CN105153294A - Recombinant insulin and insulin analogue precursor purification method - Google Patents
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- CN105153294A CN105153294A CN201510548164.2A CN201510548164A CN105153294A CN 105153294 A CN105153294 A CN 105153294A CN 201510548164 A CN201510548164 A CN 201510548164A CN 105153294 A CN105153294 A CN 105153294A
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Abstract
The invention relates to the field of insulin production methods and discloses a recombinant insulin and insulin analogue precursor purification method. The invention solves the problems that conventional chromatographic packing cannot tolerate high-salinity sample loading, the amount of used organic reagent is great, the cost is high and the product purity is not high in the purification process of recombinant expressed insulin precursors and insulin analogue precursors. The invention adopts the technical scheme that the method comprises the steps of performing pH regulation and centrifugation to centrifuged fermentation supernatant, then directly loading a sample and performing adsorption, separation, purification and elution through a chromatographic column prepared by using any packing of Capto S, Capto MMC, Uni PMM S and Uni MSP to finally obtain high-purity recombinant insulin and insulin analogue precursors. Compared with the existing purification method, the recombinant insulin and insulin analogue precursor purification method has the advantages that the operation is simple, the yield is high, the spent time is short, the environmental influence is small, and the product production cost of the existing insulin and insulin analogues can be greatly reduced.
Description
Technical field
The present invention relates to insulin production method field, particularly a kind of Recombulin and insulin analog precursor purification process.
Background technology
Diabetes are only second to cardiovascular and the third-largest fatal disease that is tumour, and within 2014, world diabetic subject has reached 3.87 hundred million, and annual in accelerating rising number.Regular Insulin be treatment diabetes specifics, particularly patients with terminal treatment time, do not have other medicines to replace, along with diabetic subject crowd constantly expand and insulin administration mode variation increase, insulin dosage just increases sharply.In the evolution of Regular Insulin, animal insulin spreads due to immunogenicity and the transmissible disease such as mad cow disease and animal foot and mouth disease, clinically eliminates gradually; Be used widely clinically with the Recombulin analogue that Lantus, insulin aspart, Insulin lispro, insulin detemir and moral paddy Regular Insulin are representative, and occupy most Regular Insulin market.
Along with the progress of recombinant DNA technology, utilize microorganism as fermentation platforms such as intestinal bacteria, pichia spp, yeast saccharomyces cerevisiaes, obtain a large amount of expression of insulin precursors to become a reality, particularly pichia pastoris phaff, it is using methyl alcohol as sole carbon source and the BSM substratum of employing cheapness carries out high density fermentation, efficient secretory expression recombinant protein, fermentation costs is low.But in pichia spp fermenting process, not only can produce a large amount of pigment, and the inorganic salt content remained in fermented liquid is very high, conductivity value can reach 40-50mS/cm, so the insulin precurosor of expressing before purification, must to fermented supernatant fluid carry out ultrafiltration change liquid or suitably dilution reduce conductance, so that proinsulin can realize separation and purification in conjunction with chromatographic stuffing.
As everyone knows, the acidic buffer linear gradient elution that insulin regular precursor purification mode is employing 0.5 ~ 1.0MNaCl, pH value is 2 ~ 4, the human insulin analogue precursor solution pH that this mode obtains is for acid, and a large amount of Na existed
+can not only the activity of inhibitory enzyme, but also need to carry out exchange buffering liquid and removing metal ion just can carry out follow-up endonuclease reaction, extend manufacture cycle, increase production cost.
Conventional cationic exchange coloum (SP-fastflow) purified insulin analogue precursor (MIP) is used disclosed in patent CN1699412A, but conventional ion displacement chromatography just need can carry out purifies and separates to the dilution of sample large volume after reducing salt concn or ultrafiltration desalting treatment, extend the production cycle, add the input of production unit; Application XAD-7 hydrophobic adsorbent filler (Sigma company) separation and purification-revolve and steam except organic reagent-gel-filtration (SephadexG25) removes pigment and par-tial polysaccharide is disclosed in this patent Example 4, then cationic exchange coloum SP-Fastflow (AmershamBiosciences company) is utilized to remove the impurity such as greasiness class, insulin precurosor (MIP) purity more than 90% of collecting, the processing step that it adopts is long, organic reagent consumption is large, hydrophobic adsorbent chromatography exchange capacity is low, desalting steps productive rate is low, is not suitable for large-scale production.
Patent CN101029077A discloses the tactful purified genes Recombulin precursor of a kind of gac in conjunction with cationic exchange (filler is: SP550EC), ultrafiltration desalination, gac after fermented liquid directly adds process, centrifugal after stirring, supernatant obtains insulin precurosor through activated carbon filtration, permeate through Cationic column chromatography, ultrafiltration desalination, freeze-drying again.This processing step also needs to carry out dialysis desalination to supernatant liquor, and processing step is loaded down with trivial details equally, is not suitable for large-scale industrial and produces.
Patent CN102816819A is by adopting ion-exchange and reversed phase chromatography method purifying successively by insulin analog precursor albumen; but the program still exists aforementioned chromatographic stuffing can not tolerate high salt loading; need sample desalting treatment, the scheme therefore proposed is difficult to be applied to large-scale industrial and produces.
In sum, the Conventional chromatography filler that recombinant expressed Regular Insulin and insulin analog precursor exist in purification process can not tolerate high salt loading, the inorganic salt that conventional dewatering filling exists in chromatography, organic reagent consumption is large, cost is high, with serious pollution problem, so purifying process has to increase the processing step reducing salt concn on strategy, liquid desalination is changed as increased ultrafiltration, the processing steps such as large volume dilution, so not only increase equipment investment, and reduce the insulin precurosor rate of recovery significantly, therefore from the high salt supernatant liquor of pichia spp fermentation, the method for extraction purification target protein is the difficult point of Regular Insulin purification technique.
Summary of the invention
The present invention is directed to the technical deficiency in existing Recombulin precursor and Recombulin analogue precursor purification preparation technology, propose a kind of easy and simple to handle, process of preparing that cost is lower.
Technical scheme of the present invention comprises the following steps:
(1) fermented liquid pre-treatment: fermentation liquor acid for adjusting pH, obtains the supernatant liquor of insulin-containing precursor or insulin analog precursor albumen by centrifugal or filtration etc.; Gained fermented liquid conductivity range is between 10-55mS/cm;
(2) balance: with the post bed of acid equilibration buffer chromatography column;
(3) loading: by the supernatant liquor loading in step (1) to the chromatography column in step (2), then uses the impurity that the acid Equilibration buffer wash in step (2) does not adsorb;
(4) wash-out: with the chromatography column in elution buffer elution step (3), obtain the elution peak of Recombulin or insulin analog precursor.
In described step (1) fermented liquid pre-treatment, regulate the acid of pH can be one or more in glacial acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, pH regulator be to 2.0-6.0; The specific conductivity of described supernatant liquor is between 10 ~ 55mS/cm; The preferred pH of described supernatant is 3.0-4.0, and preferred specific conductivity is loading absorption under 20 ~ 40mS/cm condition.
In the chromatography column adopted in described step (2), chromatographic stuffing is any one in CaptoS, CaptoMMC, UniPMMS, UniMSP, and the height of filling is 8-25cm, preferred 8-15cm.Wherein CaptoMMC is compound weak cation exchange aglucon, and CaptoS is compound strong cation exchange aglucon, the monodisperse polymer filler of UniPMMS and UniMSP to be high crosslinked polypropylene or polystyrene/divinylbenzene polymkeric substance be matrix.
In above-mentioned filler, Capto series is the new packing that a new generation is matrix with highly cross-linked sepharose; MMC is the cation exchange group of composite ligand, and S is the strong cation exchange group group of composite ligand; Its adsorption process has salt-tolerant trait, thus this aglucon can in direct injection under high conductivity and and protein binding on aglucon.
UniPMMS, UniMSP are that S, MSP represent that its cation exchange groups is strong cation exchange group group with polystyrene/divinylbenzene or polyacrylic ester matrix a new generation polymer packing.In the Uni series filler efficient analysis that is widely used in small organic molecule, albumen, polypeptide, nucleic acid, natural product and chemical synthetic drug and separation and purification process.Polystyrene/divinylbenzene or polyacrylic ester matrix make it have certain hydrophobicity, and its adsorption process has salt-tolerant trait, can in direct injection under high conductivity and and protein binding on aglucon.
Acid level pad in described step (2) and step (3) is any one in glycine-HCI damping fluid, phthalic acid-hydrochloride buffer, Sodium phosphate dibasic-citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate damping fluid, acetic acid-sodium acetate buffer solution, phosphate buffered saline buffer; Balance acidic buffer concentration is 5 ~ 200mM, preferred 10-100mM, more preferably 20-50mM; The pH of damping fluid is 2.0 ~ 6.0, and preferable range is 3.0-4.0.
In described step (4), wash-out ealkaline buffer is a kind of buffer system in Glycine-NaOH damping fluid, Tris-hydrochloride buffer, phosphate buffered saline buffer, the concentration range of wash-out ealkaline buffer is 5mM ~ 200mM, preferable range 20-100mM; PH is 8.0 ~ 10.0, preferred pH8.0-9.5.
The present invention has suitability widely to restructuring insulin precurosor, insulin analog precursor or derivatives thereof, those skilled in the art can improve or optimize the preparation method of Regular Insulin or its analogue precursor according to the present invention, its purification technique is all applicable to multiple insulin analog precursor.
Term used herein " restructuring ", refers to adopt genetic engineering means, applies the technology of recombinant DNA or recombinant RNA thus the protein of acquisition.
Term used herein " Regular Insulin " refers to the natural insulins such as insulin human, Sigma I8405, pork insulin.
Term used herein " insulin analog " refers to carry out amino acid sites sudden change, disappearance or interpolation to natural insulin, makes it have a para-insulin of new physiological property, as Lantus, insulin aspart, Insulin lispro, DesB
30regular Insulin etc.
Term used herein " precursor ", refers to the Regular Insulin after natural insulin and sudden change, as insulin human, pork insulin, Sigma I8405, insulin aspart, Lantus, Insulin lispro etc. do not form the precursor protein of Regular Insulin duplex structure.
Compared with traditional extracting and purifying method, the present invention has obvious technique effect:
(1) method removals such as dilution being adopted to inorganic salt excessive in fermented liquid supernatant, liquid is changed in ultrafiltration is not needed, chromatography column in the method directly can adsorb, be separated restructuring insulin precurosor or analogue precursor under high salt condition, one step yield more than 85%, the precursor of purity more than 90%, and can most pigment be removed, by the direct step separation and purification of original multi-step process, reduce process time and equipment investment cost;
(2) compared with hydrophilic hydrophobic exchange chromatography, do not need to use a large amount of inorganic salt to do moving phase; Compared with nonaqueous phase hydrophobic adsorbent, do not use a large amount of organic reagents, environmental pollution is little, and cost is lower;
(3) directly can efficiently carry out endonuclease reaction after utilizing the insulin precurosor of elution buffer wash-out to add trypsinase in the present invention and prepare DesB
30-Regular Insulin, process continuity is better, and operation steps is simple, consuming time short.
Accompanying drawing explanation
Fig. 1 utilizes the HPLC of the preparation figure of CaptoS column separating purification insulin precurosor and corresponding insulin precurosor elution peak to analyze collection of illustrative plates under 55mS/cm height salt condition in embodiment 1;
Fig. 2 is that the HPLC of column separating purification insulin precurosor collection of illustrative plates and corresponding insulin precurosor elution peak under CaptoMMC55mS/cm height salt condition in embodiment 2 analyzes collection of illustrative plates;
Fig. 3 is that the HPLC of column separating purification insulin precurosor collection of illustrative plates and corresponding insulin precurosor elution peak under UniPMMS55mS/cm height salt condition in embodiment 3 analyzes collection of illustrative plates;
Fig. 4 is that the HPLC of column separating purification insulin precurosor collection of illustrative plates and corresponding insulin precurosor elution peak under UniMSP55mS/cm height salt condition in embodiment 4 analyzes collection of illustrative plates;
Embodiment
The embodiment of the invention discloses a kind of Recombulin and insulin analog precursor purification process, those skilled in the art can use for reference present disclosure, suitable improving technique, it is to be noted, all similar replacements and change are easy to do for a person skilled in the art, and they are all deemed to be included in the present invention.
Embodiment 1: utilize the filler CaptoS in the application to be separated recombinant human DesB with control filler SP-Sepharose6FF
30insulin precurosor
(1) fermented liquid pre-treatment: fermented liquid derives from pichia spp fermented liquid, be 2.0 through hydrochloric acid adjustment pH, centrifugal treating 15min, 8000g, 4-10 DEG C, collect centrifuged supernatant, measures conductivity value 55mS/cm; HPLC detects supernatant purity 38%, content 3.5mg/ml;
For testing the impact of different specific conductivity on purification result, be the fermentation of 55mS/cm by above-mentioned specific conductivity, water use regulation be 40,20,3 parts of 10mS/cm, test respectively.
(2) equipment/filler/buffered soln
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatography column: XK16/20 post, determined wavelength: 280nm.
Chromatographic stuffing: filler 1: the filler CaptoS in the technical program, post bed height: 8cm; Column volume 20ml (loading voluntarily); Filler 2: common chromatographic stuffing SP-Sepharose6FF: column length: 8cm; Column volume 20ml;
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min.
Level pad: 100mM glycine-HCI damping fluid, pH2.0;
Elution buffer: 200mM Glycine-NaOH damping fluid, pH10.0
(3) operation steps
A. with equilibrating buffer balance filler 5 column volumes, 10ml/min, walks to put down or fluctuation≤10mAU to UV baseline;
B.10ml/min, sample introduction 60 ~ 100ml;
C. with equilibrating buffer balance filler 5 column volumes, 10ml/min;
D. with elution buffer eluant solution, peak is collected, HPLC detected result.
(4) experimental result
Table 1CaptoS, SP-Sepharose6FF purifies and separates recombinant human DesB
30insulin precurosor result
Comparison finds that CaptoS can adsorb under high salt condition, separation and purification by experiment, obtain the purity of sample and yield all meets industrialized requirement, and common filler SP-Sepharose6FF cannot binding purposes albumen, only ability well binding purposes albumen after reduction salt concn under high salt condition.
CaptoS is along with the reduction of salt concn simultaneously, and purification effect is better, and the purity of elution peak is higher, and the yield of product is also higher.
Embodiment 2:CaptoMMC purifies and separates recombinant human DesB
30insulin precurosor
(1) sample source: with embodiment 1.
(2) equipment/filler/buffered soln condition used in this embodiment is as follows:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatography column: XK16/20 post, determined wavelength: 280nm.
Chromatographic stuffing: CaptoMMC, post bed height: 10cm; Column volume 20ml (loading voluntarily);
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min.
Level pad: 100mM glycine-HCI damping fluid, pH2.0;
Elution buffer: 200mMTris-HCl damping fluid, pH8.5.
(3) operation steps: with embodiment 1.
(4) experimental result
Table 2CaptoMMC purifies and separates recombinant human DesB
30insulin precurosor result
Embodiment 3:UniPMMS purifies and separates recombinant human DesB
30insulin precurosor
(1) sample source: with embodiment 1.
(2) equipment/filler/buffered soln condition used in this embodiment is as follows:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatography column: XK16/20 post, determined wavelength: 280nm.
Chromatographic stuffing: UniPMMS, post bed height: 10cm; Column volume 20ml (loading voluntarily);
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min;
Level pad: 80mM citric acid-sodium citrate damping fluid, pH4.0;
Elution buffer: 100mM Glycine-NaOH damping fluid, pH8.5;
(3) operation steps: with embodiment 1.
(4) experimental result is as follows:
Table 3UniPMMS separation and purification recombinant human DesB
30insulin precurosor result
Embodiment 4:UniMSP purifies and separates recombinant human DesB
30insulin precurosor
(1) sample source: with embodiment 1.
(2) equipment/filler/buffered soln condition used in this embodiment is as follows:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatography column: XK16/20 post, determined wavelength: 280nm.
Chromatographic stuffing: UniMSP, post bed height: 14cm; Column volume 20ml (loading voluntarily);
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min;
Level pad: 80mM acetic acid-sodium acetate buffer solution, pH4.0;
Elution buffer: 150mMTris-HCl damping fluid, pH9.5
(4) other conditions are with embodiment 1, and experimental result is as follows:
Table 4UniMSP purifies and separates recombinant human DesB
30insulin precurosor result
Embodiment 5:CaptoS purifies and separates recombinant human insulin aspart precursor
(1) fermented liquid pre-treatment: derive from recombinant human insulin aspart pichia spp fermented liquid, be 4.0 through phosphoric acid adjustment pH, centrifugal treating 15min, 8000g, 4-10 DEG C, collect centrifuged supernatant, and pure water adjustment specific conductivity is to 35mS/cm; HPLC detects supernatant purity 67%, content 6.3mg/ml.
(2) equipment/filler/buffered soln:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.Chromatography column: XK16/20 post.Determined wavelength: 280nm.
Chromatographic stuffing: CaptoS, column length: 8cm; Column volume 20ml (loading voluntarily).
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min.
Level pad: 10mM phthalic acid-hydrochloride buffer, pH4.0;
Elution buffer is washed: 100mMTris-hydrochloride buffer, pH8.0.
(3) operation steps is with embodiment 1.
(4) collect insulin aspart precursor elution peak, HPLC detects purity 92.02%, yield 89.36%.
Embodiment 6:UniMSP purifies and separates restructuring Insulin lispro precursor
(1) fermented liquid pre-treatment: derive from restructuring Insulin lispro precursor pichia spp fermented liquid, be 6.0 through citric acid adjustment pH, centrifugal treating 15min, 8000g, 4-10 DEG C, collect centrifuged supernatant, and pure water adjustment conductivity value is to 20mS/cm; HPLC detects supernatant purity 54%, content 3.2mg/m.
(2) equipment/filler/buffered soln:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatography column: XK16/20 post, determined wavelength: 280nm.
Chromatographic stuffing: CaptoS, column length: 8cm; Column volume 20ml (loading voluntarily).
Level pad: 10mM Sodium phosphate dibasic-citrate buffer solution, pH3.0;
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min.
Elution buffer: 20mM Glycine-NaOH damping fluid, pH8.5
(3) operation steps: with embodiment 1.
(4) result: collect insulin precurosor HPLC purity 92.45%, insulin precurosor yield 90.11%.
Embodiment 7:UniPMMS purifies and separates recombinant human Lantus precursor
(1) fermented liquid pre-treatment: derive from pichia spp fermented liquid, be 4.0 through phosphoric acid adjustment pH, centrifugal treating 15min, 8000g, 4-10 DEG C, collect centrifuged supernatant, and pure water adjustment conductivity value is to 30mS/cm; HPLC detects supernatant purity 44%, content 5.1mg/ml, sample introduction 80ml.
(2) equipment/filler/buffered soln:
Equipment: AKTAPureM1 tomography devices (GE company of the U.S.), full wavelength UV detector, flow rates 0.01-20ml/min.
Chromatographic stuffing: UniPMMS, column volume 20ml.
Sampling volume: 70 ~ 80ml, linear rate of flow: 300cm/h, volumetric flow rate: 10ml/min.
Equilibrating buffer: 5mM SODIUM PHOSPHATE, MONOBASIC-disodium hydrogen phosphate buffer solution, pH4.0;
Elution buffer: 10mM SODIUM PHOSPHATE, MONOBASIC-disodium hydrogen phosphate buffer solution, pH8.0;
(3) operation steps is with embodiment 1.
(4) insulin precurosor HPLC purity 93.87% is collected, insulin precurosor yield 90.63%.
Claims (10)
1. Recombulin and an insulin analog precursor purification process, comprises the following steps:
(1) fermented liquid pre-treatment: fermented liquid with acid for adjusting pH, the supernatant liquor of centrifugal acquisition insulin-containing precursor or insulin analog precursor albumen; Gained fermented liquid conductivity range is between 10-55mS/cm;
(2) balance: with the post bed of acid equilibration buffer chromatography column;
(3) loading: by the supernatant liquor loading in step (1) to the chromatography column in step (2), then uses the impurity that the acid Equilibration buffer wash in step (2) does not adsorb;
(4) wash-out: with the chromatography column in ealkaline buffer elution step (3), obtain the elution peak of Recombulin or insulin analog precursor.
2. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, it is characterized in that, in step (1) fermented liquid pre-treatment, regulate the acid of pH can be one or more in glacial acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid.
3. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, is characterized in that, the centrifugal supernatant pH2.0-6.0 of fermented liquid in step (1), preferred pH is 3.0-4.0.
4. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, is characterized in that, in step (1), the preferred specific conductivity of the centrifugal supernatant of fermented liquid is 20 ~ 40mS/cm.
5. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, it is characterized in that, in the chromatography column adopted in step (2), chromatographic stuffing is any one in CaptoS, CaptoMMC, UniPMMS, UniMSP.
6. a kind of Recombulin and insulin analog precursor purification process according to claim 1 or 5, is characterized in that, in step (2), the height of chromatography column chromatographic stuffing filling is 8-25cm, preferred 8-15cm.
7. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, it is characterized in that, the acid level pad in step (2) and step (3) is any one in glycine-HCI damping fluid, phthalic acid-hydrochloride buffer, Sodium phosphate dibasic-citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate damping fluid, acetic acid-sodium acetate buffer solution, phosphate buffered saline buffer.
8. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, it is characterized in that, balance acidic buffer concentration in step (2) and step (3) is 5 ~ 200mM, preferred 10-100mM, more preferably 20-50mM; The pH of damping fluid is 2.0 ~ 6.0, and preferable range is 3.0-4.0.
9. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, it is characterized in that, step (4) neutral and alkali damping fluid is a kind of buffer system in Glycine-NaOH damping fluid, Tris-hydrochloride buffer, phosphate buffered saline buffer.
10. a kind of Recombulin according to claim 1 and insulin analog precursor purification process, is characterized in that, in step (4), the concentration range of wash-out ealkaline buffer is 5mM ~ 200mM, preferable range 20-100mM; PH is 8.0 ~ 10.0, preferred pH8.0-9.5.
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CN112430252A (en) * | 2020-11-13 | 2021-03-02 | 宜昌东阳光长江药业股份有限公司 | Chromatography method for improving recovery rate of target protein |
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