CN114316019A - Method for preparing PEG (polyethylene glycol) -modified IL-2 through ion exchange chromatography - Google Patents
Method for preparing PEG (polyethylene glycol) -modified IL-2 through ion exchange chromatography Download PDFInfo
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Abstract
The invention relates to the technical field of protein modification, and particularly discloses a method for preparing PEG (polyethylene glycol) -modified IL-2 by ion exchange chromatography. The method for preparing the PEG-modified IL-2 through the ion exchange chromatography adopts the anion exchange chromatography to perform PEG solid phase modification on the IL-2, and then uses eluent containing surfactant to perform elution to obtain the PEG-modified IL-2. The method for preparing the PEG-modified IL-2 by the ion exchange chromatography provided by the invention uses the traditional anion exchange filler as a solid support, and then completely changes the traditional pH elution or high-salt elution method, thereby realizing the PEG solid phase modification of the IL-2 molecule based on the ion exchange chromatography, the operation process is simple, and the activity of the obtained PEG-modified IL-2 molecule is not obviously different from that of the modified molecule obtained by the liquid phase modification method.
Description
Technical Field
The invention relates to the technical field of protein modification, in particular to a method for preparing PEG-modified IL-2 by ion exchange chromatography.
Background
Interleukin-2 (IL-2) is an important cytokine in humans, which plays an important role in the proliferation and differentiation of T cells. At present, high-dose IL-2 has been approved by pristinate ministry in tumor clinical treatment, and the role of low-dose IL-2 in diseases such as systemic lupus erythematosus and the like is also proved in clinical research. However, due to the narrow safety window of IL-2, severe adverse reactions such as pulmonary edema and gastrointestinal hemorrhage are easily caused especially under high dosage. Adverse reactions occur mainly in relation to affinity preferences of IL-2 and its receptor different subunits. Meanwhile, the IL-2 has small molecular weight, can be quickly eliminated by glomerular filtration in vivo, and has a half-life period of only 7-15min in vivo, so that the development of the IL-2 molecule with long-acting effect and low adverse reaction has important significance.
At present, the long-acting property of IL-2 is mainly improved by a method of modifying IL-2 by PEG, and the method is divided into random modification and fixed-point modification according to different modification sites of PEG in IL-2 molecules. The modification methods all adopt a liquid phase modification method, namely, the modification reaction occurs in a liquid phase environment. And purifying the IL-2 molecule modified by PEG by a chromatographic technique after the modification is finished. The purification process of PEG modified IL-2 generally needs to obtain modified molecules with higher purity through a plurality of steps such as concentration, solution changing, ion exchange chromatography, gel filtration chromatography and the like, and the process is complicated and the yield is low. In addition, due to the poor stability of PEG-modified IL-2, a certain amount of SDS was required to be added to the system as a stabilizer. SDS as an ionic detergent can be combined with IL-2 through hydrophobic interaction to enable molecules to carry negative charges, and then aggregation of PEG modified IL-2 molecules is inhibited through the action of charge repulsion of the same species, so that precipitation and denaturation of the modified molecules are prevented. However, once the SDS concentration in the system is reduced or removed, irreversible aggregate denaturation of the IL-2 molecules occurs.
Due to the weak hydrophobicity of the PEG modified IL-2 molecule, the hydrophobic filler is bound in a high-salt environment, but SDS is separated out in the high-salt environment, so that the hydrophobic chromatography is not suitable for the PEG modification of the IL-2 molecule. Meanwhile, since SDS is negatively charged under all pH conditions, the cation exchange chromatography system also fails to accomplish the capture of IL-2 molecules. Therefore, only anion exchange chromatography can be used to capture the IL-2 molecules, but due to the strongly negative charge nature of SDS stably bound to solid phase fillers, elution of the captured molecules cannot be achieved with the high salt used in ion exchange and with elution modes that change pH. Therefore, conventional solid phase modification methods based on ion exchange chromatography or hydrophobic chromatography are not suitable for solid phase modification with IL-2 due to the presence of SDS.
Disclosure of Invention
Aiming at the problems of the existing PEG modified IL-2 preparation method, the invention provides a method for preparing PEG modified IL-2 by ion exchange chromatography, which realizes PEG solid phase modification of IL-2 molecules based on ion exchange chromatography, and the obtained PEG modified IL-2 molecules are easy to elute and purify, PEG modified IL-2 with high yield and high purity is easy to obtain for analysis, the operation method is simple, and the activity of the obtained PEG modified IL-2 for analysis is not obviously different from that of a liquid phase modification method.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method of preparing PEG-modified IL-2 by ion exchange chromatography comprising: and (3) performing PEG solid phase modification of the IL-2 by adopting an anion exchange chromatography method, and then performing elution by using an eluent containing a surfactant to obtain the PEG modified IL-2.
Compared with the prior art, the method for preparing the PEG-modified IL-2 by the ion exchange chromatography provided by the invention uses the traditional anion exchange filler as a solid support, then completely changes the traditional pH elution or high-salt elution method, adopts the surfactant for elution, changes the hydrophobic effect between SDS and the PEG-modified IL-2 molecules in the elution process, and ensures that the PEG-modified IL-2 molecules can be smoothly eluted. The method realizes PEG solid phase modification of the IL-2 molecule based on ion exchange chromatography, and the PEG modified IL-2 molecule obtained by elution can obtain high-purity modified molecules only by one-step gel filtration chromatography, so that compared with the conventional liquid phase method, the method saves a large number of experimental steps, and the activity of the obtained PEG modified IL-2 molecule (PEG-IL2) is not obviously different from that of the modified molecules obtained by the liquid phase modification method.
Preferably, the solid phase modification method is as follows: sequentially adding equilibrium liquid containing IL-2 and equilibrium liquid containing mPEG-SC (monomethoxy polyethylene glycol succinimide carbonate) into the anion exchange chromatographic column after equilibrium liquid is balanced to perform a solid phase modification process; wherein the retention time of the equilibrium solution containing IL-2 after being added into an anion exchange chromatographic column is more than or equal to 2 min; the retention time of the equilibrium liquid containing mPEG-SC after being added into an anion exchange chromatographic column is more than or equal to 0.5 h. The concentration of IL-2 and mPEG-SC in the equilibration fluid may be such that IL-2 and mPEG-SC are dissolved.
Preferably, the equilibration process by adding equilibration fluid to the chromatography column is carried out between the addition of equilibration fluid comprising IL-2, the addition of equilibration fluid comprising mPEG-SC, and the addition of eluent.
Preferably, the molar ratio of the IL-2 to the mPEG-SC is 1: 5-40.
Preferably, the equilibration solution is phosphate buffer containing SDS.
Preferably, the phosphate buffer in the equilibration solution is PB buffer, the concentration of SDS is 0.01 wt% -0.1 wt%, and the concentration of PB buffer is 10mM-50 mM.
Preferably, the eluent is phosphate buffer containing surfactant.
Preferably, the phosphate buffer in the eluent is a PB buffer, the concentration of the surfactant is 0.1-2 wt%, and the concentration of the PB buffer is 10-50 mM.
Further preferably, the concentration of the surfactant is 0.1 wt% to 1 wt%.
Preferably, the surfactant is one of a nonionic surfactant and a cationic surfactant.
The selection of the nonionic surfactant and the cationic surfactant can further improve the elution efficiency of the PEG-IL2, and further obtain the PEG-IL2 with high purity and high yield.
Preferably, the nonionic surfactant is Triton X-100 or Tween-20.
Preferably, the cationic surfactant is CTAB.
Drawings
FIG. 1 is an electrophoretic image of a chromatography liquid obtained in example 1 of the present invention at various stages;
FIG. 2 is an electrophoretic image of a chromatography liquid obtained in example 2 of the present invention at various stages;
FIG. 3 is an electrophoretic image of a chromatographic solution at various stages obtained in example 3 of the present invention;
FIG. 4 is an electrophoretic image of a chromatographic solution at various stages obtained in example 4 of the present invention;
FIG. 5 is an electrophoretic image of a chromatography liquid obtained in comparative example 1 of the present invention at various stages;
FIG. 6 is an electrophoretic image of the chromatography liquid of different stages obtained in comparative example 2 of the present invention, in which the packing: directly taking out the filler for electrophoresis detection.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A method for preparing PEG-modified IL-2 by ion exchange chromatography comprising the steps of:
a. preparing a relevant solution:
balance liquid: SDS was added to 20mM PB buffer (pH 8) to adjust the concentration of SDS to 0.05 wt%, thereby obtaining an equilibrium solution.
Eluent: triton X-100 was added to 20mM PB buffer to give a Triton X-100 concentration of 0.1 wt%, to obtain an eluate.
Equilibrium containing IL-2: IL-2 is added to the equilibrium solution to completely dissolve IL-2.
Equilibrium liquid containing mPEG-SC (modification liquid): to the above-mentioned equilibrium solution, mPEG-SC (monomethoxypolyethylene glycol succinimide carbonate) was added to completely dissolve the mPEG-SC.
b. Solid phase modification:
fully balancing a Q-FF prepacked column (HiTrap Q FF, GE Healthcare) by using a 10-column volume of balancing liquid, then loading a sample by using the balancing liquid containing IL-2, keeping the sample for 2min, balancing by using 5-column volume of balancing liquid after the sample loading is finished, and then modifying by using the balancing liquid (modifying liquid) containing mPEG-SC, and keeping for 5 h; wherein the molar ratio of the IL-2 for loading to the mPEG-SC for modification is 1: 5.
c. Elution and purification
And after the solid phase modification process is finished, washing the column by using 5 column volumes of equilibrium solution, then adding the eluent for elution, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
Electrophoresis is used for detecting the modification effect of the modified molecules, the detection result is shown in figure 1, IL-2 can be effectively modified on the solid phase carrier, and the modified molecules can be fully eluted by combining the eluent.
Example 2
A method for preparing PEG-modified IL-2 by ion exchange chromatography comprising the steps of:
a. preparing a relevant solution:
balance liquid: SDS was added to 10mM PB buffer (pH 8) to adjust the concentration of SDS to 0.01 wt%, thereby obtaining an equilibrium solution.
Eluent: tween-20 was added to 10mM PB buffer to give a Tween-20 concentration of 1 wt%, giving an eluate.
Equilibrium containing IL-2: IL-2 is added to the equilibrium solution to completely dissolve IL-2.
Equilibrium liquid containing mPEG-SC (modification liquid): to the above-mentioned equilibrium solution, mPEG-SC (monomethoxypolyethylene glycol succinimide carbonate) was added to completely dissolve the mPEG-SC.
b. Solid phase modification:
fully balancing a Q-FF prepacked column (HiTrap Q FF, GE Healthcare) by using 10 column volumes of balancing liquid, then loading the sample by using the balancing liquid containing IL-2, keeping the sample for 4min, balancing by using 5 column volumes of balancing liquid after the sample loading is finished, and then modifying by using the balancing liquid containing mPEG-SC (modifying liquid), and keeping for 1 h; wherein the molar ratio of the IL-2 for loading to the mPEG-SC for modification is 1: 20.
c. elution and purification
And after the solid phase modification process is finished, washing the column by using 5 column volumes of equilibrium solution, then adding the eluent for elution, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
And (3) detecting the modification effect of the modified molecules by electrophoresis, wherein the detection result is shown in figure 2, IL-2 can be effectively modified on the solid phase carrier, and the modified molecules can be sufficiently eluted by combining the eluent.
Example 3
A method for preparing PEG-modified IL-2 by ion exchange chromatography comprising the steps of:
a. preparing a relevant solution:
balance liquid: SDS was added to 50mM PB buffer (pH 8) to adjust the concentration of SDS to 0.1 wt%, thereby obtaining an equilibrium solution.
Eluent: CTAB was added to 50mM PB buffer solution so that the concentration of CTAB became 2 wt%, and an eluate was obtained.
Equilibrium containing IL-2: IL-2 is added to the equilibrium solution to completely dissolve IL-2.
Equilibrium liquid containing mPEG-SC (modification liquid): to the above-mentioned equilibrium solution, mPEG-SC (monomethoxypolyethylene glycol succinimide carbonate) was added to completely dissolve the mPEG-SC.
b. Solid phase modification:
fully balancing a Q-FF prepacked column (HiTrap Q FF, GE Healthcare) by using 10 column volumes of balancing liquid, then loading the sample by using the balancing liquid containing IL-2, keeping the sample for 10min, balancing by using 5 column volumes of balancing liquid after loading is finished, and then modifying by using the balancing liquid (modifying liquid) containing mPEG-SC, wherein the keeping time is 0.5 h; wherein the molar ratio of the IL-2 for loading to the mPEG-SC for modification is 1: 40.
c. elution and purification
And after the solid phase modification process is finished, washing the column by using 5 column volumes of equilibrium solution, then adding the eluent for elution, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
Electrophoresis is used for detecting the modification effect of the modified molecules, the detection result is shown in figure 3, IL-2 can be effectively modified on the solid phase carrier, and the modified molecules can be fully eluted by combining the eluent.
Example 4
A high purity PEG-IL2 molecule was obtained by using a DEAE pre-packed column (HiTrap DEAE FF, GEHealthcare) in place of the Q-FF pre-packed column of example 1, and the procedure and parameters were the same as those of example 1.
Electrophoresis detection modified molecule PEG-IL2 modification effect, detection results as shown in figure 4, IL-2 can be effectively modified on solid phase carrier, and the modified molecule can be sufficiently eluted by combining with specific eluent.
Comparative example 1
The hydrophobic chromatography solid phase modification of IL-2 is carried out by using a Hitrap Phanyl prepacked column (Hitrap phenyl FF, GEHealthcare) instead of a Q-FF prepacked column, and the specific method is as follows:
a. preparing a relevant solution:
balance liquid: SDS and ammonium sulfate were added to 10mM PB buffer (pH 8) so that the concentration of SDS became 0.01 wt% and the concentration of ammonium sulfate became 200mM, thereby obtaining an equilibrium solution.
Eluent: SDS was added to 10mM PB buffer solution so that the concentration of SDS became 1 wt%, to obtain an eluate.
Equilibrium containing IL-2: IL-2 is added to the equilibrium solution to completely dissolve IL-2.
Equilibrium liquid containing mPEG-SC (modification liquid): to the above-mentioned equilibrium solution, mPEG-SC (monomethoxypolyethylene glycol succinimide carbonate) was added to completely dissolve the mPEG-SC.
b. Solid phase modification:
after the Hitrap Phanyl pre-packed column (HiTrap phenyl FF, GEHealthcare) was sufficiently equilibrated with 10 column volumes of the equilibration solution, the sample was applied with the IL-2-containing equilibration solution, the sample was retained for 4min, and after the application was completed, the sample was equilibrated with 5 column volumes of the equilibration solution, and then modified with the mPEG-SC-containing equilibration solution (modification solution) for 1h, and the molar ratio of IL-2 to mPEG-SC in the system was 1: 20.
c. Elution and purification
And after the solid phase modification process is finished, washing the column by using 5 column volumes of equilibrium solution, then adding the eluent for elution, adding 5X loading buffer into the eluted sample, and performing denaturation at 95 ℃ for 5min to obtain the sample.
The effect of modification in the sample was detected by electrophoresis, and the result is shown in FIG. 5, in which IL-2, although normally bound to the Hitrap Phanyl filler, was eluted by PEG during the modification process, resulting in failure of solid phase modification.
Comparative example 2
The eluate of example 1 was replaced with a high-salt eluate (20mM PBS, 0.05% SDS, 1M NaCl) for conventional anion exchange chromatography, and the other methods and parameters were the same as those of example 1, to obtain an eluted sample.
Electrophoresis detection is carried out on the obtained elution sample, the detection result is shown in figure 6, IL-2 can be effectively modified on a solid phase carrier, but the modified molecules are stably combined with a filler, and the conventional anion exchange eluent can not effectively elute the modified molecules.
Test example 1
Taking CTLL-2 cells in logarithmic growth phase of subculture 24h, and adjusting the concentration of the CTLL-2 cells to 1 × 10 by using a culture solution5and/mL. Then, the cell fluid of the above-mentioned specific concentration was inoculated into 96-well culture plates, and 0.1mL of the cell fluid was inoculated per well. The modified sample (sample to be tested) obtained in example 1-4 and the IL-2 standard were diluted in multiple ratios, and then 0.1mL of the sample to be tested or the IL-2 standard was added to each well, and 3 wells were provided for each dilution concentration. A control well of culture medium (100. mu.L of cells + 100. mu.L of culture medium) was provided. The above plates were incubated at 37 ℃ with 5% CO2Incubated for 40h under the conditions of (1).Pipetting 100. mu.L of supernatant, adding 100. mu.L of MTT (1mg/mL), 37 ℃ and 5% CO2Incubate for 2 h. And measuring the light absorption value by using an enzyme-labeled measuring instrument at the wavelength of 570nm, and recording the measurement result.
The test data is processed by a computer program or a four-parameter regression calculation method, and the result is calculated according to the following formula:
in the formula PrThe biological activity of the standard substance is IU/ml; dsPre-diluting for the sample; drPre-diluting the standard substance by multiple times; esThe dilution factor is half effective amount of the standard substance for the test substance; erIs the dilution multiple of the half-effect amount of the standard product.
The calculated activity values of the samples to be tested are shown in table 1.
TABLE 1 sample Activity values
As is clear from the activity values measured in Table 1, the activity of PEG-IL2 obtained by solid phase modification in examples 1 to 4 was substantially equivalent to that of PEG-IL2 obtained by liquid phase modification.
Test example 2
The PEG-IL2 samples obtained by solid phase modification in examples 1-4 were assayed for protein purity by liquid phase assay. Measuring OD280 reading by using an ultraviolet-visible spectrophotometer, dividing by an extinction coefficient to obtain the concentration of the modified molecules, multiplying by the volume to calculate the total amount, and dividing by the total amount of IL-2 added before modification to obtain the yield, wherein the measurement and calculation results are shown in Table 2.
Note: wherein the PEG-IL2 obtained by liquid phase modification is specifically selected from the group consisting of "NANDINI V.KATRE", et al (1987), "Chemical modification of recombinant interleukin 2 by polyethylene glycol secretion sites in the hormone Meth A sarcoma model.Proc Natl Acad Sci U.S. 1987; 84(6) 1487-91, by the liquid phase modification method disclosed in.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for preparing PEG-modified IL-2 by ion exchange chromatography, characterized in that: the method comprises the following steps: and (3) performing PEG solid phase modification of the IL-2 by adopting an anion exchange chromatography, and then eluting by using an eluent containing a surfactant to obtain the PEG modified IL-2.
2. The method of claim 1 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the solid phase modification method comprises the following steps: and (3) adding the equilibrium solution containing IL-2 and the equilibrium solution containing mPEG-SC into the anion exchange chromatographic column after equilibrium solution equilibrium to perform a solid phase modification process.
3. The method of claim 2 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the molar ratio of the IL-2 to the mPEG-SC is 1: 5-40.
4. The method of claim 2 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the equilibrium solution is a phosphate buffer solution containing SDS.
5. The method of claim 4 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the phosphate buffer solution in the equilibrium solution is a PB buffer solution, and the concentration of SDS is 0.01 wt% -0.1 wt%.
6. The method of claim 1 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the eluent is phosphate buffer solution containing surfactant.
7. The method of claim 6 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the phosphate buffer solution in the eluent is a PB buffer solution, and the concentration of the surfactant is 0.1-2 wt%.
8. The method of preparing PEG-modified IL-2 by ion exchange chromatography according to any one of claims 1, 6, 7, wherein: the surfactant is at least one of a nonionic surfactant and a cationic surfactant.
9. The method of claim 8 for preparing PEG-modified IL-2 by ion exchange chromatography, wherein: the non-ionic surfactant is Triton X-100 or Tween-20;
and/or the cationic surfactant is CTAB.
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Citations (5)
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| CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| CN102485742A (en) * | 2010-12-02 | 2012-06-06 | 山东新时代药业有限公司 | Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor |
| CN105802947A (en) * | 2014-12-29 | 2016-07-27 | 江苏众红生物工程创药研究院有限公司 | Method for modification of proteins with PEG and purification of modified products |
| CN106632588A (en) * | 2015-08-27 | 2017-05-10 | 江苏众红生物工程创药研究院有限公司 | A purifying process for polyethylene glycol modified protein |
| CN111212661A (en) * | 2018-09-11 | 2020-05-29 | 润俊(中国)有限公司 | Interleukin-2 polypeptide conjugates and uses thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| CN102485742A (en) * | 2010-12-02 | 2012-06-06 | 山东新时代药业有限公司 | Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor |
| CN105802947A (en) * | 2014-12-29 | 2016-07-27 | 江苏众红生物工程创药研究院有限公司 | Method for modification of proteins with PEG and purification of modified products |
| CN106632588A (en) * | 2015-08-27 | 2017-05-10 | 江苏众红生物工程创药研究院有限公司 | A purifying process for polyethylene glycol modified protein |
| CN111212661A (en) * | 2018-09-11 | 2020-05-29 | 润俊(中国)有限公司 | Interleukin-2 polypeptide conjugates and uses thereof |
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