CN113750571A - Method for prolonging service life of ion exchange filler - Google Patents
Method for prolonging service life of ion exchange filler Download PDFInfo
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- CN113750571A CN113750571A CN202111061358.1A CN202111061358A CN113750571A CN 113750571 A CN113750571 A CN 113750571A CN 202111061358 A CN202111061358 A CN 202111061358A CN 113750571 A CN113750571 A CN 113750571A
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- Prior art keywords
- filler
- ion exchange
- sodium hypochlorite
- strong cation
- cation exchange
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- 239000000945 filler Substances 0.000 title claims abstract description 103
- 238000005342 ion exchange Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 30
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 38
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 37
- 230000003647 oxidation Effects 0.000 claims abstract description 33
- 238000012799 strong cation exchange Methods 0.000 claims abstract description 29
- 239000007800 oxidant agent Substances 0.000 claims abstract description 26
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000011543 agarose gel Substances 0.000 claims abstract description 8
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- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 description 2
- XWNSFEAWWGGSKJ-UHFFFAOYSA-N 4-acetyl-4-methylheptanedinitrile Chemical compound N#CCCC(C)(C(=O)C)CCC#N XWNSFEAWWGGSKJ-UHFFFAOYSA-N 0.000 description 2
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- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N Aminoantipyrine Natural products CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
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- 241000206572 Rhodophyta Species 0.000 description 1
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Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/362—Cation-exchange
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention relates to the technical field of ion exchange, in particular to a method for prolonging the service life of an ion exchange filler, which comprises the step of carrying out oxidation treatment on the filler, namely treating the ion exchange filler by using an oxidant to remove residual reductive ligands, wherein the oxidant comprises potassium dichromate, sodium hypochlorite, bromine water and the like, particularly the oxidant is the sodium hypochlorite, the ion exchange filler is a strong cation exchange filler, particularly, the base material of the strong cation exchange filler is agarose gel, the oxidation step is carried out before the strong cation exchange filler is used, the purification efficiency and the regeneration capacity of the oxidized ion exchange filler are improved, the service life can be effectively prolonged, the cost is saved, and the method has a better market application prospect.
Description
Technical Field
The invention relates to the technical field of ion exchange, in particular to a method for prolonging the service life of an ion exchange filler.
Background
In recent years, with the rapid development of life science and bioengineering technology, a series of different kinds of biomacromolecule drugs are continuously generated, people have increasingly increased demands for protein drugs, the separation, analysis and purification of active biomacromolecules in a complex life system become an important task facing analytical chemistry and separation science, downstream protein separation and purification face serious challenges, and whether the realization of rapid and efficient separation and purification of biomacromolecules is a key factor restricting the yield of biological products (Golay. preparation of cation exchange chromatography medium and protein adsorption performance evaluation [ D ] Beijing petrochemical institute, 2020.).
In the downstream technologies of bioengineering, both high separation efficiency of the separation medium and as fast a separation speed as possible are required to increase the space-time efficiency of the production. Currently, Ion Exchange Chromatography (IEC) is an effective separation method in the multi-stage purification of protein molecules in the downstream of biopharmaceuticals, because of its good biocompatibility, mild elution mode, and ability to maintain the activity of biomolecules to the maximum extent during the separation process, and plays an important role in the separation and purification of biomacromolecules (liu giu, huang yan, yanbo, jie hua, liu guo, zhui. preparation of highly hydrophilic strong cation exchange chromatography packing and its application in protein analysis [ J ] chromatography, 2013,31(04):310 cake 316 ]. The separation method selectively adsorbs or releases valuable ions by utilizing the difference of the acting force of an ion exchanger and ions, thereby achieving the purposes of removing impurities, enriching or purifying target biochemical substances.
In the biological medicine industry, ion exchange chromatography is widely applied to extraction of biochemical micromolecular substances such as antibiotics, amino acids, organic acids and the like, and is also widely applied to separation and purification of biomacromolecules such as enzymes, polysaccharides and the like. In principle, under certain conditions, the target biomass can be separated and purified by ion exchange technology as long as it can be ionized.
The selection of the ion exchange packing plays a decisive role in the separation and purification of the biomacromolecule. Ion exchange packing can be classified into strong cation exchange packing, weak cation exchange packing, strong anion exchange packing, and weak anion exchange packing according to the type of exchange ligand. Among them, strong cation exchange chromatography consisting of strong cation exchange packing is widely used in downstream purification process of biological products as one of high-efficiency separation and purification technologies. Ion exchange fillers can be classified into polysaccharides, inorganic and organic polymers according to the type of matrix. Polysaccharide fillers are widely applied in protein separation because of the advantages of high water-based property, good biomolecule compatibility, a net structure and the like (Sun ocean, preparation and application of chitosan ion exchange medium [ D ] Zhejiang university of industry, 2011.).
Agarose is a polysaccharide with gel property from marine red algae, is a linear polymer, and has a basic structure of a copolymer formed by alternately connecting 1, 3-beta-D-galactopyranose and 1,4-3, 6-anhydro-alpha-L-galactopyranose (Wangjialin, Lefu, Huanghuanghuanghuang, Xiongjie, Zhao Peng. agarose and high-resolution agarose preparation method research progress [ J ]. Guangzhou chemical industry, 2016,44(11): 19-22.). Agarose has special gel property, certain pore size and molecular sieve function, can separate and purify biological macromolecules (such as protein and nucleic acid), is generally heated to more than 90 ℃ in water to be dissolved, and forms good semisolid gel when the temperature is reduced to 35-40 ℃, which is the main characteristic and the basis of multiple purposes.
The agarose gel filler has strong hydrophilicity, weak nonspecific adsorption, strong acid and alkali resistance, good biocompatibility, high column loading capacity up to 90 percent of pore volume, high recovery rate of separated molecules and the like due to the basic structure of the polysaccharide, can be directly used for filling columns, has strong selectivity, good chemical stability and the like, and can be widely used for gel filtration media, ion exchange media, hydrophobic chromatography media, affinity chromatography media and metal chelate chromatography media and used for separating and analyzing different substances.
In the separation and purification process, the particle impurities of the biomacromolecules easily cause serious pollution to the chromatographic medium, which leads to the increase of the operation pressure, the great shortening of the service life of the medium and the great reduction of the purification effect in the subsequent chromatographic separation, thereby bringing adverse effects to the separation and purification of the biomacromolecules (Sun West Yan, Zhang Yan, Liyan, Rojia, Qinbergen, Suzhou. the pollution mechanism of the medium in the chromatographic separation process of the expression product of the mammary gland bioreactor and the regeneration strategy thereof [ J ]. Bioengineering report, 2011,27(11): 1645-. At present, the price of the ion exchange packing in the market is higher, and the prolonging of the service life of the ion exchange packing is particularly important in view of research and development and production costs.
The effective regeneration of chromatographic column usage is disclosed in all of the methods of He Ling Ice, Zhang Ming Lian, etc., which can prolong the lifetime of the filler (He Ling Ice, Li le, Dang Yi xi, Mongolian, Yu Gen. several comparison of the effects of Protein A affinity chromatography filler purification recombinant monoclonal antibody [ J ]. Chinese journal of bioengineering, 2015,35(12):72-77.) (the application of Zhang Ming Lian, Hu ding, Liu Ru, Kong Xia. ion exchange chromatography in medicinal plasmid analysis [ J ]. chemical progress 2010,22(Z1):482 one 488.), so that the stronger the regeneration ability of the filler in the chromatographic column, the longer the lifetime thereof.
The applicant has found that treatment with an oxidant before use of an ion exchange filler can improve the regeneration capability of the filler and prolong the service life of the filler, and the prior art has no report on the regeneration capability.
Disclosure of Invention
In view of the shortcomings of the prior art, the present invention provides a method for prolonging the service life of an ion exchange packing, which is characterized in that: the method is characterized by comprising the following steps of oxidizing the filler: the filler is treated with an oxidizing agent.
The ion exchange filler is strong cation filler.
Preferably, the substrate of the strong cation exchange packing is agarose gel.
The oxidant comprises potassium dichromate, sodium hypochlorite and bromine water.
Preferably, the oxidant is sodium hypochlorite.
Preferably, the concentration of the sodium hypochlorite is 10-50 times of the dilution of the stock solution.
Preferably, the solid-to-liquid ratio of the strong cation exchange packing to the sodium hypochlorite is 1: 2-10.
The action time of the oxidation treatment is 1-2 h.
The oxidation treatment according to the invention is carried out before the use of the filler.
The invention provides a method for prolonging the service life of ion exchange packing, which comprises the steps of treating the ion exchange packing with an oxidant before using the ion exchange packing, to remove any reducing ligands which may be present, preferably, the ion exchange packing is a strong cation exchange packing, the base material of the strong cation exchange filler is agarose gel, the oxidant comprises potassium dichromate, sodium hypochlorite, bromine water and the like, preferably, the oxidant is sodium hypochlorite, the concentration of the sodium hypochlorite is 10-50 times of the dilution of the stock solution, the solid-liquid ratio of the strong cation exchange filler to the sodium hypochlorite is 1:2-10, the action time is 1h-2h, preferably, the optimal oxidation condition is that the concentration of the sodium hypochlorite is 10 times of the dilution of the stock solution, the solid-liquid ratio of the strong cation exchange filler to the sodium hypochlorite is 1:2, and the action time of the oxidation treatment is 2 hours. The purification efficiency and the regeneration capacity of the strong cation exchange filler after oxidation treatment are improved, the service life of the filler is prolonged, the cost is saved, and the method has a wide application prospect.
Drawings
FIG. 1 comparison of oxidizable reducing ligands of fillers.
Detailed Description
The invention provides a method for prolonging the service life of an ion exchange filler, which is characterized by comprising the following steps: the method is characterized by comprising the following steps of adding oxidation treatment in the preparation process: the filler is treated with an oxidizing agent.
The ion exchange filler is strong cation exchange filler.
Preferably, the substrate of the strong cation exchange packing is agarose gel.
The oxidant comprises potassium dichromate, sodium hypochlorite and bromine water.
Preferably, the oxidant is sodium hypochlorite.
Preferably, the concentration of the sodium hypochlorite is 10-50 times of the dilution of the stock solution.
Preferably, the solid-to-liquid ratio of the strong cation exchange packing to the sodium hypochlorite is 1: 2-10.
The acting time of the sub-oxidation treatment is 1h-2 h.
The oxidation treatment according to the invention is carried out before the use of the filler.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and the like added in the examples are commercially available unless otherwise specified.
The method for prolonging the service life of the ion exchange filler is simple and economic, and due to the chemical reaction process in the preparation process of the filler, ligands on the surface of the filler are not all sulfonic acid groups, and the method also comprises groups generated by side reactions or incomplete reactions, wherein most of the groups have reducibility, and can react with biopolymer substances in a covalent bond forming mode to cause the oxidation-reduction reaction of impurities and the ligands of a sample in use, so that the situation that the filler is difficult to regenerate or incomplete to regenerate is generated, the oxidation step is increased, the existing reducibility ligands can be removed, the regeneration capacity and the purification efficiency are increased, and the service life of the filler is prolonged.
This section of the examples further illustrates the content of the invention but should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The filler treated by the oxidant comprises potassium dichromate, sodium hypochlorite, bromine water and the like. After the comprehensive economic benefit, operation process and comparative experiment, sodium hypochlorite is preferably used as the oxidant in the embodiment.
There are strong cation exchange packing, weak cation exchange packing, strong anion exchange packing and weak anion exchange packing according to the type of exchange ligand. Among them, strong cation exchange chromatography consisting of strong cation exchange packing is widely used in downstream purification process of biological products as one of high-efficiency separation and purification technologies. So strong cation exchange packing was chosen for the experiments.
And because the sepharose is a common matrix of the traditional ion exchange purification medium, the sepharose has better advantages on the separation medium, is stable, is simple, convenient and flexible to operate and has wide application range, the base material of the strong cation exchange filler is sepharose 6 FF.
EXAMPLE 1 preparation of Strong cation exchange Filler
1.1 reagents
Preparing a storage solution: adding 20mL of absolute ethyl alcohol into 80mL of purified water, and uniformly mixing to prepare a 20% ethyl alcohol solution; and weighing 0.1g of sodium azide and 1.6406g of sodium acetate solid to dissolve in the 20% ethanol solution.
1.2 washing of Sepharose 6FF starting Material
Taking a proper amount of agarose gel 6FF suspension, and carrying out suction filtration and washing; when the first suction filtration is carried out, the agarose gel 6FF storage liquid is drained, and then the solid is suspended and soaked for 2min by purified water and drained; repeating the operation for 5 times; and finally, draining (keeping constant weight for 10 min).
1.3 Filler preparation
1.3.1, using a water bath to keep the temperature of an elliptical single-layer glass reaction kettle (hereinafter referred to as a reaction kettle) constant, and adjusting the temperature of a water bath kettle to 50 ℃; 1500g of sepharose gel 6FF are weighed and placed in a reaction kettle (the reaction kettle is provided with a polytetrafluoroethylene cross double-layer stirring rod and can be mechanically stirred).
1.3.2 taking a 2L beaker, adding sodium hydroxide, sodium borohydride and anhydrous sodium sulfate into the 2L beaker according to the feeding ratio of 40:1:25, adding 750mL of purified water, cooling to room temperature after stirring and dissolving, adding into a 1.3.1 reaction kettle, starting a stirrer (the rotating speed is controlled at 230rpm/min), and uniformly stirring.
1.3.3 measuring 650mL of allyl glycidyl ether by using a measuring cylinder, adding the allyl glycidyl ether into a 1.3.1 reaction kettle, uniformly stirring, and carrying out activation reaction for 16 h.
1.3.4 suction filtration washing of intermediate: pouring the intermediate suspension into a suction filtration cup, using 50% ethanol purified water solution to trickle wash the reaction kettle for 2 times, and pumping to dry; washing with 3.5L anhydrous ethanol for three times, fully suspending each time, and draining; neutralizing the reactant with 10% acetic acid solution (until the pH of the filtrate is 5.0-7.0), draining, repeatedly cleaning with purified water until the pH test paper detects that the cleaning solution is 6.0 + -0.2, and draining; a sample (about 10.0g) was left for intermediate activation ligand density determination.
The temperature of the water bath is preheated to 37 ℃ and the activated product in the suction filter cup of 1.3.4 is transferred to a reaction kettle.
1.3.6 weighing 1500g of sodium metabisulfite, adding 4000mL of purified water for dissolving, adding into a 1.3.5 reaction kettle, starting a stirrer (the rotating speed is controlled at 230rpm/min), stirring for reacting for 30min, monitoring a reaction system, controlling the pH to be 5.0 +/-0.5 (regulating by adopting sodium hydroxide or hydrochloric acid solution), and stirring for reacting for 24 h.
1.3.7 after the reaction is finished, carrying out suction filtration washing on a reaction product, firstly, repeatedly washing the reaction product for 5 times by using purified water, suspending and soaking the reaction product for 2min by using the purified water during each washing, draining the product, and reserving a sample for carrying out full exchange capacity determination; finally, leaching and draining the solution by using a storage solution; transferring the reaction product into a 5L white sterile filling bottle, adding the storage solution, storing, sealing, and storing in a refrigerator at 4 ℃. The preparation of the strong cation exchange packing is completed, and is hereinafter referred to as GES.
Example 2 sequence of Oxidation steps
The sodium hypochlorite is used for carrying out preliminary experiments on the prepared GES, and the discovery shows that after the used filler is combined with the sodium hypochlorite, no obvious impurity removal effect is achieved and no oxidation reaction is observed, while after the finished filler is subjected to oxidation treatment before use, an obvious oxidation reaction can be observed, so the oxidation step is determined to be added before the filler is used.
Example 3 measurement of Oxidation conditions
3.1 Experimental mechanism
NaClO reacts with KI in an acid solution to quantitatively generate an iodine simple substance, starch is used as an indicator, sodium thiosulfate standard solution is used for titration, and blue disappearance is used as an end point, so that the amount of residual NaClO in a solution system is determined. The reaction formula is as follows:
2H++ClO-+2I-＝I2+Cl-+H2O
I2+2S2O3 2-＝2I-+S4O6 2-
3.2 reagents
Oxidizing agent: 100% sodium hypochlorite (active chlorine is more than or equal to 5.2%, free alkali is 6% -8%, trade name is antipyrine), and water can be added for dilution according to the requirement;
reagents for titration: 20% KI; 0.1M sodium thiosulfate standard solution; 0.5M H2SO4(ii) a 0.5% starch solution.
3.3 operating procedure
3.3.1 weighing M g GES in a 500mL iodine bottle, diluting with respect to the original solution of sodium hypochlorite by N, adding sodium hypochlorite V1mL, at 37 degrees, 200rpm shaking table under full reaction.
3.3.2 get V3Adding 50mL of water, 50mL of 0.5M sulfuric acid, 5mL of 0.5% starch indicator and 20% of potassium iodide into the reacted supernatant; titrating with sodium thiosulfate until the blue color fades and does not change for 30s, and recording the volume V of the blank control consuming the sodium thiosulfate0And volume V of test specimen consuming sodium thiosulfate2(ii) a Blank control refers to the addition of M g untreated blank filler.
3.3.3 formula for calculating degree of oxidation of filler:
c- -sodium thiosulfate concentration V0Consumption of sodium thiosulfate volume by blank
M- -Filler quality V1Volume of sodium hypochlorite
V2- -volume of sodium thiosulfate consumed by the sample after oxidation V3Volume of supernatant taken
X- -amount of sodium hypochlorite consumed per unit of filler
3.4 Experimental design and results
By changing experimental conditions, the GES oxidation conditions and the difference between the GES oxidation conditions and theoretical values are explored, so that parameters required to be controlled by the preparation process and the implementation method are evaluated. The experiment has 9 groups of data, and the specific numerical values are shown in Table 1. Ligand density of GES used in this experiment: 0.19-0.25mmol/g, particle size 35-165 μm.
TABLE 1 data sheet of the results of the oxidative titration experiment
3.5 analysis of the results of the experiment table 1 shows that group 1, in order to end the reaction, adds an excessive amount of oxidant, after the reaction is terminated, when the oxidation degree of the filler is measured, a large amount of iodine vapor is generated, which brings a large measurement error, and at the same time, since sodium hypochlorite consumed by the filler accounts for the remaining 6.2%, a large amount of reagents are wasted, and the accuracy of the measured data is not sufficient, the data needs to be readjusted.
The data of group 2 and group 3 were modified based on group 1 by diluting the sodium hypochlorite concentration by 100 times, at this time, the volume of sodium thiosulfate consumed after oxidation was very small, indicating that the oxidation reaction was not complete and needs to be readjusted.
The concentration of sodium hypochlorite is diluted by 50 times in groups 4 and 5, and the volume of sodium thiosulfate consumed after oxidation is also proper when the oxidation reaction is measured, the solid-to-liquid ratio of the filler to the oxidant is 1:10, and the oxidation degree is also good at this moment, but the volume ratio of the filler to the oxidant is reduced because the solid-to-liquid ratio of the oxidant to the filler is too large.
And the quantity of the filler is increased to 25g in the 6 th group, the 7 th group and the 8 th group, the solid-liquid ratio of the filler to the oxidant is 1:2, the concentration of the sodium hypochlorite is diluted by 10 times, the volume of the sodium thiosulfate consumed after oxidation is smaller, and the final oxidation degree is not greatly different in a titration test by sucking supernatant liquid for 1h and 2 h.
In group 9, the amounts of filler and sodium hypochlorite added were increased at the same time in equal proportions, and the amount of sodium hypochlorite consumed per filler was not much different from those in groups 7 and 8, and it was found that the reducing groups on the filler were sufficiently oxidized.
The finally determined oxidation conditions are that the solid-to-liquid ratio of GES to oxidant is 1:2-10, diluting the sodium hypochlorite by 10-50 times as the stock solution, reacting at 37 ℃ for 1-2 h at 200rpm, and optimally oxidizing under the conditions that the solid-to-liquid ratio of GES to oxidant is 1:2, the sodium hypochlorite concentration is 10 times of the original solution, and the reaction is carried out for 2 hours at 37 ℃ and 200 rpm.
Example 4 simulation of the use of the packing material because the actual sample purification process requires the steps of loading, elution, regeneration, etc., the time spent in the whole process is long, the used amount of the packing material is large, and the cost is too high, a simulation experiment is performed by using a static adsorption and analysis method.
SP focus 6FF D1 (abbreviated as summary SP): h+Load 161.3umol/mL gel, production lot number: y21061601, Wuhan Hui research Biotech GmbH, is an ion exchange filler commonly used in industry; GE Sepharose 6FF (GE SP for short): h+The loading capacity of 180-250mmol/mL gel, the production batch number of 10274788, GE Healthcare in America, foreign products, are used as benchmarking products of similar products. The two ion exchange fillers have certain representativeness, and parameters of the strong cation exchange filler before and after oxidation treatment can be checked as a reference.
For convenience of statistics, the oxidized strong cation exchange packing is abbreviated as GESII in the invention.
Polypeptide feed liquid: dissolving the powder with purified water, and performing suction filtration with a particle size of 0.45 μm to obtain a polypeptide concentration of about 14.5 g/L;
resolving the solution: 2M NaCl solution.
1) The GES, GESII, convergent SP and GE SP fillers are respectively cleaned by ultrapure water, then are drained, and about 8.5g of fillers are weighed.
2) 40mL of the polypeptide solution was added to each of the cells and adsorbed for 0.5h at 200rpm in a shaker.
3) Taking out the adsorbed filler, drying the filler by using a vacuum pump filter, washing the filler by using a small amount of ultrapure water, drying the filler again, adding 40mL of analysis solution into the filler, and analyzing the solution for 0.5h at 200rpm of a shaking table; the resolved solution needs to be rinsed several times with a small amount of ultrapure water to ensure complete removal of the salt solution, and the filler is drained again.
4)2) to 3) are repeated for 2 times.
5) And (3) taking the filler subjected to cyclic adsorption-desorption for 3 times as a second group of detection samples, weighing 4g of each filler, and subpackaging 2 tubes, wherein each tube contains 2g of each filler, so as to detect the subsequent filler regeneration capacity.
6) About 4g of each remaining filler was added to the polypeptide solution, 30mL was added, and the mixture was adsorbed for 0.5h at 200rpm in a shaker.
7) Taking out the adsorbed filler, drying by using a vacuum pump filter, washing the filler by using a small amount of ultrapure water, drying again, adding 30mL of analysis solution into the filler, and analyzing for 0.5h at 200rpm of a shaking table; the resolved solution needs to be rinsed several times with a small amount of ultrapure water to ensure complete removal of the salt solution, and the filler is drained again.
8)6) to 7) were repeated 2 times.
9) And (3) taking the filler subjected to cyclic adsorption-desorption for 6 times as a third group of detection samples, weighing 4g of each filler, and subpackaging 2 tubes, wherein each tube contains 2g of each filler, so as to detect the subsequent filler regeneration capacity.
Example 5 determination of regeneration capacity of filler because of the existence of residual reductive ligands such as carbon-carbon double bonds in the strong cation exchange filler, the instability of such ligands may generate redox reaction with part of impurities in the material to form covalent connection, even if strong regeneration steps such as acid-base oscillation and alcohol cleaning are added, a large amount of bound impurities cannot be removed, and at the same time, steric hindrance of impurities also affects adsorption of adjacent sulfonic acid groups to the target, resulting in gradual reduction of loading capacity. Therefore, GESII performs a sealing operation on the basis of GES. The testing of the reducing value of the filler is to actually measure the density of the residual reducing ligand, and the value is higher and is difficult to regenerate. This experiment compares the regeneration capacity between several fillers by calculating the density of the oxidizable ligands in the filler.
5.1 solution preparation
1)0.1mol/L potassium bromide-potassium bromate mixed solution: preparing 1L, weighing 6g of potassium bromate and 50g of potassium bromate, dissolving in ultrapure water, and completely dissolving for later use.
2)6mol/L hydrochloric acid solution: 500mL of concentrated hydrochloric acid was mixed with 500mL of water.
3)0.1mol/L sodium thiosulfate solution: preparing 1L, weighing 26g of sodium thiosulfate pentahydrate and 0.2g of anhydrous sodium carbonate, and dissolving in 1L of ultrapure water for later use.
4) Starch indicator: adding water 5mL of 0.5g of soluble starch, stirring, slowly pouring into 100mL of boiling water, stirring with water, boiling for 2min, cooling, and collecting supernatant. The solution should be prepared fresh temporarily.
5) Preparing a 20% KI solution (for use in the preparation): weighing 20g of KI, and adding 80mL of ultrapure water for dissolving.
The experimental principle is as follows: after activation, bromine was prepared and reacted with C ═ C double bond addition, and the density of ligand generated by filler activation was determined by back titration with 0.1mol/L sodium thiosulfate.
5.2 Experimental procedures
The 3 groups of samples in example 3 were cleaned with ultrapure water, after draining, a certain amount of sample was accurately weighed, the specific weight W (accurate to 0.0001g) was recorded, placed in a 250mL iodine vial, and a blank sample was set.
Adding 15mL of 0.1mol/L potassium bromide-potassium bromate mixed solution into the filler, adding 50mL of ultrapure water, adding 10mL of 6mol/L hydrochloric acid, quickly covering a bottle cap, fully shaking and uniformly mixing, sealing the bottle mouth with water, standing in the dark for 25min, and shaking uniformly once every 10 min; after standing for 25min, 10mL of 20% KI solution was added quickly and shaken up. Adding 3.0ml of starch indicator, and titrating with 0.1mol/L sodium thiosulfate standard solution until the blue-purple color disappears, wherein the titration end point is obtained when the blue color does not change for 30 s.
The ligand density is calculated as mmol/g (wet weight of gel) as follows:
c: allyl Density μmol/g
Concentration mol/L of standard solution of sodium thiosulfate
W: mass g V of sample after draining0: volume mL of standard solution of sodium thiosulfate consumed by blank
V: sample consumption of sodium thiosulfate Standard solution volume mL
5.3 results of the experiment
TABLE 2 comparison of the four fillers with oxidizable and reducible ligands
5.4 analysis of results
As can be seen from Table 2 and FIG. 1, the oxidizable and reducible ligands of GES are significantly reduced in GES II compared with GES, and the products of the same type also have certain advantages. From the analysis of the data in the same group, the results of the first group of fillers before use (new fillers) show that the GESII is reduced in the surface reducing ligands after oxidation treatment; the results of the second and third groups (3 and 6 purifications, respectively) show that the GESII surface oxidizable substance or functional group is significantly lower than that of other fillers, especially GES, and therefore we believe that the GESII impurities after oxidation treatment have better adsorption and regeneration capacity.
Example 6 comparative testing of filler purified polypeptide before and after Oxidation
GES column: 20mL of GES packing is filled into the column;
GESII column: packed GESII 20mL
Polypeptide feed liquid: pH4.0, 0.35 g/L.
And (3) purification process:
balancing: balancing the column by 200mL of purified water to restore the state of the column before sample loading;
loading: the two columns are respectively loaded with 800mL, and the flow-through liquid is collected in a front section and a rear section;
washing: 20mM Tris, 0.5M NaCl, 80mL of solution washing with pH8.0, 1 sample collection of 40mL, respectively marked as "washing 1" and "washing 2";
and (3) elution A: 20mM Tris, 1.0M NaCl, 80mL of pH8.0 solution elution, and the collected liquid is marked as "elution A";
and (3) elution B: 20mM Tris, 2.0M NaCl, 80mL of pH8.0 solution elution, and the collected liquid is marked as "elution B";
regeneration: 80mL of 0.5M NaOH and 1.0M NaCl solution was washed and the cartridge was stored in 20% ethanol solution.
The results are shown in Table 3.
TABLE 3 comparison of the purification purities of two fillers
Therefore, as can be seen from table 3, in the eluent with lower salt concentration (elution a), GESII is improved by about 7% in purity compared with the target substance obtained by GES separation, and in the eluent with higher salt concentration (elution B), GESII is also improved in purity, so that the oxidized strong cation exchange filler GESII can improve the purification purity to a certain extent and the purification efficiency.
In conclusion, compared with GES, the oxidized strong cation exchange filler GESII of the present invention has a higher regeneration capacity, and at the same time, improves the purification purity, and is beneficial to improving the accuracy and efficiency of analysis, prolonging the service life of the ion exchange filler, saving the cost, and improving the purification efficiency, and has a wide commercial application prospect.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (10)
1. A method for extending the useful life of an ion exchange packing, characterized in that it comprises subjecting the packing to an oxidation treatment: the filler is treated with an oxidizing agent.
2. The method of claim 1, characterized in that the ion exchange packing is a strong cation exchange packing.
3. The method according to claim 2, characterized in that the substrate of the strong cation exchange packing is agarose gel.
4. The method of claim 3, wherein the oxidizing agent comprises potassium dichromate, sodium hypochlorite, and bromine water.
5. The method according to claim 4, characterized in that the oxidizing agent is sodium hypochlorite.
6. The method of claim 5, wherein the concentration of sodium hypochlorite is 10-50 times the dilution of the stock solution.
7. The method of claim 6, wherein the solid to liquid ratio of the strong cation exchange packing to sodium hypochlorite is 1: 2-10.
8. A method according to any one of claims 2 to 7, characterized in that the action time of the oxidation treatment is between 1h and 2 h.
9. A method according to any of claims 1-7, characterized in that the oxidation treatment is carried out before the use of the ion exchange packing.
10. The method according to claim 8, wherein the oxidation treatment is carried out before the use of the ion exchange packing.
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