Preparation of recombinant human insulin - study on downstream purification process

Preparation of recombinant human insulin - study on downstream purification process

Abstract by a set of E. coli expression (H is) 6

2A rg2A rg2 human proinsulin [ (H is) 6

2A rg2A rg2human p ro insulin,

RRhP I] A process for preparing recombinant human insulin. RRhP I expressed in the form of inclusion bodies in E. coli was sequentially subjected to DEA E2 agarose fast flow anion exchange chromatography, Sephadex G225 chromatography, recombinant refolding and Superdex 75 molecular sieve to prepare recombinant human insulin. The results of SDS2PA GE, HPLC, amino acid composition analysis and mouse convulsion experiment proved that the prepared recombinant human insulin has natural biological activity and high purity.

Keywords purified recombinant human insulin DEA E2 Sepharose Fast Flow anion exchanger Superdex75

1 Introduction

Separation and purification is an extremely important part of the production of genetic engineering drugs. Because of the large-scale cultivation of engineered bacteria, the content of active ingredients is very low, and the content of impurities is very high. In addition, since genetic engineering drugs are derived from transformed cells, It is not produced from normal cells, so the purity requirements of the products are higher than those of traditional products. Therefore, in order to obtain genetically engineered drugs that meet medical requirements, separation and purification are much more difficult than traditional products. In the genetic engineering production of recombinant human insulin (Recom b inan t hum anin su lin, rh I), it also faces a very complicated problem of downstream processing. In order to effectively purify the target product, methods such as affinity chromatography and HPLC are commonly used in genetic engineering production. These separation and purification techniques are expensive and highly specific, but they are very expensive. Not conducive to reducing costs. In order to reduce the production cost, this study established a downstream purification process for the preparation of recombinant human insulin from (H is) 62A rg2A rg2 human proinsulin [ (H is) 62A rg2A rg2 hum an p ro in su lin, RRhP I ]. The nature and purity of the product were identified.

2 Materials and methods

2.1 engineered bacteria

RRhP Iö pQ E240 Escherichia coli M 15 strain.

2 . 2 main reagents

DEA E2 agarose fast flow anion exchanger, Sephadex, Superdex75, trypsin and carboxypeptidase B, low molecular weight protein, human insulin, glutathione (oxidized and reduced), DTT, other chemical reagents are domestic or Import flight is pure.

2 . 3 　 Preliminary purification of RRhP I

DEA E2 agarose fast flow anion exchange chromatography column with 30mmo löL T ris2HCl, 8 mo löL urea, pH 8. 0 balance, inclusion body with 30 mmo löL T ris2HCl, 8 mo löL urea, 50mmo löL DTT, pH 8. 0 dissolved After the upper column, elution with a suitable sodium chloride gradient, SDS2PA GE determines the RRhP I fraction, and collects the eluate containing RRhP I. 2. 6 Refolding and restriction enzyme digestion The preliminary purified RRhP I was dehydrogenated on a Sephadex G225 column, and the transformation buffer was used for recombinant renaturation. Then, the RRhP I was transformed by trypsin and carboxypeptidase B. The reaction was terminated and precipitated for human insulin (Hum an in su lin, h I), 0.1 mol to L ZnCl 2 .

2 . 4 　 Purification of h I

The crude h I was dissolved in 30 mmo löL T ris2HCl, 8 mo löL urea, pH 8. 0, and purified on Superdex 75. The pH of the solution is 0. 2 mo löL sodium acetate 2 acetic acid, pH 4. 0.

2 . 5 　 HPLC analysis

The analysis was carried out on an HPLC apparatus using an ODS C18 reverse phase column (150 mm × 6. 0 mm), and the mobile phase was 0. 2mol löL(NH4) 2SO 4 solution (1 mo löL H3PO 4 adjusted pH to 3.5) and 50% The acetonitrile aqueous solution was mixed at a ratio of 1:1 (vöv) at a flow rate of 1.0 m löm in, a detection wavelength of 214 nm, and a loading of 20 ul.

2. 10 amino acid composition analysis

The sample was weighed 2. 5 mg, added with 6 mo löL HCl 2 ml, hydrolyzed at 110 ° C for 24 h, evaporated to dryness, dissolved in 2 ml of ion-free water, directly injected, and analyzed for amino acid composition on an amino acid analyzer.

2 . 11 　 Identification of h I overall biological activity

The overall activity of h I was identified by the method of convulsion in mice according to the 2000 edition of the Pharmacopoeia of the People's Republic of China.

3 results

3 . 1 　 Preliminary purification of RRhP I

The chromatogram is shown in Figure 1, and RRhP I is mainly concentrated in peak 2. Collect peak 2 for SDS2PA GE analysis, see Figure 2. The results showed that the electrophoresis bands were mainly concentrated near 13 KD, and the peak 1 was the breakthrough peak. This peak contained a protein with a p I higher than 8.0 and some hydrophobic proteins, and most of the proteins with a lower p I than RRhP I It is firmly bound to the column with nucleic acid impurities and requires a higher salt concentration to elute.

Table 1 shows the results of repeated experiments on 6 chromatograms. It can be seen that the recovery rate of RRhP I is relatively high and is stable at around 75% to 80%.

Table 1 RRhP I recovery of ion exchange chromatography

Table 1 RRhP I recovery of DEAE-Sepharrose Fast Flow ion exchange chromatography

Test number ( n = 6) RRhP I recovery (% )

1 75. 2

2 77. 0

3 75. 5

4 78. 2

5 79. 6

6 79. 8

x ± s 77. 55±1. 81

3.2 renaturation and refolding

The preliminary purified RRhP I was dehydrogenated by Sephadex G225 column chromatography and the conversion buffer was 50 mmo löL glycine 2N aOH, pH 9.5. The chromatogram (see Figure 3) has two chromatographic peaks, and peak 2 is mainly the monomer RRhP I that folds toward the correct one, and peak 2 is collected. Perform an SDS2PA GE analysis. The results (see Figure 2) show that the amount of high molecular weight hybrid protein in the collected RRhP I samples is reduced. Peak 1 is a dimer or multimer of high molecular weight hybrid proteins and RRhP I and an incomplete or incorrect RRhP I fold.

3 . 3 　 RRhP I digestion and transformation h I

In the collected RRhP I monomer component, 2. 5 mmo löL reduced glutathione and 0.25 mmo löL oxidized glutathione were added to control the 2SH ratio, and stable formation by exchange of disulfide bonds Natural structure of RRhP I. Co-digestion with trypsin (1ö500, enzyme öRRhP I substrate) and carboxypeptidase B (1ö1000, enzyme öRRhP I substrate) at 37 ° C 30- 40 m in, (H is) 62A rg2A rg and C The peptide was accurately excised and RRhP I was converted to h I . The results of SDS2PA GE showed that RRhP I was almost completely digested into h I , and the position of the main electrophoresis band was moved from 13 KD to a position parallel to the human insulin standard, as shown in Figure 2.

3 . 4 　 Purification of h I

The crude h I product precipitated with 0.1 mol LöL ZnCl2 can be purified by Superdex 75 as shown in Fig. 4, and h I is mainly concentrated at peak 3. Peak 3 was collected for SDS2PA GE analysis and the results showed that, as shown in Figure 2, the obtained h I components were homogeneous in the SDS2PA GE analysis with only one protein band. The peak 3 fraction is dialyzed, concentrated and lyophilized to finally yield h I .

Characterization of 3.5 products

3 . 5 . 1 　 The results of the SDS2PA GE analysis (see Figure 2) indicate that the product obtained in this study has the same molecular weight as the h I standard and is of higher purity with a protein band in the SDS2PA GE. 3. 5. 2 HPLC analysis The retention time of the main peak of the recombinant insulin preparation prepared by this process (27. 757 m in) is basically consistent with the retention time of the standard h I (27. 968 m in), and the retention time with porcine insulin (25. 437 m in) There are significant differences.

Figure 4 Superdex 75 purification hI

Fig 4 Gel f iltration of hI on Superdex 75Peak a and peak b: impurities, Peak c: h I

3.5.. 3 amino acid composition analysis results in Table 2, the results show that recombinant insulin acid samples prepared according to the process of composition is consistent with h I. The sample does not contain Me, and the (H is) 62A rg2A rg expressed together with proinsulin has been accurately excised. Only one A rg and two H is left in the sample, indicating that RRhP I has been successfully converted into H I.

3 . 5 . 4 h I Identification of the overall biological activity 5 Kunming clean mice (male 20 ~ 24 g), subcutaneous injection of h I sample 1. 25 U per 20 g body weight, appeared within 2 h after injection Positive hypoglycemic reaction, including 4 convulsions. Immediately, 1 ml of 5% glucose injection was injected intraperitoneally, and the convulsions disappeared. This indicates that the h I sample prepared by the process has good hypoglycemic activity.

4 Discussion

4 . 1 　 Preliminary purification of RRhP I

In order to capture target products quickly and accurately from a large number of heteroproteins, highly specific affinity chromatography is an ideal choice. By specifically recognizing the six histidine residues in RRhP I, the target product RRhP I[ 3 ] can be purified in one step by N i2+ 2N TA affinity chromatography. However, the N i2+ 2N TA affinity medium is relatively expensive, which is bound to increase the production cost, so it is difficult to promote the application in actual production, and can only be used for expression screening. In order to reduce the cost, this study used the relatively inexpensive DEA E2 agarose fast flow anion exchange chromatography to purify the target product RRhP I. 5%±1. 81% (n= 6). The yield of the electrophoresis band was mainly concentrated at MW 13 KD, and the yield was 77.5%±1.81% (n=6). At present, there is no report on the purification of recombinant insulin products by domestic DEA E2 agarose fast flow anion exchange chromatography.

4.2 Refolding and enzyme conversion

RRhP I expressed in the form of inclusion bodies is poorly water-soluble due to mismatched disulfide bonds. In order to increase the solubility of RRhP I in the DEA E2 agarose fast flow anion exchange chromatography, a large amount of urea was added to the buffer. At this time, RRhP I is in a denatured environment. Therefore, urea must first be removed prior to double digestion to allow RRhP I to renature. In this study, Sephadex G225 was used to remove the denaturing agent, which could effectively prevent protein aggregation and induce the denatured protein RRhP I to fold into its natural state. Chromatography was carried out under the conditions we used to obtain two chromatographic peaks, and peak 2 was the monomer RRhP I which was folded to be correct. Among them, the amount of high-scoring heteroprotein is reduced. Peak 1 is a high molecular weight heteroprotein, poly RRhP I and an incomplete or incorrect RRhP I fold. Re-chromatography and folding reactions of this fraction can increase the overall yield of folding. It can be seen that this step chromatography can achieve the triple effect of purification, removal of denaturant and induction of renaturation. In vivo, there are two Ca2+-dependent endopeptidases and carboxypeptidase H involved in the proinsulin transformation process. In vitro, pro-proteolysis with trypsin and carboxypeptidase B can also convert proinsulin to insulin [4, 5], which makes it possible to prepare human insulin by expressing human proinsulin with E. coli. In the expression product constructed in this study, two Arg were inserted between the leader peptide (H is) 6 and proinsulin, which is the same as the connection between the C peptide and insulin, and thus is converted in double digestion. At the same time, (H is) 62A rg2A rg can be accurately removed without additional processing, thus reducing the post-processing. Under the conditions of the enzyme digestion selected in this study, RRhP I was accurately and efficiently converted into h I . SDS2PA GE analysis showed that the enzyme digestion was more complete and the main electrophoresis band was transferred from 13 KD to a position parallel to the human insulin standard.

4 . 3 insulin purification

Preparative HPLC is currently commonly used for the final purification of genetically engineered products [6-8]. This is because HPLC has the advantages of convenient operation, high resolution, and high purity of the product to meet clinical requirements at one time. However, HPLC has the disadvantages of high requirements on hardware and software, large initial investment, limited purification, and the like, so that the production capacity is affected. In this study, Superdex 75 molecular sieve chromatography was used in the final purification of human insulin. It has high mechanical strength, good stability, good reproducibility, small particle size (24-44L) and high resolution (13 000 p latesöm). Fast flow rate (0. 85～4.4 4 m löm in, 25°C), and can be operated at low pressure, wide pH range (3~12), wide separation range (spherical protein, 3～70KD), non-specific adsorption It has the advantages of large loading capacity, easy control of chromatographic conditions, etc. It is an ideal chromatographic method for purifying genetic engineering products. Use 0.2 mol löL sodium acetate 2 acetic acid, pH 4.0 buffer as the balance solution and

The eluate, after the crude h I was subjected to Superdex 75 column chromatography, we obtained a higher purity human insulin preparation, and the SDS2PA GE analysis sample had only one band. There have been no reports on the use of Superdex 75 purified genetic engineering products. Insulin is a natural biologically active substance that must maintain its natural secondary structure in order to exert its biological activity of lowering blood sugar. The positive results of the mouse convulsion experiment fully indicate that the chromatographic conditions selected in this study not only effectively purify the expression product, but also effectively prevent the inactivation and degeneration of the target product h I , and finally the preparation of the higher purity has Naturally active genetically engineered human insulin.