CN111676261A - Preparation process of high-purity recombinant interleukin-2 - Google Patents
Preparation process of high-purity recombinant interleukin-2 Download PDFInfo
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- CN111676261A CN111676261A CN202010592543.2A CN202010592543A CN111676261A CN 111676261 A CN111676261 A CN 111676261A CN 202010592543 A CN202010592543 A CN 202010592543A CN 111676261 A CN111676261 A CN 111676261A
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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Abstract
The invention relates to a preparation process of high-purity recombinant interleukin-2, belonging to the technical field of biological medicines. The preparation process of the high-purity recombinant interleukin-2 comprises the following steps: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, extracting an inclusion body from the interleukin-2 fermentation liquor, cracking the inclusion body, and performing renaturation treatment to obtain interleukin-2 renaturation liquor; adding interleukin-2 renaturation liquid into a hydrophobic high-performance liquid chromatography column for adsorption and elution, collecting eluent, namely a crude pure product, adding the crude pure product into a reversed-phase high-performance liquid chromatography column for adsorption and elution, and collecting the eluent, namely a fine pure product; adding the refined pure product into a weak anion high-efficiency liquid chromatographic column for adsorption and elution, and collecting the eluent, namely the high-purity recombinant interleukin-2; the high-purity recombinant interleukin-2 prepared by the method has high recovery rate, high activity and high purity.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a preparation process of high-purity recombinant interleukin-2.
Background
IL-2 is produced primarily by activated CD4+ T cells and CD8+ T cells (especially CD4+ T cells), is a glycoprotein with broad biological activity that enables long-term survival of T cells in vitro. With the continuous development of the research, especially the introduction of the genetic engineering (recombinant) IL-2 in 1983, the research of IL-2 has been advanced more. IL-2 has been found to act on a variety of immune effector cells, including B cells, macrophages, NK cells, in addition to T cells. IL-2 can stimulate the proliferation of B cells capable of producing antibodies and induce the production of novel killer cells, namely lymphokine activated killer cells (LAK), so that IL-2 becomes an important factor for regulating immune response, and has the effects of resisting viruses, resisting tumors and improving and enhancing the immune function of the organism. Can improve the immune response of human body to virus, bacteria, protozoon and other infections, and eliminate tumor cells and virus infected cells in vivo. Can also promote the secretion of antibody and interleukin-2, and stimulate the production of IFN-gamma and TNF-alpha, beta; promoting proliferation and activation of Th; activating neutrophils; stimulation of NK and Tumor Infiltrating Lymphocyte (TIL) proliferation. IL-2 has been used to treat tumors, immunodeficiency and infectious diseases, and has achieved significant results.
Currently, commercially available lL-2 mainly takes recombinant human interleukin-2 as a main component, takes genetic engineering bacteria as a basis, and finally obtains high-purity recombinant human interleukin-2 protein by utilizing a biological fermentation method and matching with a large-scale separation and purification technology. Because the recombinant protein is mainly obtained through fermentation expression, the components in the fermentation liquor are often complex, and substances such as living cells, dead cells, extracellular products, intracellular release substances, residual substrates and the like exist, so that the target protein is obtained from the complex environment, the biological activity of the protein is maintained, and the separation and purification work is relatively complex. The existing separation and purification needs to dissolve and renature the inclusion body, then carries out multi-step membrane separation and chromatography, and often has the defects of complicated steps, relatively low recovery rate and the like. The recombinant interleukin-2 expressed by the genetic engineering bacteria usually exists in the form of inclusion bodies, the inclusion bodies need to be denatured, cracked, purified and renatured, and disulfide bonds are easy to generate mismatching in the renaturation process, so that the activity of the interleukin-2 is reduced. Denaturant is often used in the denaturing and cracking process, and the activity of the product is inevitably affected when the product is further concentrated if the denaturant is not removed in the subsequent process.
And IL-2 is easily contaminated by endotoxin, which limits its therapeutic application. Endotoxin, also known as lipopolysaccharide, lipid a, a pyrogen, is a unique structure on the outer wall layer of the cell wall of gram-negative bacteria (GNB), the outer lipid component of the outer membrane of the cell wall is composed entirely of endotoxin molecules, which are released in large quantities after dissolution or artificial destruction of the bacterial cells when the bacteria die, and one escherichia coli cell contains about 200 million LPS molecules. Endotoxin has extremely high thermal stability and is decomposed only after 1 hour under dry heat at 250 ℃. Endotoxin is also insensitive to pH changes, but can be inactivated by high concentrations of acids or bases. Endotoxin molecules are negatively charged due to the phosphate group and can therefore theoretically be removed by positively charged adsorbents, such as anion exchange chromatography. However, in practice, this bonding effect is not good. The reason for this is, on the one hand, that the phosphate group (negative charge) carried on naturally occurring endotoxin molecules has been positively charged (Ca)2+，Mg2+，Na+，K+Etc.) or polyamines (cadaverine, spermine, spermidine, ethanolamine), etc.; on the other hand, in nature, endotoxin does not exist as a single molecule but exists as aggregates with different relative molecular masses, the endotoxin molecule and negative charges carried by the endotoxin molecule participate in the formation of aggregates, and few free negative charges are exposed on the appearance of the aggregates; furthermore, other negatively charged species are often present in the solution, making complete endotoxin separation difficult, e.g., diethylamine ethyl (DEAE) anion exchange media are only suitable for endotoxin removal from alkaline proteins. Thus, the removal of endotoxin contamination or elimination in solution has been a problem in biological research.
Disclosure of Invention
The invention aims to provide a preparation process of high-purity recombinant interleukin-2 with high recovery rate, high activity and high purity aiming at the problems in the prior art.
The purpose of the invention can be realized by the following technical scheme:
a preparation process of high-purity recombinant interleukin-2 comprises the following steps:
s1, preparing interleukin-2 renaturation liquid: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, extracting an inclusion body from the interleukin-2 fermentation liquor, cracking the inclusion body, and performing renaturation treatment to obtain interleukin-2 renaturation liquor;
s2, coarse purification treatment: adding the interleukin-2 renaturation solution into a hydrophobic high performance liquid chromatography column for adsorption and elution, and collecting eluent, namely a crude pure product;
s3, fine purification treatment: adding the crude pure product into a reversed-phase high performance liquid chromatography column for adsorption and elution, and collecting eluent to obtain a pure product;
s4, final purification treatment: and adding the refined pure product into a weak anion high-efficiency liquid chromatographic column for adsorption and elution, and collecting the eluent to obtain the high-purity recombinant interleukin-2.
The invention effectively removes the impurities such as nucleic acid, foreign protein, endotoxin and the like in the recombinant interleukin-2 fermentation liquor through three steps of separation and purification treatment, has high removal efficiency and can finally obtain the high-purity interleukin-2 product. The crude purification treatment is carried out by adopting a hydrophobic high-performance liquid chromatographic column, so that substances such as nucleic acid and the like in fermentation liquor can be effectively removed, the fine purification treatment is carried out by adopting a reversed-phase high-performance liquid chromatographic column, so that impurities such as hetero-protein, IL-2 polymer, isomer and the like which have smaller difference with IL-2 polarity can be effectively removed, and the final purification treatment is carried out by adopting a weak-anion high-performance liquid chromatographic column, so that endotoxin can be effectively removed, and the use safety of products is improved.
Preferably, the inclusion body extraction method in step S1 includes performing ultrasonic bacteria-breaking treatment on the recombinant interleukin-2 fermentation broth, then centrifuging, and washing with 40-60 mmol/L, PH 8.5.5-9.0 PBS buffer solution;
the inclusion body lysis is carried out in a lysis agent comprising the following components: 0.8-1.2 wt% of Sodium Dodecyl Sulfate (SDS), 8-13 mmol/L of Dithiothreitol (DTT) and 40-60 mmol/L of PB buffer solution, wherein the PH of the PB buffer solution is 7.0-8.0;
the renaturation treatment method comprises the following steps: firstly, 0.8-1.2 wt% Sodium Dodecyl Sulfate (SDS) is adopted to dilute the inclusion body lysate until the protein concentration is not more than 4mg/ml, then renaturation agent is added to stir and dilute until the protein concentration in the inclusion body lysate is 0.1-0.2 mg/ml and the SDS concentration is 0.05-0.06 wt%.
Preferably, the time for the inclusion body cracking is 5-7 h.
Preferably, the renaturation agent is 40-60 mmol/L PB buffer solution, and the pH value is 7.0-8.0.
When the Escherichia coli genetic engineering bacteria are adopted to produce IL-2, because IL-2 exists in an inner cell membrane of the Escherichia coli genetic engineering bacteria and has strong hydrophobicity, when the IL-2 gene is expressed in the Escherichia coli genetic engineering bacteria, the IL-2 is accumulated in cells to form insoluble protein polymers, namely inclusion bodies, because the Escherichia coli is a prokaryotic expression system and is not subjected to post-translational processing and modification, and the IL-2 generally exists in the cells of the Escherichia coli genetic engineering bacteria in the form of inclusion bodies. The inclusion bodies are spherical and are 0.5-1 mu m refractile corpuscles, most (more than 50 percent) of the inclusion bodies are products of clone expression, the primary structures of the products are correct, but the products are not folded correctly to form natural high-level structures, so that the biological activity is not generated. The inclusion body is dissolved and renatured to obtain the protein with biological activity. The inclusion bodies have relatively high relative hardness, are insoluble in water under common conditions, can be dissolved in the presence of strong denaturants such as guanidine hydrochloride, urea and the like, and the high-order structure of the protein is opened and exists in a denatured state, which brings great difficulty to the separation and purification of the IL-2. Therefore, in the case of isolation and purification, the cells are first disrupted to extract inclusion bodies, and then renaturation and isolation and purification are carried out.
The SDS in the cracking agent can effectively break various chemical bonds in the inclusion body protein molecules and among the molecules, destroy the secondary structure and the tertiary structure of the protein molecules, extend the polypeptide, and open all disulfide bonds in the inclusion body protein by using the DTT as a reducing agent, so that the inclusion body is fully cracked to form the polypeptide chain.
SDS is not only a cracking agent of an inclusion body (namely a denaturant of protein) but also a cosolvent of IL-2, IL-2 molecules and SDS are fully combined to form an IL-2-SDS complex with negative charges, the negative charges of the complex greatly exceed the original charge amount of the IL-2 molecules, and the original charge difference between different IL-2 molecules is eliminated, so that the solubility of the IL-2 is increased.
In the renaturation treatment process, SDS is added to dilute the inclusion body lysate to a specific concentration (not more than 4mg/ml), so that when renaturation agent is further added, the protein concentration and the SDS concentration in the inclusion body lysate are simultaneously controlled within a specific range, and IL-2 is prevented from polymerizing in the renaturation process; if the protein concentration in the inclusion body lysate is higher than 0.2mg/ml after renaturation treatment, or the SDS concentration is lower than 0.05 wt%, the IL-2 is easy to polymerize during renaturation.
Preferably, the packing of the hydrophobic high performance liquid chromatography column in step S2 is silica gel as a matrix, and hydrophobic groups are bonded on the surface of the silica gel, and the hydrophobic groups are phenyl groups.
Preferably, the column size of the hydrophobic high performance liquid chromatography column is: diameter 40mm L500 mm.
Preferably, the granularity of the filler of the hydrophobic high performance liquid chromatography column is 4-6 μm.
The invention adopts silica gel matrix chromatographic packing bonded with phenyl groups to adsorb the recombinant interleukin-2, and the phenyl groups have strong hydrophobic effect and excellent adsorption performance on the recombinant interleukin-2. In the coarse purification step, the hydrophobic high performance liquid chromatography column is adopted and the hydrophobic filler bonded with phenyl groups is preferably adopted to effectively separate the components by utilizing the different polarities of the components in the renaturation liquid, so that macromolecular nucleic acid substances and the like in the renaturation liquid and buffer salts and SDS in the renaturation liquid influencing the next separation are effectively removed, and the recombinant interleukin-2 with higher concentration and higher purity is obtained.
Preferably, the elution in step S2 includes performing a first elution with eluent i, and then performing a second elution with eluent ii, and collecting the effluent of the second elution as the crude pure product, where eluent i includes the following components: 8-12 v% ethanol, 1.8-2.2 mo1/L urea and 0.07-0.13 v% trifluoroacetic acid (TFA), wherein the eluent II comprises the following components: 30-50 v% ethanol, 0.07-0.13 v% trifluoroacetic acid (TFA).
The invention adopts a mode of two-time elution to elute the filler adsorbed with interleukin-2, wherein urea is added in the eluent I of the first elution and matched with ethanol with lower content, the aim is to weaken the adsorption effect of SDS and the filler, thereby washing out the SDS adsorbed on the filler and reserving the interleukin-2, the effluent of the first elution is detected by Lowry method, and the protein content is not detected, thereby avoiding that the SDS remained on the surface of the filler increases the reserving strength of the interleukin-2 on the filler, causing the dissociation effect of the interleukin-2 to be reduced, further reducing the recovery rate of the interleukin-2, simultaneously eliminating the SDS in the interleukin-2 and avoiding that the stability of the activity of the interleukin-2 is influenced by overhigh SDS concentration (more than 0.1%).
Preferably, the packing of the reversed-phase high performance liquid chromatography column in the step S3 takes silica gel as a matrix, and C4 groups are bonded on the surface.
The invention adopts the reversed-phase high performance liquid chromatography column and preferably selects the silica gel filler with the surface bonded with the C4 group to carry out fine purification treatment on the interleukin-2 after the coarse purification treatment, thereby removing impurities with smaller difference with IL-2 polarity, such as heteroprotein, IL-2 polymer, isomer and the like in the coarse purification product, and further improving the purity of the interleukin-2 product and the specific activity of the IL-2 product in unit mass.
The fine purification treatment of the invention adopts the silica gel filler of which the surface is bonded with the C4 group, but does not adopt the silica gel filler bonded with the C8 or C18, because the strength of IL-2 reserved by the C4 group is weaker, the IL-2 can be eluted from the filler by using the organic solvent eluent with lower solubility, thereby not influencing the activity of the IL-2; the retention of the C8 or the C18 on the IL-2 is too strong, and the IL-2 can be eluted from the filler by high-concentration organic solvent eluent; high concentration organic solvent eluent can damage the activity of IL-2, which is not favorable for the stability of the activity of IL-2.
The crude pure product contains components such as ethanol and TFA, the pH value of the crude pure product is 2-3, and IL-2 is soluble in the pH range and contains ethanol (the ethanol can be used as a cosolvent), so that the crude pure product can be directly added into a reversed-phase high-performance liquid chromatography column for fine purification treatment to remove impurities such as heteroprotein, IL-2 polymer, isomer and the like.
Preferably, the column size of the reversed phase high performance liquid chromatography column is: diameter 40mm L500 mm.
Preferably, the granularity of the filler of the reversed-phase high-performance liquid chromatography column is 4-6 μm.
The invention limits the filler granularity of the reversed-phase high-performance liquid chromatography column in a smaller range, improves the surface area of the filler and further improves the cut-off amount.
Preferably, the elution in the step S3 includes performing a preliminary elution with a solution a, and then increasing a solution B and gradually increasing a ratio of the solution B to the solution a to perform a gradient elution, where the solution a is a mixture of 8-12 v% ethanol and 0.08-0.12 v% trifluoroacetic acid, and the solution B is a mixture of 40-60 v% ethanol and 0.08-0.12 v% trifluoroacetic acid.
Preferably, the time of the preliminary elution in the step S3 is 0-60 min, the time of the gradient elution is 60-180 min, and the flow rate of the elution is 25-35 ml/min.
Preferably, before the purified product is added into the weak anion high performance liquid chromatography column for adsorption and elution in step S4, a cosolvent Sodium Dodecyl Sulfate (SDS) is added into the purified product, the PH is adjusted to 7.0-7.5, and then sodium chloride is added.
Preferably, the addition amount of the cosolvent Sodium Dodecyl Sulfate (SDS) is 0.03-0.07 wt% of the pure product; the concentration of the sodium chloride in the refined pure product is 0.8-1.2 mol/L.
Preferably, the step of dialysis is carried out after the pH is adjusted to 7.0-7.5, and the dialysate comprises the following components in percentage by weight: 8 to 12mmol/L, PH of 7.0 to 7.5 Tris-HCl buffer solution and 0.03 to 0.07 wt% SDS.
Ethanol is introduced in the steps of coarse purification and fine purification, and if the ethanol is not removed, the adsorption function of the weak anion high performance liquid chromatography column on IL-2 and endotoxin is weakened, so the ethanol is removed by a dialysis method.
Preferably, the anion high performance liquid chromatography column of step S4 uses DEAE anion exchange packing.
According to the invention, DEAE anion exchange filler is preferably used as adsorption filler, the adsorption filler has better adsorption performance on IL-2 and endotoxin under the adsorption environment condition of the invention, and different reagents are adopted during elution to respectively elute IL-2 and endotoxin.
Preferably, the DEAE anion exchange filler of the step S4 has a particle size of 4 to 6 μm.
The invention limits the filler particle size in a smaller range, improves the surface area of the filler, and improves the cut-off amount of IL-2 and endotoxin.
Preferably, the weak anion high performance liquid chromatography column of step S4 has a column size of 40mm × L250mm in diameter.
Preferably, the elution in the step S4 includes performing a first elution on the weak anion high performance liquid chromatography column with a balancing solution, where the balancing solution contains sodium chloride with a concentration of 0.8-1.2 mol/L, performing a second elution with a Tris-HCL buffer solution, and collecting an effluent of the second elution, that is, the high-purity recombinant interleukin-2.
Preferably, the equilibrium liquid comprises the following components and the contents thereof,
Tris-HCl buffer: 8 to 12mmol/L, pH 7.0 to 7.5,
NaCl：0.8～1.2mol/L。
according to the invention, the DEAE anion exchange filler is adopted to adsorb IL-2 and endotoxin in the IL-2 sample at the same time, in order to improve the solubility of IL-2, a cosolvent SDS is added in the IL-2 sample, SDS will be attached, then a balancing solution is adopted to wash out the SDS, and a Tris buffer solution is adopted to dissociate IL-2, so that endotoxin and IL-2 are separated, and the IL-2 product with low endotoxin is obtained. After obtaining an IL-2 product with low endotoxin, the column is subjected to a regeneration treatment, thereby making the column recyclable.
The crude pure product of the invention contains components such as ethanol and TFA, the pH value is 2-3, under the pH condition, the IL-2 is below the isoelectric point (the isoelectric point of the IL-2 is about 6.5) and can not be adsorbed by DEAE anion exchange filler, so the pH of the crude pure product needs to be adjusted to be neutral and alkalescent firstly, and both endotoxin and IL-2 can be adsorbed by the DEAE anion exchange filler.
IL-2 is highly hydrophobic, and precipitates in neutral or alkaline aqueous solutions, and is not easily dissolved, and precipitates more easily when the concentration of IL-2 is higher. According to the invention, SDS is added into the recombinant interleukin-2 sample I, the IL-2 molecules and the SDS are fully combined to form an IL-2-SDS compound with negative charges, the negative charges of the compound greatly exceed the original charge quantity of the IL-2 molecules, and the original charge difference between different IL-2 molecules is eliminated, so that the solubility of IL-2 under a weak alkaline condition is increased, the adsorption of IL-2 on anion fillers is also increased, and the retention strength of endotoxin on the anion fillers under a NaCl-containing condition is also increased. Without the addition of SDS, IL-2 of the present application was difficult to dissolve and the next step was performed.
In addition, 0.8-1.2 mol/L sodium chloride is added into a recombinant interleukin-2 sample before sample loading adsorption, the sodium chloride is an ionic compound, and sodium ions with positive charges can effectively weaken strong adsorption of SDS with negative charges and the surface of DEAE anion filler, so that the situation that the SDS preferentially occupies the adsorption area to reduce the adsorption quantity of IL-2 is avoided, and meanwhile, the situation that high-concentration SDS is eluted along with IL-2 after the SDS and the filler are adsorbed and accumulated and dissociated to influence the stability of IL-2 activity is avoided, and the situation that the retention strength of the IL-2 in the filler is increased due to the SDS remaining in the filler is also avoided, so that the recovery rate of the IL-2 is influenced.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, through three steps of separation and purification treatment, impurities such as nucleic acid, foreign protein, endotoxin and the like in the recombinant interleukin-2 fermentation liquor are effectively removed, the removal efficiency is high, and a high-purity interleukin-2 product can be finally obtained;
(2) according to the invention, by adopting the hydrophobic high performance liquid chromatography column for crude purification treatment, substances such as nucleic acid in recombinant interleukin-2 fermentation liquor can be effectively removed;
(3) the invention is carried out by adopting the reverse phase high performance liquid chromatography column for fine purification, and can effectively remove impurities with smaller difference with IL-2 polarity, such as heteroprotein, IL-2 polymer, isomer and the like in the recombinant interleukin-2 fermentation liquor;
(4) according to the invention, the weak anion high-efficiency liquid chromatographic column is adopted for final purification treatment, so that endotoxin in recombinant interleukin-2 fermentation liquor can be effectively removed, and the use safety of the product is improved;
(5) the invention effectively removes the influence of the cosolvent and ensures the recovery rate of the interleukin-2 by optimizing the auxiliary reagent in the coarse purification and final purification treatment.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.
Example 1
The preparation of high purity recombinant interleukin-2 in this example was as follows:
(1) preparing interleukin-2 renaturation solution: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, carrying out ultrasonic bacterium breaking treatment on the recombined interleukin-2 fermentation liquor, then centrifuging, and washing by using 5mmol/L, PH 9.0.0 PBS buffer solution to obtain an inclusion body; adding a cracking agent (the components of the cracking agent are 1.0 wt% SDS, 10mmol/L DTT and 50mmol/L, PH 7.5.5 PB buffer solution) into the washed inclusion body, and carrying out cracking treatment for 6h to obtain an inclusion body cracking solution; then 1.0 wt% SDS is adopted to dilute the inclusion body lysate until the protein concentration is not more than 3mg/ml, then 50mmol/L, PH PB buffer solution of 7.5 is added, and the mixture is stirred and diluted until the protein concentration in the inclusion body lysate is 0.2mg/ml and the SDS concentration is 0.05 wt%, thus obtaining the interleukin-2 renaturation solution.
(2) Coarse purification treatment: adding interleukin-2 renaturation solution into a hydrophobic high-performance liquid chromatography column for adsorption, wherein the hydrophobic high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded phenyl groups, and the column size is 40mm multiplied by L500 mm; then, carrying out first elution by adopting an eluent I, wherein the eluent I comprises the following components: 10 v% ethanol, 2mo1/L urea and 0.1 v% TFA, and then carrying out secondary elution by using an eluent II, wherein the eluent II comprises the following components: 40% ethanol and 0.1% TFA, and collecting effluent of the second elution to obtain the crude pure product.
(3) Fine purification treatment: adding the crude pure product into a reversed-phase high-performance liquid chromatography column for adsorption, wherein the reversed-phase high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded C4 groups, and the column size is 40mm multiplied by L500 mm; and then, carrying out primary elution by adopting the solution A in 0-60 min, then, increasing the solution B in 60-180 min and gradually increasing the ratio of the solution B to the solution A to carry out gradient elution, wherein the elution flow rate is 25-35ml/min, the solution A comprises 10 v% ethanol and 0.1 v% TFA, and the solution B comprises 50 v% ethanol and 0.1 v% TFA.
(4) Final purification treatment: adding 0.05 wt% of cosolvent SDS into the refined pure product, adjusting the pH to 7.5, and dialyzing (the components of the dialyzate are 10mmol/L, PH 7.5.5 Tris-HCl buffer solution and 0.05 wt% SDS); adding sodium chloride into the obtained dialysate to a concentration of 1.0mol/L, and adsorbing in an anion high performance liquid chromatography column, wherein the anion high performance liquid chromatography column adopts DEAE anion exchange filler with a particle size of 5 μm, and the column size is 40mm × L250 mm; after adsorption, the anion high performance liquid chromatography column is eluted for the first time by adopting a balance liquid, and the balance liquid comprises the following components: Tris-HCl buffer solution with pH7.5 and 10mmol/L, and NaCl 1.0 mol/L; and then carrying out secondary elution by adopting a Tris-HCL buffer solution with the pH value of 7.5, and collecting effluent liquid of the secondary elution, namely the high-purity recombinant interleukin-2.
Example 2
The preparation of high purity recombinant interleukin-2 in this example was as follows:
(1) preparing interleukin-2 renaturation solution: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, carrying out ultrasonic bacterium breaking treatment on the recombined interleukin-2 fermentation liquor, then centrifuging, and washing by using 40mmol/L, PH 9.0.0 PBS buffer solution to obtain an inclusion body; adding a cracking agent (the components of the cracking agent are 1.2 wt% SDS, 13mmol/L DTT and 60mmol/L, PH 8.0.0 PB buffer solution) into the washed inclusion body, and carrying out cracking treatment for 5h to obtain an inclusion body lysate; then diluting the inclusion body lysate to 4mg/ml protein concentration by adopting 1.2 wt% SDS, adding 40mmol/L, PH PB buffer solution of 7.0-8.0, stirring and diluting until the protein concentration in the inclusion body lysate is 0.19mg/ml and the SDS concentration is 0.05 wt%, and obtaining interleukin-2 renaturation solution;
(2) coarse purification treatment: adding interleukin-2 renaturation solution into a hydrophobic high-performance liquid chromatography column for adsorption, wherein the hydrophobic high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded phenyl groups, and the column size is 40mm multiplied by L500 mm; then, carrying out first elution by adopting an eluent I, wherein the eluent I comprises the following components: 8 v% ethanol, 1.5mo1/L urea and 0.07 v% TFA, and then carrying out secondary elution by using an eluent II, wherein the eluent II comprises the following components: 30 v% ethanol and 0.07 v% TFA, and collecting effluent of the second elution to obtain the crude pure product.
(3) Fine purification treatment: adding the crude pure product into a reversed-phase high-performance liquid chromatography column for adsorption, wherein the reversed-phase high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded C4 groups, and the column size is 40mm multiplied by L500 mm; and then, carrying out primary elution by adopting the solution A in the 0-60 min period, then, increasing the solution B in the 60-180 min period and gradually increasing the ratio of the solution B to the solution A to carry out gradient elution, wherein the elution flow rate is 25-35ml/min, the solution A comprises 8 v% ethanol and 0.12 v% TFA, and the solution B comprises 40 v% ethanol and 0.12 v% TFA.
(4) Final purification treatment: 0.07 wt% of SDS as a cosolvent was added to the purified product, and the pH was adjusted to 7.3, and dialysis was performed (composition of dialysate: 12mmol/L, PH 7.3.3 Tris-HCl buffer, 0.07 wt% SDS); adding sodium chloride into the obtained dialysate to a concentration of 1.2mol/L, and adsorbing in an anion high performance liquid chromatography column, wherein the anion high performance liquid chromatography column adopts DEAE anion exchange filler with a particle size of 5 μm, and the column size is 40mm × L250 mm; after adsorption, the anion high performance liquid chromatography column is eluted for the first time by adopting a balance liquid, and the balance liquid comprises the following components: Tris-HCl buffer solution with pH7.3 and 12mmol/L and NaCl 1.2 mol/L; and then carrying out secondary elution by adopting a Tris-HCL buffer solution with the pH value of 7.3, and collecting effluent liquid of the secondary elution, namely the high-purity recombinant interleukin-2.
Example 3
The preparation of high purity recombinant interleukin-2 in this example was as follows:
(1) preparing interleukin-2 renaturation solution: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, carrying out ultrasonic bacterium breaking treatment on the recombined interleukin-2 fermentation liquor, then centrifuging, and washing by using 45mmol/L, PH 8.5.5 PBS buffer solution to obtain an inclusion body; adding a cracking agent (the components of the cracking agent are 1.1 wt% SDS, 12mmol/L DTT and 55mmol/L, PH 8.0.0 PB buffer solution) into the washed inclusion body, and carrying out cracking treatment for 6h to obtain an inclusion body lysate; then diluting the inclusion body lysate to a protein concentration of 3mg/ml by adopting 1.1 wt% SDS, adding 55mmol/L, PH PB buffer solution of 8.0, stirring and diluting until the protein concentration in the inclusion body lysate is 0.18mg/ml and the SDS concentration is 0.06 wt%, and obtaining interleukin-2 renaturation solution;
(2) coarse purification treatment: adding interleukin-2 renaturation solution into a hydrophobic high-performance liquid chromatography column for adsorption, wherein the hydrophobic high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded phenyl groups, and the column size is 40mm multiplied by L500 mm; then, carrying out first elution by adopting an eluent I, wherein the eluent I comprises the following components: 9 v% ethanol, 2.2mo1/L urea, 0.11 v% TFA, and then carrying out second elution by using an eluent II, wherein the eluent II comprises the following components: 43 v% ethanol, 0.11 v% TFA, and collecting the effluent of the second elution as the crude pure product.
(3) Fine purification treatment: adding the crude pure product into a reversed-phase high-performance liquid chromatography column for adsorption, wherein the reversed-phase high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded C4 groups, and the column size is 40mm multiplied by L500 mm; and then, carrying out primary elution by adopting the solution A in the 0-60 min period, then, increasing the solution B in the 60-180 min period and gradually increasing the ratio of the solution B to the solution A to carry out gradient elution, wherein the elution flow rate is 25-35ml/min, the solution A comprises 9 v% ethanol and 0.11 v% TFA, and the solution B comprises 53 v% ethanol and 0.09 v% TFA.
(4) Final purification treatment: 0.06 wt% of cosolvent SDS was added to the purified product, and the pH was adjusted to 7.4, and dialysis was performed (composition of dialysate: 9mmol/L, PH 7.4.4 Tris-HCl buffer, 0.06 wt% SDS); adding sodium chloride into the obtained dialysate to a concentration of 1.1mol/L, and adsorbing in an anion high performance liquid chromatography column, wherein the anion high performance liquid chromatography column adopts DEAE anion exchange filler with a particle size of 5 μm, and the column size is 40mm × L250 mm; after adsorption, the anion high performance liquid chromatography column is eluted for the first time by adopting a balance liquid, and the balance liquid comprises the following components: Tris-HCl buffer solution with pH7.4 and 9mmol/L, and NaCl 1.1 mol/L; and then carrying out secondary elution by adopting a Tris-HCL buffer solution with the pH value of 7.4, and collecting effluent liquid of the secondary elution, namely the high-purity recombinant interleukin-2.
Example 4
The preparation of high purity recombinant interleukin-2 in this example was as follows:
(1) preparing interleukin-2 renaturation solution: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, carrying out ultrasonic bacterium breaking treatment on the recombined interleukin-2 fermentation liquor, then centrifuging, and washing by using 60mmol/L, PH 9.0.0 PBS buffer solution to obtain an inclusion body; adding a cracking agent (the components of the cracking agent are 0.8 wt% SDS, 8mmol/L DTT and 40mmol/L, PH 7.5.5 PB buffer solution) into the washed inclusion body, and carrying out cracking treatment for 7h to obtain an inclusion body lysate; then 0.8 wt% SDS is adopted to dilute the inclusion body lysate to the protein concentration of 2mg/ml, then 60mmol/L, PH PB buffer solution of 7.5 is added, and the mixture is stirred and diluted until the protein concentration in the inclusion body lysate is 0.15mg/ml and the SDS concentration is 0.05 wt%, thus obtaining interleukin-2 renaturation solution;
(2) coarse purification treatment: adding interleukin-2 renaturation solution into a hydrophobic high-performance liquid chromatography column for adsorption, wherein the hydrophobic high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded phenyl groups, and the column size is 40mm multiplied by L500 mm; then, carrying out first elution by adopting an eluent I, wherein the eluent I comprises the following components: 12 v% ethanol, 2.5mo1/L urea, 0.13 v% TFA, and then carrying out second elution by using an eluent II, wherein the eluent II comprises the following components: 50 v% ethanol and 0.13 v% TFA, and collecting effluent of the second elution to obtain the crude pure product.
(3) Fine purification treatment: adding the crude pure product into a reversed-phase high-performance liquid chromatography column for adsorption, wherein the reversed-phase high-performance liquid chromatography column adopts filler with the granularity of 5 mu m, silica gel matrix and surface bonded C4 groups, and the column size is 40mm multiplied by L500 mm; and then, carrying out primary elution by adopting the solution A in 0-60 min, then, increasing the solution B in 60-180 min and gradually increasing the ratio of the solution B to the solution A to carry out gradient elution, wherein the elution flow rate is 25-35ml/min, the solution A comprises 12 v% ethanol and 0.08 v% TFA, and the solution B comprises 60 v% ethanol and 0.08 v% TFA.
(4) Final purification treatment: 0.03 wt% of a cosolvent SDS was added to the purified product, and the pH was adjusted to 7.5, and dialysis was performed (composition of dialysate: 8mmol/L, PH 7.5.5 Tris-HCl buffer, 0.03 wt% SDS); adding sodium chloride into the obtained dialysate to reach concentration of 0.8mol/L, and adsorbing in anion high performance liquid chromatography column with DEAE anion exchange filler with particle size of 5 μm and diameter of 40mm × L250 mm; after adsorption, the anion high performance liquid chromatography column is eluted for the first time by adopting a balance liquid, and the balance liquid comprises the following components: Tris-HCl buffer solution with pH of 7.5 and 8mmol/L and NaCl of 0.8 mol/L; and then carrying out secondary elution by adopting a Tris-HCL buffer solution with the pH value of 7.5, and collecting effluent liquid of the secondary elution, namely the high-purity recombinant interleukin-2.
Comparative example 1
The crude purification treatment was carried out using a silica gel packing bonded with a butyl group, and the amount of the packing and the column size were increased by 50%, and the rest was the same as in example 1.
Comparative example 2
The first elution in the crude purification treatment was carried out without addition of urea, and the rest was the same as in example 1.
Comparative example 3
The purification treatment was carried out using a silica gel filler bonded with a C8 group, and the rest was the same as in example 1.
Comparative example 4
The purification treatment was carried out using a silica gel filler bonded with a C18 group, and the rest was the same as in example 1.
Comparative example 5
In the final purification treatment, sodium chloride is not added into the pure product, and the pure product is directly adsorbed by a weak anion high performance liquid chromatography column, and the rest is the same as the example 1.
Comparative example 6
Sodium chloride was added to the purified product in the final purification treatment to give a concentration of 0.7mol/L, and the other steps were the same as in example 1.
Comparative example 7
The endotoxin in the recombinant interleukin-2 sample I is removed by adopting a gel permeation method, and specifically, the recombinant interleukin-2 sample I is added into a gel S200 column for separation, the size of the column is 50mm multiplied by L1200mm, the sample injection volume is 50ml, the sample injection amount is not more than 200mg, the mobile phase is 10% acetonitrile and 0.01% TFA, and the separation flow rate is 120 ml/hr.
The characteristics of the crude pure products obtained in examples 1 to 4 of the present invention and comparative examples 1 to 2 were compared, and the comparison results are shown in table 1. The specific activity is determined by CTLL-2 dependent cell strain/MTT colorimetric method, the protein content is determined by Lowry micro-method, the protein recovery rate is total protein content of the crude pure product/total protein content of renaturation liquid is multiplied by 100%, and the purity of interleukin-2 in the crude pure product is determined by SDS-PAGE electrophoresis method.
Table 1: comparison of characteristics of crude pure products in examples 1 to 4 and comparative examples 1 to 2
Comparing the comparative example 1 with the example 1, it can be known that the interleukin-2 can be completely adsorbed by adopting other hydrophobic groups, and the renaturation liquid protein amount which is the same as that in the example 1 can be completely adsorbed only when the filler amount is increased by 50%, so that the recovery rate of the interleukin-2 is not obviously reduced, and the cost is obviously increased, which shows that the silica gel filler bonded with the phenyl group used in the invention has obviously excellent adsorption performance on the interleukin-2.
As is clear from comparison between comparative example 2 and example l, the recovery of interleukin-2 in the crude pure product was significantly reduced without urea addition in the first elution because SDS remaining on the surface of the filler increased the retention strength of interleukin-2 in the filler, and the dissociation effect was reduced without increasing the solvent concentration in the eluent II, thereby reducing the recovery of interleukin-2.
The pure treatment adopts C8 silica gel filler of comparative example 3 or C18 silica gel filler of comparative example 4, the interleukin-2 is difficult to elute by adopting the solution A and the solution B with the same concentration as the solution A and the solution B of example 1, and only trace amount of protein can be detected in the effluent.
The characteristics of the high-purity recombinant interleukin-2 samples prepared in examples 1 to 4 and comparative examples 5 to 7 of the present invention were compared, and the comparison results are shown in table 2. The endotoxin content of the high-purity recombinant interleukin-2 samples in examples 1 to 4 and comparative examples 5 to 7 was detected by limulus reagent, the result of endotoxin content in 100 ten thousand IU of recombinant interleukin-2, the endotoxin content in comparative example 7 was the endotoxin content in 10 ten thousand IU of recombinant interleukin-2, the recovery rate was total amount of injected sample/total amount of high-purity recombinant interleukin-2 × 100%, and the purity was detected by SDS-PAGE electrophoresis and HpLC-C18.
Table 2: comparison of characteristics of high-purity recombinant Interleukin-2 in examples 1 to 4 and comparative examples 5 to 7
As can be seen from Table 2, the detection results of the recombinant interleukin-2 sample for removing endotoxin prepared by the invention show that the endotoxin content in 100 ten thousand IU of the recombinant interleukin-2 is less than 0.25EU, the split-charging dose endotoxin which is superior to the national standard requirement is less than 10EU/ml, and the detection method is superior to other methods, for example, the gel permeation method is adopted in the comparative example 1, the endotoxin removal effect can only reach the maximum of 10 ten thousand IU which is less than 0.25EU of limulus reagent, the separation speed is slow, the sample injection amount is limited, and the maximum interleukin-2 concentration of 5mg/ml can only reach 2-5% of the column volume. Meanwhile, the method has higher recovery rate of interleukin-2, which can be more than 95%. The invention finally obtains the high-quality interleukin-2 product through a three-step purification process, the quality indexes of the product are superior to the national standard, and the purity of SDS-PAGE electrophoresis detection and HpLC-C18 detection can reach more than 98 percent.
The technical scope of the invention claimed by the embodiments of the present application is not exhaustive, and new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the invention claimed by the present application; in all the embodiments of the present invention, which are listed or not listed, each parameter in the same embodiment only represents an example (i.e., a feasible embodiment) of the technical solution, and there is no strict matching and limiting relationship between the parameters, wherein the parameters may be replaced with each other without departing from the axiom and the requirements of the present invention, unless otherwise specified.
The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and the technical scheme also comprises the technical scheme formed by any combination of the technical characteristics. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.
Claims (9)
1. A preparation process of high-purity recombinant interleukin-2 is characterized by comprising the following steps:
s1, preparing interleukin-2 renaturation liquid: forming interleukin-2 fermentation liquor by using escherichia coli gene recombination, extracting an inclusion body from the interleukin-2 fermentation liquor, cracking the inclusion body, and performing renaturation treatment to obtain interleukin-2 renaturation liquor;
s2, coarse purification treatment: adding the interleukin-2 renaturation solution into a hydrophobic high performance liquid chromatography column for adsorption and elution, and collecting eluent, namely a crude pure product;
s3, fine purification treatment: adding the crude pure product into a reversed-phase high performance liquid chromatography column for adsorption and elution, and collecting eluent to obtain a pure product;
s4, final purification treatment: and adding the refined pure product into a weak anion high-efficiency liquid chromatographic column for adsorption and elution, and collecting the eluent to obtain the high-purity recombinant interleukin-2.
2. The process of claim 1, wherein the step S1 of extracting the inclusion bodies comprises subjecting the fermentation broth of recombinant interleukin-2 to ultrasonic disruption, centrifuging, and washing with 40-60 mmol/L, PH 8.5.5-9.0 PBS buffer;
the inclusion body lysis is carried out in a lysis agent comprising the following components: 0.8-1.2 wt% of Sodium Dodecyl Sulfate (SDS), 8-13 mmol/L of Dithiothreitol (DTT) and 40-60 mmol/L of PB buffer solution, wherein the PH of the PB buffer solution is 7.0-8.0;
the renaturation treatment method comprises the following steps: firstly, 0.8-1.2 wt% of Sodium Dodecyl Sulfate (SDS) is adopted to dilute the inclusion body lysate until the protein concentration is not more than 4mg/ml, then renaturation agent is added to stir and dilute until the protein concentration in the inclusion body lysate is 0.1-0.2 mg/ml, and the concentration of the Sodium Dodecyl Sulfate (SDS) is 0.05-0.06 wt%.
3. The process of claim 1, wherein the hydrophobic HPLC column of step S2 is filled with silica gel as a matrix, and hydrophobic groups are bonded to the surface of the silica gel, and the hydrophobic groups are phenyl groups.
4. The process of claim 1, wherein the step S2 comprises eluting with eluent I, and then eluting with eluent II, and collecting the eluate of the second elution as a crude product, wherein the eluent I comprises the following components: 8-12 v% ethanol, 1.8-2.2 mo1/L urea and 0.07-0.13 v% trifluoroacetic acid (TFA), wherein the eluent II comprises the following components: 30-50 v% ethanol, 0.07-0.13 v% trifluoroacetic acid (TFA).
5. The process of claim 1, wherein the packing material of the reversed-phase HPLC column of step S3 is silica gel-based and has C4 group bonded to its surface.
6. The process of claim 1, wherein the step S3 of eluting includes performing a preliminary elution with solution A, and then increasing solution B and gradually increasing the ratio of the amount of solution B to the amount of solution A to perform a gradient elution, wherein the solution A is a mixture of 8-12 v% ethanol and 0.08-0.12 v% trifluoroacetic acid, and the solution B is a mixture of 40-60 v% ethanol and 0.08-0.12 v% trifluoroacetic acid.
7. The process for preparing high-purity recombinant interleukin-2 according to claim 1, wherein step S4 is further performed by adding Sodium Dodecyl Sulfate (SDS) as a cosolvent to the purified product, adjusting the PH to 7.0-7.5, and then adding sodium chloride before adding the purified product to the weak anion high performance liquid chromatography column for adsorption and elution.
8. The process of claim 1, wherein the anion hplc column of step S4 uses DEAE anion exchange packing.
9. The process of claim 8, wherein the step S4 of eluting comprises eluting the weak anion hplc column with a balance solution containing sodium chloride at a concentration of 0.8-1.2 mol/L for the first time, eluting with Tris-HCL buffer solution for the second time, and collecting the eluate of the second elution, i.e. the high purity recombinant interleukin-2.
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