CN114316019B - Method for preparing PEG modified IL-2 by ion exchange chromatography - Google Patents
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Abstract
The invention relates to the technical field of protein modification, and particularly discloses a method for preparing PEG modified IL-2 through ion exchange chromatography. The method for preparing the PEG modified IL-2 by ion exchange chromatography adopts an anion exchange chromatography method to carry out PEG solid phase modification of the IL-2, and then uses eluent containing a surfactant to carry out elution to obtain the PEG modified IL-2. The method for preparing the PEG modified IL-2 by 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 ion exchange chromatography, having simple operation process, and having no obvious difference between the activity of the obtained PEG modified IL-2 molecule and the modified molecule obtained by a 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 that plays an important role in the proliferation and differentiation of T cells. At present, high-dose IL-2 is approved by the regulatory authorities in clinical treatment of tumors, and the effect of low-dose IL-2 in diseases such as systemic lupus erythematosus and the like is also confirmed in clinical researches. However, due to the narrow safety window of IL-2, serious adverse reactions such as pulmonary edema and gastrointestinal hemorrhage are easy to cause especially at high doses. Adverse reactions occur primarily in association with affinity preferences of IL-2 and its receptor for different subunits. Meanwhile, because the molecular weight of the IL-2 is smaller, the IL-2 can be quickly cleared by glomerular filtration in vivo, and the half-life in vivo is only 7-15min, so that the development of the IL-2 molecule with long-acting and low adverse reaction has important significance.
At present, the long-acting property of IL-2 is improved mainly by a method of modifying IL-2 by PEG, and the modification is divided into random modification and fixed-point modification according to different modification sites of the PEG in IL-2 molecules. The modification methods all adopt liquid phase modification methods, namely, modification reaction occurs in a liquid phase environment. And purifying the PEG modified IL-2 molecule by chromatography after the modification is finished. The purification process of PEG modified IL-2 molecules generally needs to obtain modified molecules with higher purity through a plurality of steps such as concentration liquid exchange, ion exchange chromatography, gel filtration chromatography and the like, and has the advantages of complex process and low yield. In addition, due to the poor stability of PEG-modified IL-2, it is necessary to add a certain amount of SDS as a stabilizer to the system. SDS as an ionic detergent can combine with IL-2 through hydrophobic action to make the molecule negatively charged, and further inhibit aggregation of PEG modified IL-2 molecule through the action of homogeneous charge repulsion to prevent modified molecule from precipitating and denaturing. However, once the SDS concentration in the system is reduced or removed, irreversible aggregation denaturation of IL-2 molecules occurs.
Because of the relatively weak hydrophobicity of PEG-modified IL-2 molecules, hydrophobic fillers need to be bound in high salt environments, but SDS can precipitate in high salt environments, rendering hydrophobic chromatography unsuitable for PEG modification of IL-2 molecules. Meanwhile, since SDS is negatively charged under all pH conditions, the cation exchange chromatography system cannot complete the capture of IL-2 molecules. Therefore, only anion exchange chromatography can be used to capture IL-2 molecules, but neither the high salt used in ion exchange nor the pH-altering elution mode can achieve elution of the capture molecules due to the strongly negative charge nature of SDS which forms a stable bond with the solid-phase packing. Therefore, due to the presence of SDS, conventional solid phase modification methods based on ion exchange chromatography or hydrophobic chromatography are not suitable for solid phase modification with IL-2.
Disclosure of Invention
The invention provides a method for preparing PEG modified IL-2 by ion exchange chromatography, which aims at the problems of the existing preparation method of the PEG modified IL-2, realizes the PEG solid-phase modification of the IL-2 molecule based on the ion exchange chromatography, and the obtained PEG modified IL-2 molecule is easy to elute and purify, and is easy to obtain high-yield and high-purity PEG modified IL-2 analysis, the operation method is simple, and the activity of the obtained PEG modified IL-2 analysis is not obviously different from that of a liquid phase modification method.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
A method of preparing PEG-modified IL-2 by ion exchange chromatography, comprising: and carrying out PEG solid phase modification of the IL-2 by adopting an anion exchange chromatography method, and then eluting 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 ion exchange chromatography provided by the invention uses the traditional anion exchange filler as a solid support, then completely changes the traditional method of pH elution or high-salt elution, adopts a surfactant for elution, and changes the hydrophobic effect between SDS and the PEG modified IL-2 molecule in the elution process, so that the PEG modified IL-2 molecule can be smoothly eluted. The method realizes the PEG solid phase modification of the IL-2 molecule based on ion exchange chromatography, and the eluted PEG modified IL-2 molecule can obtain high-purity modified molecules through one-step gel filtration chromatography, compared with the conventional liquid phase method, a large number of experimental steps are saved, and the activity of the obtained PEG modified IL-2 molecule (PEG-IL 2) is not obviously different from that of the modified molecules obtained by the liquid phase modification method.
Preferably, the method for solid phase modification is as follows: sequentially adding a balancing solution containing IL-2 and a balancing solution containing mPEG-SC (monomethoxy polyethylene glycol succinimidyl carbonate) into the anion exchange chromatographic column after balancing the balancing solution for carrying out a solid phase modification process; wherein the retention time of the balance liquid containing IL-2 after being added into the anion exchange chromatographic column is more than or equal to 2min; the retention time of the balance liquid containing mPEG-SC after being added into an anion exchange chromatographic column is more than or equal to 0.5h. The concentration of IL-2 and mPEG-SC in the equilibration solution is sufficient to solubilize the IL-2 and mPEG-SC.
Preferably, equilibration by addition of equilibration liquid to the column is performed between addition of equilibration liquid containing IL-2, addition of equilibration liquid containing mPEG-SC and addition of elution liquid.
Preferably, the molar ratio of IL-2 to mPEG-SC is between 1:5 and 40.
Preferably, the equilibration solution is a phosphate buffer containing SDS.
Preferably, the phosphate buffer in the balance solution is PB buffer, the concentration of SDS is 0.01-0.1 wt%, and the concentration of PB buffer is 10-50 mM.
Preferably, the eluent is phosphate buffer containing a surfactant.
Preferably, the phosphate buffer in the eluent is 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 0.1wt% to 1wt%.
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, so that the PEG-IL2 with high purity and high yield is obtained.
Preferably, the nonionic surfactant is Triton X-100 or Tween-20.
Preferably, the cationic surfactant is CTAB.
Drawings
FIG. 1 is an electrophoresis detection chart of the chromatographic liquid at different stages obtained in example 1 of the present invention;
FIG. 2 is an electrophoresis detection chart of the chromatographic liquid at different stages obtained in example 2 of the present invention;
FIG. 3 is an electrophoresis detection chart of the chromatographic liquid at different stages obtained in example 3 of the present invention;
FIG. 4 is an electrophoresis detection chart of the chromatographic liquid at different stages obtained in example 4 of the present invention;
FIG. 5 is an electrophoresis detection chart of the chromatographic liquid at different stages obtained in comparative example 1 of the present invention;
FIG. 6 is an electrophoretically detected graph of the chromatographic liquid at different stages obtained in comparative example 2 of the present invention, wherein the packing: and directly taking out the filler for electrophoresis detection.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A method for preparing PEG-modified IL-2 by ion exchange chromatography comprising the steps of:
a. Preparing related solution:
Balancing solution: SDS was added to 20mM PB buffer (pH=8) to make the concentration of SDS 0.05wt%, thereby obtaining a balance solution.
Eluent: triton X-100 was added to 20mM PB buffer to give a Triton X-100 concentration of 0.1wt%, to give an eluate.
IL-2 containing equilibration solution: IL-2 was added to the equilibration solution to completely solubilize the IL-2.
Balance (modification) solution containing mPEG-SC: to the above-mentioned equilibration solution, mPEG-SC (monomethoxy polyethylene glycol succinimidyl carbonate) was added to completely dissolve mPEG-SC.
B. modification of solid phase:
Fully balancing a Q-FF preassembled column (HITRAP Q FF, GE HEALTHCARE) by using a balancing solution with a volume of 10 columns, loading the sample by using a balancing solution containing IL-2, keeping the loading for 2min, balancing the sample by using a balancing solution with a volume of 5 columns after loading, and then modifying the sample by using a balancing solution (modifying solution) containing mPEG-SC, wherein the retention time is 5h; wherein, the molar ratio of IL-2 for loading and 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 balance liquid, adding the eluent for eluting, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
The modification effect of the modified molecule is detected by electrophoresis, the detection result is shown in figure 1, IL-2 can be effectively modified on a solid phase carrier, and the modified molecule 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 related solution:
Balancing solution: SDS was added to 10mM PB buffer (pH=8) to make the concentration of SDS 0.01wt%, thereby obtaining a balance solution.
Eluent: tween-20 was added to 10mM PB buffer to achieve a concentration of Tween-20 of 1wt%, to give an eluate.
IL-2 containing equilibration solution: IL-2 was added to the equilibration solution to completely solubilize the IL-2.
Balance (modification) solution containing mPEG-SC: to the above-mentioned equilibration solution, mPEG-SC (monomethoxy polyethylene glycol succinimidyl carbonate) was added to completely dissolve the mPEG-SC.
B. modification of solid phase:
Fully balancing a Q-FF preassembled column (HITRAP Q FF, GE HEALTHCARE) by using a balancing solution with a volume of 10 columns, loading the balancing solution containing IL-2 for 4min, balancing the balancing solution with a volume of 5 columns after loading, and then modifying the balancing solution with a volume of mPEG-SC (modifying solution) for 1h; wherein, the molar ratio of IL-2 for loading and 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 balance liquid, adding the eluent for eluting, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
The modification effect of the modified molecule is detected by electrophoresis, the detection result is shown in figure 2, IL-2 can be effectively modified on a solid phase carrier, and the modified molecule can be fully 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 related solution:
Balancing solution: SDS was added to 50mM PB buffer (pH=8) to make the concentration of SDS 0.1wt%, thereby obtaining a balance solution.
Eluent: CTAB was added to 50mM PB buffer to a concentration of 2wt% CTAB, to thereby obtain an eluate.
IL-2 containing equilibration solution: IL-2 was added to the equilibration solution to completely solubilize the IL-2.
Balance (modification) solution containing mPEG-SC: to the above-mentioned equilibration solution, mPEG-SC (monomethoxy polyethylene glycol succinimidyl carbonate) was added to completely dissolve the mPEG-SC.
B. modification of solid phase:
Fully balancing a Q-FF preassembled column (HITRAP Q FF, GE HEALTHCARE) by using a balancing solution with a volume of 10 columns, loading the balancing solution containing IL-2 for 10min, balancing the balancing solution with a volume of 5 columns after loading, and then modifying the balancing solution with a volume of mPEG-SC (modifying solution) for 0.5h; wherein, the molar ratio of IL-2 for loading and 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 balance liquid, adding the eluent for eluting, and performing Superdex 200 gel filtration chromatography on the eluted sample to obtain the high-purity PEG-IL2 molecule.
The modification effect of the modified molecule is detected by electrophoresis, the detection result is shown in figure 3, IL-2 can be effectively modified on a solid phase carrier, and the modified molecule can be fully eluted by combining the eluent.
Example 4
The Q-FF pre-packed column of example 1 was replaced with DEAE pre-packed column (HITRAP DEAE FF, GEHEALTHCARE) and the other methods and parameters were the same as in example 1 to obtain high purity PEG-IL2 molecules.
The modification effect of the modified molecule PEG-IL2 is detected by electrophoresis, the detection result is shown in figure 4, the IL-2 can be effectively modified on a solid phase carrier, and the modified molecule can be fully eluted by combining with a specific eluent.
Comparative example 1
The IL-2 is subjected to hydrophobic chromatography solid phase modification by using HITRAP PHANYL pre-packed columns (HITRAP PHENYL FF, GEHEALTHCARE) instead of Q-FF pre-packed columns, and the specific method is as follows:
a. Preparing related solution:
Balancing solution: SDS and ammonium sulfate were added to 10mM PB buffer (pH=8) to make the concentration of SDS 0.01wt% and the concentration of ammonium sulfate 200mM, to obtain a balance.
Eluent: SDS was added to 10mM PB buffer to make the concentration of SDS 1wt%, thereby obtaining an eluate.
IL-2 containing equilibration solution: IL-2 was added to the equilibration solution to completely solubilize the IL-2.
Balance (modification) solution containing mPEG-SC: to the above-mentioned equilibration solution, mPEG-SC (monomethoxy polyethylene glycol succinimidyl carbonate) was added to completely dissolve the mPEG-SC.
B. modification of solid phase:
After fully balancing HITRAP PHANYL pre-packed columns (HITRAP PHENYL FF, GEHEALTHCARE) by using 10 column volumes of balancing solution, loading the sample by using the balancing solution containing IL-2, keeping the sample for 4min, balancing by using 5 column volumes of balancing solution after loading, and then modifying by using the balancing solution (modifying solution) containing mPEG-SC, wherein the keeping time is 1h, and the molar ratio of IL-2 to mPEG-SC in the system 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 balancing solution, adding the eluent for eluting, and adding 5X loading buffer,95 ℃ for denaturation for 5min to the eluted sample to obtain the sample.
The modification effect in the sample is detected by electrophoresis, and the detection result is shown in fig. 5, and the IL-2 can be normally combined on HITRAP PHANYL filler, but can be eluted by PEG in the modification process, so that the solid phase modification fails.
Comparative example 2
The eluent in example 1 was replaced by a conventional anion exchange chromatography using a high-salt eluent (20mM PBS,0.05%SDS,1M NaCl), and the other methods and parameters were the same as those in example 1, to obtain eluted samples.
The obtained eluted sample is subjected to electrophoresis detection, the detection result is shown in fig. 6, IL-2 can be effectively modified on a solid phase carrier, but the modified molecule is stably combined with a filler, and conventional anion exchange eluent cannot effectively elute the modified molecule.
Test example 1
And taking CTLL-2 cells in logarithmic growth phase after subculturing for 24 hours, and regulating the concentration of the CTLL-2 cells to 1X 10 5/mL by using a culture solution. The cell fluid of the above specified concentration was then inoculated into 96-well culture plates with 0.1mL of the cell fluid per well. The modified samples (samples to be tested) obtained in examples 1 to 4 and IL-2 standard were subjected to double dilution, and then 0.1mL of the sample to be tested or IL-2 standard was added to each well, and 3 wells were provided for each dilution concentration. Culture control wells (100. Mu.L of cells+100. Mu.L of culture) were additionally provided. The above-mentioned culture plate was incubated at 37℃under 5% CO 2 for 40 hours. 100. Mu.L of the supernatant was aspirated, 100. Mu.L of MTT (1 mg/mL) was added thereto, and incubated at 37℃for 2 hours with 5% CO 2. The absorbance was measured at 570nm using an enzyme-labeled analyzer, and the measurement result was recorded.
The test data are processed by a computer program or a four-parameter regression calculation method, and the result is calculated according to the following formula:
Wherein P r is the biological activity of a standard substance and IU/ml; d s is the pre-dilution of the test sample; d r is the standard pre-dilution; e s is the dilution multiple of the half-effective amount of the sample corresponding to the standard; e r is the dilution of the standard half-dose.
The calculated activity values of the samples to be tested are shown in table 1.
TABLE 1 sample Activity values
From the activity values measured in Table 1, it was found that the activity of PEG-IL2 obtained by solid phase modification in examples 1-4 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 subjected to liquid phase method to determine the protein purity. The OD280 reading was measured by an ultraviolet-visible spectrophotometer and divided by the extinction coefficient to obtain the concentration of the modified molecule, the total amount was calculated by multiplying the concentration by the volume, the yield was obtained by dividing the total amount of IL-2 added before modification, and the measurement and calculation results are shown in Table 2.
TABLE 2 sample purity and yield
Note that: the PEG-IL2 obtained by liquid phase modification is specifically obtained by adopting a liquid phase modification method disclosed in "NANDINI V.KATRE,, et al.(1987).Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model.Proc Natl Acad Sci U S A.1987;84(6):1487-91.".
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (4)
1. A method for preparing PEG-modified IL-2 by ion exchange chromatography, characterized in that: comprising the following steps: carrying out PEG solid phase modification of IL-2 by adopting an anion exchange chromatography method, and then eluting by using an eluent containing a surfactant to obtain the PEG modified IL-2;
The solid phase modification method comprises the following steps: sequentially adding an IL-2-containing equilibrium solution and an mPEG-SC-containing equilibrium solution into an anion exchange chromatographic column after equilibrium of the equilibrium solution to carry out a solid phase modification process, wherein the equilibrium solution contains SDS with the concentration of 0.01-0.1 wt%;
The concentration of the surfactant is 0.1-2 wt%;
the surfactant is Triton X-100, tween-20 or CTAB.
2. The method of preparing PEG-modified IL-2 by ion exchange chromatography according to claim 1, wherein: the molar ratio of IL-2 to mPEG-SC is 1:5-40.
3. The method of preparing PEG-modified IL-2 by ion exchange chromatography according to claim 1, wherein: the balancing solution is phosphate buffer solution containing SDS.
4. The method of preparing PEG-modified IL-2 by ion exchange chromatography according to claim 1, wherein: the eluent is phosphate buffer containing surfactant.
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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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