finished, washing the chromatographic column by using 25mM sodium acetate buffer solution (pH5.2), and stopping collecting the flow-through solution when the A280 absorption value is reduced to 20 mAU; then washing the column with 25mM sodium acetate buffer (pH5.2) containing 1M NaCl; the results are shown in FIG. 2 (FIG. 2 is a liquid phase purification of anti-human nerve growth factor by affinity chromatography followed by Capto Q anion exchange chromatography). As can be seen from fig. 2, the first peak is an anti-human nerve growth factor penetration peak, and a fraction with a280 absorption higher than 20mAU was collected. The second peak was an elution peak (component not detected) obtained by washing the column with 25mM sodium acetate buffer (pH5.2) containing 1M NaCl.
The obtained flow-through liquid is collected and frozen at-20 ℃ for later use.
The applicant verified that when the pH adjusted values obtained in example 1 were 4.8, 4.9, 5.0, 5.1, 5.3, 5.5, 5.6, and 5.7, respectively, the main peak areas were found to be 41.63%, 45.22%, 50.38%, 65.77%, 68.21%, 72.62%, 73.16%, and 73.89%, respectively; total protein recovery was 89.50%, 88.12%, 86.0%, 84.28%, 80.32%, 76.57%, 63.93%, 50.27%, respectively. It can be seen that the combined consideration of the area of the main peak and the recovery of total protein is the best when the pH is 5.0-5.5.
Example 3: capto S cation exchange chromatography of anti-human nerve growth factor antibody
Cation exchange chromatography column: a Hitrap Capto S5 ml pre-column was purchased from GE Healthcare and the chromatography system was AKTA Purifier.
The anti-human nerve growth factor antibody feed liquid after the affinity chromatography with the pH adjusted to 5.0 obtained in example 1 is adjusted to the conductivity of 5.0ms/cm by 25mM sodium acetate buffer (pH 5.0); equilibrating the Hitrap Capto S5 ml pre-column with 100ml of 25mM sodium acetate buffer (pH 5.0); 5ml of the feed liquid with the adjusted pH and conductivity is loaded to a Capto S chromatographic column; after the sample loading is finished, washing the chromatographic column by using 30ml of 25mM sodium acetate buffer solution (pH5.0), after the absorption value of A280 is leveled, carrying out gradient elution by using 25mM sodium acetate buffer solution (pH5.0) as solution A and 25mM sodium acetate buffer solution (pH5.0) containing 0.5M NaCl as solution B, carrying out gradient elution for 100min from 100% solution A to 100% solution B, starting to collect an elution peak when the absorption value of A280 reaches 20mAU, and stopping collection when the absorption value of A280 is reduced to 20 mAU; the results are shown in FIG. 3 (FIG. 3 is a liquid phase diagram of the purification of anti-human nerve growth factor by affinity chromatography followed by Capto S cation exchange chromatography). As can be seen from FIG. 3, the peak of elution of anti-human nerve growth factor appeared during gradient elution, and the fraction with A280 absorption value higher than 20mAU was collected.
The samples obtained were collected and frozen at-20 ℃ until use.
Example 4: capillary isoelectric focusing (cIEF) detection of anti-human nerve growth factor antibodies
The capillary electrophoresis apparatus manufacturer is AB Sciex with the model of P \ ACE MDQ Plus; the coating capillary tube manufacturer is Agilent; ampholyte manufacturer GE Healthcare; the cIEF Peptide Marker manufacturer is Genscript, and the isoelectric point range is 10.0-4.1; the 10KD ultrafiltration membrane manufacturer is Millipore.
Sample concentration and desalting treatment of the affinity chromatography feed (obtained in example 1), Capto Q permeate (obtained in example 2) and Capto S eluate (obtained in example 3): taking a 10KD ultrafiltration membrane, adding 400uL of 20mM Tris (pH8.0) solution, centrifuging at the rotation speed of 14000rpm at 15 ℃ for 15min, discarding the lower filtrate, and repeating the steps until the concentration of the sample is 2.5mg/mL by using an ultraviolet spectrophotometer; taking 60ug of concentrated sample, adding 10KD of ultrafiltration membrane upper layer, adding 400uL of 10mM Tris (pH8.0) solution, rotating at 14000rpm, centrifuging at 15 deg.C for 15min, and discarding lower layer filtrate; adding 400uL of 20mM Tris (pH8.0) solution into the upper layer of the 10KD ultrafiltration membrane, centrifuging at the rotating speed of 14000rpm and the temperature of 15 ℃ for 15min, discarding filtrate at the lower layer, repeating twice, and taking out all samples at the upper layer of the membrane for later use.
cIEF sample preparation: 200uL of 3M Urea-cIEF Gel,12uL ampholyte, 20uL cathodically stabilized solution (500mM arginine), 2.0uL anodically stabilized solution (20mM phosphoric acid), pI Markers standards, each at 2.0uL, were mixed thoroughly: adding the desalted sample, fully mixing and loading. The cIEF separation process is divided into two steps: focusing and migration. In the focusing process, voltage is applied to two ends of the capillary tube, a pH gradient is formed in the capillary tube under the action of ampholyte, after the gradient is formed, pI markers and separated samples are detected by a chemical migration method of acetic acid, the pI value of the sample to be detected is determined according to the linear relation of the pH gradient, an ultraviolet detector with the wavelength of 280nm is used for detection, and data are processed by 32Karat software.
The results are shown in Table 1 and FIGS. 4-6. Wherein, FIG. 4 is the capillary isoelectric focusing detection diagram after the anti-human nerve growth factor fermentation liquor affinity chromatography. FIG. 5 is a capillary isoelectric focusing detection graph of a sample after affinity chromatography of anti-human nerve growth factor fermentation broth and Capto Q anion exchange chromatography. FIG. 6 is a capillary isoelectric focusing detection graph of a sample after affinity chromatography of anti-human nerve growth factor fermentation broth and then cation exchange chromatography of Capto S. As can be seen from Table 1 and FIGS. 4-6, the isoelectric points of the purified substances obtained by different separation methods are all 6.81, and on the area of the main peak, the isoelectric point of the purified substances is only 35.01% after affinity chromatography, the isoelectric point of the purified substances is 65.77% after affinity chromatography and is the highest after anion exchange chromatography, and the isoelectric point of the purified substances is 48.06% after affinity chromatography and cation exchange chromatography, so that the separation efficiency of the antihuman nerve growth factor fermentation broth after affinity chromatography and anion exchange chromatography is the highest and is as high as 65.77%, which is far greater than the effect of cation exchange chromatography.
TABLE 1 table of isoelectric points and percent of main peak area of samples determined by cIEF
Example 5: detection of cell biological activity of anti-human nerve growth factor antibody
TF-1 cell line (purchased from Shanghai Bailey Biotechnology Co., Ltd.) was cultured in RPMI1640 containing 10% fetal bovine serum at 37 ℃ under 5% carbon dioxide, the cell concentration was controlled to 1ml containing 1.0X 10E 5-7.0X 10E5 cells, and the cell line was used for biological activity measurement 24-36 hours after passage.
The above-mentioned affinity chromatography, Capto Q, Capto S anti-human nerve growth factor antibodies were added to 96-well plates at an initial concentration of 1000ng/ml, respectively, and diluted in a basal medium (RPMI 1640 containing 10% fetal bovine serum) at an equal ratio gradient of 1:3 volume, maintaining the medium volume per well at 100 μ l, followed by addition of 8ng of recombinant human nerve growth factor to a final concentration of 40ng/ml to each test well. Mixing, and placing in incubator.
Remove appropriate amount of 5 × 10E4 cells per wellTF-1 cells were post-centrifuged and the cells were washed three times with RPMI1640 resuspension to replace the original serum-containing medium. After washing, 100. mu.l of a medium containing 5X 10E4 TF-1 cells was added to each well of a 96-well plate to which a test article was added, mixed well, and cultured at 37 ℃ under 5% carbon dioxide for 72 hours. After 72 hours, 20. mu.l of MTS solution was added to each well, and the reaction was carried out at 37 ℃ under 5% carbon dioxide for 2 hours, followed by measuring the absorbance at a wavelength of 490nm and recording the measurement result. The data were processed by four-parameter regression method, and the data analysis results are shown in FIG. 7, the cell activity assay curve (y: 0.14549+ (1.46313-0.14549)/(1+ (x/102.38897) ^1.68317), R) of the samples after affinity chromatography20.9985. Cell activity assay curve of sample after Capto Q chromatography (y ═ 0.13+ (1.43986-0.13)/(1+ (x/50.35025) ^2.18332), R20.9977. Cell viability assay Curve (y-0.13799 + (1.44771-0.13799)/(1+ (x/78.94306) ^1.63991), R for samples after Capto S chromatography20.9996. The cell activity IC50 of the anti-human nerve growth factor antibody after affinity chromatography is calculated to be 102.4ng/ml, the cell activity IC50 of the anti-human nerve growth factor antibody after Capto Q anion exchange chromatography is 50.3ng/ml, and the cell activity IC50 of the anti-human nerve growth factor antibody after Capto S cation exchange chromatography is 78.9 ng/ml. It can be seen that the cell activity of the anti-human nerve growth factor antibody is the highest after the Capto Q anion exchange chromatography of the invention is adopted, and is far higher than the cell activity after Capto S cation exchange chromatography or without affinity chromatography.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
SEQUENCE LISTING
<110> Unfamous biomedical Co., Ltd
<120> method for separating charge isomer of anti-human nerve growth factor antibody
<130> WMSW-18007-CNI
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1391
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<213> Artificial Synthesis
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aaggtgtcgg cttcaccctg accgagctgt ccatccactg ggtgaggcag gcccctggaa 180
agggcctgga gtggatgggc ggcttcgatc ccgaggatgg cgagaccatc tacgcccaga 240
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ttgccctcct tgtcctgccc ctgagttcct gggcggcccc tcgtgttcct gttccccccc 780
aagcccaagg acaccctgat gatctccagg acccctgagg tgacctgcgt ggtggtggac 840
gtgtcccagg aggaccccga ggtgcagttc aactggtacg tggacggcgg gaggtgcaca 900
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Claims (9)
1. A method for separating the electric charge isomer of anti-human nerve growth factor antibody is characterized by comprising the following steps,
affinity chromatography: performing affinity chromatography column chromatography on cell fermentation liquid containing the anti-human nerve growth factor antibody, and collecting A280 to obtain an elution peak; adjusting the pH of the collected elution peak to 5.0-5.50; the conductance is adjusted to 4.90-5.10 ms/cm;
anion exchange chromatography: after passing through an anion exchange chromatography column, collecting flow-through liquid when a penetration peak appears and the A280 absorption value reaches 20mAU, wherein the collected flow-through liquid is the antihuman nerve growth factor after the charge isomers are removed;
the heavy chain sequence of the anti-human nerve growth factor antibody is shown as SEQ ID NO. 1, and the light chain sequence is shown as SEQ ID NO. 2.
2. The method for separating a charge isomer of an anti-human nerve growth factor antibody according to claim 1, wherein the cell fermentation broth containing the anti-human nerve growth factor antibody is a fermentation broth of recombinant anti-human nerve growth factor antibody CHO cells.
3. The method for separating charge isomers of an anti-human nerve growth factor antibody according to claim 1, wherein the pH adjustment is performed using a sodium acetate buffer.
4. The method for separating charge isomers of an anti-human nerve growth factor antibody according to claim 3, wherein the pH is adjusted to 5.2.
5. The method for separating charge isomers of an anti-human nerve growth factor antibody according to claim 1, wherein the adjustment of the electric conductance is performed by using a sodium acetate buffer solution having a pH of 5.0 to 5.50.
6. The method for separating charge isomers of an anti-human nerve growth factor antibody according to claim 1, wherein the anion exchange chromatography step comprises the steps of equilibrating 15 to 25 column volumes of an anion exchange chromatography column with 25mM sodium acetate buffer solution having a pH of 5.00 to 5.50; loading the feed liquid with the adjusted pH and conductivity to an anion exchange chromatographic column, and collecting the flow-through liquid when a penetration peak appears and the A280 absorption value reaches 15-25 mAU; after the sample loading is finished, washing the chromatographic column by using 25mM sodium acetate buffer solution with pH of 5.00-5.50, and when the absorption value of A280 is reduced to 15-25 mAU; stopping collecting the flow-through liquid, wherein the collected flow-through liquid is the antihuman nerve growth factor after the charge isomers are removed.
7. The method for separating charge isomers of an anti-human nerve growth factor antibody according to claim 6, wherein the anion exchange chromatography column is equilibrated with 25mM sodium acetate buffer solution having a pH of 5.00 to 5.50 for 20 column volumes.
8. The method of claim 6, wherein the pH and conductivity of the feed solution are adjusted and the flow through is started when the breakthrough peak occurs and the A280 absorption reaches 20 mAU.
9. The method for separating the charged isomers of the anti-human nerve growth factor antibody according to claim 6, wherein after the sample loading is completed, the chromatography column is washed with 25mM sodium acetate buffer solution with pH5.00-5.50, when the A280 absorption value is reduced to 20mAU, the flow-through solution is stopped to be collected, and the collected flow-through solution is the anti-human nerve growth factor after the charged isomers are removed.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928341A (en) * | 2010-06-30 | 2010-12-29 | 深圳科兴生物工程有限公司 | Purifying process for separating isomers of recombined human interferon alpha1b and detection method thereof |
| CN105175548A (en) * | 2015-08-13 | 2015-12-23 | 齐鲁制药有限公司 | Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
| CN108059676A (en) * | 2017-12-28 | 2018-05-22 | 未名生物医药有限公司 | A kind of anti human nerve growth factor scFv antibody and preparation method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928341A (en) * | 2010-06-30 | 2010-12-29 | 深圳科兴生物工程有限公司 | Purifying process for separating isomers of recombined human interferon alpha1b and detection method thereof |
| CN105175548A (en) * | 2015-08-13 | 2015-12-23 | 齐鲁制药有限公司 | Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
| CN108059676A (en) * | 2017-12-28 | 2018-05-22 | 未名生物医药有限公司 | A kind of anti human nerve growth factor scFv antibody and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone;B Houston;《J Endocrinol》;19900531;第125卷(第2期);全文 * |
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