WO2017114216A1 - Method for preparing rhcnb dimer - Google Patents

Abstract

Provided is a method for preparing recombinant human calcineurin subunit B (rhCNB) dimer. The method comprises the following steps: step 1, preparing an rhCNB coarse pure liquid; and step 2, on the reaction condition that the pH value ranges from 6.0 to 7.0 and the temperature ranges from 2ºC to 15ºC, taking the rhCNB coarse pure liquid and mixing the rhCNB coarse pure liquid with an oxidizing agent, conducting oxidation for 12h to 24 h, and after oxidation is ended, conducting purification and separation. In the method, a final product is rhCNB dimer, substances such as β mercaptothanolb, DTT, EGTA are not contained; according to a purity detection conducted on a collected protein solution, the content of the dimer is higher than 98%, and the content of a polymer is smaller than 0.5%.

Description

[Name of invention made by ISA according to Rule 37.2] Method for preparing rhCNB dimer

The present application claims priority to Chinese Patent Application No. 201511024337.7, entitled "A Method for Preparing a RhCNB Dimer", filed on December 30, 2015, the entire contents of which is incorporated herein by reference. In the application.

Technical field

The invention relates to the field of biotechnology, and in particular to a method for preparing a rhCNB dimer.

Background technique

CNB is a regulatory subunit of calcineurin, CN). CN is the only protein phosphatase that is known to be dependent on Ca2+/CaM, which has a relatively narrow substrate specificity. In the late 1970s and early 1980s, Chinese scholar Wang Xuejing, American Chinese Zhang Yiyao and American Claude B. Klee discovered and purified from the cattle brain. The enzyme consists of A and B disubunits in a ratio of 1:1. The A subunit (CNA) is a catalytic subunit with a relative molecular mass of 61×10 ³ ; the B subunit (CNB) is a regulatory subunit with a relative molecular mass of 19×10 ³ .

Professor Wei Qun found in the study of CNB for about 20 years that CNB has a good effect of inhibiting tumor growth at the animal and cell level, and it is at a higher level than other anticancer drugs, and the toxic and side effects are extremely low. Wide range of applications. And in 1998 applied for the patent "pharmaceutical composition containing calcineurin B subunit", patent number 98117642.9, which discloses a method for separating and purifying a pharmaceutical composition containing calcineurin B subunit Specifically, the method is as follows: “The crushed bacteria are subjected to a boiling water bath at 100° C. for 30-40 minutes, then centrifuged at 12000 rpm for 20 minutes, and the supernatant is taken, which is a crude extract of the CaNB subunit. Add 3 mmol/L CaCl ₂ by volume, 1 mmol. Phenel-sepharose CL-4B chromatography equilibrated with /Lβ thiol ethanol and 0.5 mol/L NaCl in a buffer (20 mmol/L Tris, pH 7.4, 0.5 mmol/L CaCl ₂ 1 mmol/L β-mercaptoethanol) The column was washed with the same buffer, and finally eluted with buffer 20 mmol/L tris, pH 7.4, 1 mmol/L EGTA, 0.5 mmol/L DTT. Electrophoresis pure CaNB subunit was obtained per liter of bacterial solution. ～120 mg, the obtained product can be stored in a freeze-dried manner.

The product obtained by the purification process described in Patent No. 98117642.9 is a pharmaceutical composition of CaNB, and the substance with β-mercaptoethanol, DTT, EGTA and the like in the process is not removed in the next step.

Therefore, a method for providing a high-purity rhCNB dimer has important practical significance.

Summary of the invention

In view of this, the present invention provides a method of preparing a rhCNB dimer. The final product of the process of the invention is rhCNB dimer, and the process is innovative and reproducible, and is suitable for pilot test and production scale.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a method for preparing a rhCNB dimer comprising the following steps:

Step 1: preparing a rhCNB solution;

Step 2: Under the reaction conditions of pH 6.0-7.0 and 2-15 ° C, the rhCNB solution is mixed with an oxidizing agent, oxidized for 12-24 hours, and the oxidation is terminated, and then purified and separated.

In the present invention, the crude pure liquid is a protein solution obtained by preliminary purification of the cells by cell wall breaking and separation, and further purification is required.

In some embodiments of the invention, the oxidizing agent is added in a stepwise manner every 1 to 5 hours.

In some embodiments of the invention, the mass ratio of the rhCNB solution to the oxidizing agent is from 20:1 to 30:1.

In some embodiments of the invention, the oxidizing agent is a mixture of one or more of hydrogen peroxide, iodine, oxidized glutathione, glutathione redox couple or oxygen.

In some embodiments of the invention, the concentration of the oxidizing agent is from 0.05% to 1% (w/w).

In some embodiments of the present invention, the oxidative-terminated reagent is an oxidative stop solution, and the oxidative stop solution is one or two of sodium hydrogen sulfite, sodium sulfite, 0.1% DTT or reduced glutathione. The above mixture.

In some embodiments of the invention, the concentration of the oxidative stop solution is from 0.05% to 1% (w/w).

In some embodiments of the invention, the mass ratio of the rhCNB solution to the oxidation terminating liquid is from 50:1 to 100:1.

In some embodiments of the invention, the separation and purification are performed by size exclusion chromatography using 10 mM sodium citrate-sodium citrate buffer at a pH of 5.8-7.0. Elution was carried out under conditions, and the rhCNB dimer protein peak was collected.

In some embodiments of the invention, the size exclusion chromatography employs a medium having a separation range of 3000 to 70,000 Da.

Specifically, the method for preparing the rhCNB protein solution comprises: culturing the fermentation liquid containing the target protein through a suitable medium, collecting the wet cells containing the target protein by centrifugation in a tube centrifuge, and adding 5 to 20 times of the resuspension buffer. Wherein the resuspension buffer comprises a reducing substance, an enzyme inhibitor; uniformly stirring and accumulating the heteroprotein at a suitable temperature, and collecting the supernatant after centrifugation. The addition of the reducing substance is to maintain a high content of the rhCNB single-chain structure in the process, and the recovery rate is improved as compared with the case where no reducing substance is added. Among them, suitable medium contains: Tryptone 20～50g, Yeast Extract 9～35g, sodium chloride 4～12g, glycerol 12～50g, potassium dihydrogen phosphate 2～10g, dipotassium hydrogen phosphate trihydrate 2 to 10 g and 2 g of sodium hydroxide.

The supernatant was collected and applied to a hydrophobic medium chromatography column equilibrated by the equilibration solution A, Phenyl Sepharose 6 Fast Flow (low sub) packing, and the unbound protein was washed away by the equilibration solution A, and the heteroprotein eluent B was The hydrophobic protein is washed away, and finally the target protein is eluted with the target protein eluent C to obtain a protein of interest containing the CNB composition. Among them, each 1 kg of the balance liquid A contains: TRIS 1.5 to 3.5 g, sodium chloride 30 to 70 g, calcium chloride 0.2 to 1.0 g, and hydrochloric acid 1.0 to 1.5 g. Each 1 kg of the heteroprotein eluent B contains: TRIS 1.5 to 3.5 g, calcium chloride 0.2 to 1.0 g, and hydrochloric acid 1.0 to 1.5 g. Each 1 kg of the protein eluent C contained: TRIS 1.5 to 3.5 g, EGTA 0.1 to 0.5 g, and hydrochloric acid 1.0 to 1.5 g.

The invention provides a method for preparing a rhCNB dimer, comprising the following steps: Step 1: preparing a rhCNB solution; Step 2: taking the rhCNB under a reaction condition of pH 6.0-7.0, 2-15 ° C The solution is mixed with an oxidizing agent and oxidized for 12 to 24 hours. After the oxidation is terminated, it is separated and purified.

The invention forms more than 90% of the monomers under the reduction of the reducing agent DTT, and then converts more than 99% of the monomers into dimers by using a special redox pair, and effectively controls the polymerization by concentration, temperature and time. The formation of the body gives a high content of dimer rhCNB. The process route is a reduction-oxidation-reduction termination oxidation.

1. The preliminary purified rhCNB solution contains more than 90% of the monomers. In this step, the pH is 6.0-7.0, the temperature is 2-15 °C, and the low-concentration oxidant (0.05%~1% hydrogen peroxide solution) is added in stages. In order to slowly convert the rhCNB monomer to the dimer, when the dimer reaches 75% or more and the polymer is 1%, the low concentration oxidation stop solution (0.1% sodium hydrogen sulfite) is added to neutralize the oxidant to avoid further oxidation. The formation of trimers and above polymers. The principle of the conversion is that the rhCNB monomer contains a free sulfhydryl group. Under the oxidizing environment, the two free sulfhydryl groups form a disulfide bond to achieve the purpose of conversion to a dimer, taking into account the pH value of the buffer and the reaction rate of the temperature. influences.

2. The CNB composition containing the higher content of the dimer in the step 2 still contains the monomer and a small amount of the trimer. In this step, the CNB composition is applied to the molecular exclusion chromatography column equilibrated by the equilibrium liquid (including The molecular exclusion filler) is eluted sequentially by the buffer according to the molecular weight, and the purity of the dimer is more than 98%, and the trimer and the above polymer are <0.5%.

Based on the above experimental results, the final product of the process of the invention is rhCNB dimer, the process is innovative and reproducible, and the collected protein solution is tested for purity, the dimer content is above 98%, and the multimer content is less than 0.5%. .

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art description will be briefly described below.

Figure 1 shows the results of the ratio of protein components after oxidation in Example 2;

2 shows the results of the ratio of protein components after separation by size exclusion chromatography after oxidation in Example 2;

Figure 3 shows the results of the ratio of protein components after oxidation in Example 3;

Figure 4 shows the results of the ratio of protein components after separation by size exclusion chromatography after oxidation in Example 3;

Figure 5 shows the results of the ratio of protein components after oxidation in Example 4;

Figure 6 shows the results of the ratio of protein components after separation by size exclusion chromatography after oxidation in Example 4;

Figure 7 shows the results of the ratio of protein components after oxidation in Example 5;

Figure 8 shows the results of the ratio of protein components after separation by size exclusion chromatography after oxidation in Example 5;

Figure 9 shows the results of the ratio of protein components after oxidation in Example 6;

Figure 10 shows the results of the ratio of protein components after separation by size exclusion chromatography after oxidation in Example 6;

Figure 11 shows the results of screening the test 1 protein component ratio in Example 7;

Figure 12 shows the results of the screening test 2 oxidized 0h protein component ratio in Example 7;

Figure 13 shows the results of Example 7 screening test 2 oxidation 5h protein component ratio;

Figure 14 shows the results of Example 7 screening test 2 oxidation 9h protein component ratio;

Figure 15 shows the results of the comparison of the ratio of the 13h protein component of the test 2 in the screening test of Example 7;

Figure 16 shows the results of Example 7 screening test 2 oxidation of 24h protein component ratio;

Figure 17 shows the results of the screening test 3 oxidized 0h protein component ratio in Example 7;

Figure 18 shows the results of Example 7 screening test 3 oxidation of 5h protein component ratio;

Figure 19 shows the results of Example 7 screening test 3 oxidation 9h protein component ratio;

Figure 20 shows the results of the comparison of the ratio of the 13h protein component of the test 3 in the screening test of Example 7;

Figure 21 shows the results of Example 7 screening test 3 oxidation of 24h protein component ratio;

Figure 22 shows the results of the screening test 4 oxidized 0h protein component ratio in Example 7;

Figure 23 is a graph showing the results of the comparison of the ratio of the 13h protein component of the test 4 in the screening test of Example 7;

Figure 24 shows the results of Example 7 screening test 4 oxidation of 15h protein component ratio;

Figure 25 shows the results of Example 7 screening test 4 oxidation of 24h protein component ratio;

Figure 26 shows the first batch of dimer and multimer content profiles of Example 8 repeated test;

Figure 27 shows the second batch of dimer and multimer content maps of Example 8 repeated test;

Figure 28 shows the third batch of dimer and multimer content profiles of Example 8 repeated experiments.

detailed description

The invention discloses a method for preparing a rhCNB dimer, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The anti-tumor drug rhCNB active ingredient is a monomer and a dimer, the multimer is an impurity, the dimer is better than the monomer, and the stability is better than the monomer, and the invention is formed under the reduction of the reducing agent DTT. More than 90% of the monomers, and then use a unique redox pair to convert the monomers into dimerization At the same time, the formation of multimers is effectively controlled by concentration, temperature and time, and the high-density dimer rhCNB is obtained by molecular exclusion chromatography. The route is a reduction-oxidation-reduction termination oxidation-separation dimer.

1. The preliminary purified rhCNB solution contains more than 90% of the monomers. In this step, the pH is 6.0-7.0, the temperature is 2-15 ° C, and 0.05% to 1% oxidant (such as hydrogen peroxide solution) is added in stages. When the rhCNB monomer is slowly converted to a dimer, when the dimer reaches 75% or more and the polymer is 1%, a low concentration oxidation stop solution (0.1% sodium hydrogen sulfite) is added to neutralize the oxidant to avoid further oxidation. Formation of trimers and above polymers. The principle of the conversion is that the rhCNB monomer contains a free sulfhydryl group. Under the oxidizing environment, the two free sulfhydryl groups form a disulfide bond to achieve the purpose of conversion to a dimer, taking into account the pH value of the buffer and the reaction rate of the temperature. influences.

2. The rhCNB composition containing a higher content of dimer in step 2 still contains a monomer and a small amount of trimer. In this step, the CNB composition is applied to a molecular exclusion chromatography column equilibrated by an equilibrium solution (including The molecular exclusion filler) is eluted sequentially by the buffer according to the molecular weight, and the purity of the dimer is more than 98%, and the trimer and the above polymer are <0.5%.

In order to better illustrate the present invention, the above described process steps for achieving the objectives are further described:

1. The pH condition of the oxidation is preferably 6.0 to 7.0. When the pH is high, the sulfhydryl group is easily oxidized, and it is difficult to control the polymer. Especially under alkaline conditions, the polymer is easily formed, forming the process technology of the present invention. The experimental data is in line with this theory. Furthermore, according to the physicochemical properties of rhCNB, it can be precipitated to different degrees in a solution having a pH of 4.3 to 5.5. Therefore, the oxidizing pH condition is 6.0 to 7.0.

2. The oxidizing agent comprises a hydrogen peroxide solution, an oxidized glutathione, a glutathione redox pair, ozone, pure oxygen, and the like. The present invention tests the five oxidizing agents or redox couples, and after the comparison, the hydrogen peroxide solution is selected.

3. Another innovative technique of the present invention is segmental oxidation. The amount of hydrogen peroxide used is calculated according to the oxygen ions consumed by forming a dimer per equivalent of monomer, and then added in proportion to time, and the oxidation rate is strictly controlled to form Reaction curve.

4. In the step of molecular exclusion chromatography, according to the characteristics of rhCNB, a medium having a separation range of 3000 to 70,000 is selected.

The materials and reagents used in the method for preparing the rhCNB dimer provided by the present invention are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Example 1 prepared to obtain crude crude liquid of rhCNB protein

The fermentation broth containing the target protein is cultured in a suitable medium, and the wet cells containing the target protein are collected by centrifugation in a tube centrifuge, and 5 to 20 times of the resuspension buffer is added, wherein the resuspension buffer includes a reducing substance and an enzyme. Inhibitor; homogenize and accumulate the heteroprotein at a suitable temperature, and collect the supernatant after centrifugation. Each 1kg of medium contains: Tryptone 20～50g, Yeast Extract9～35g, sodium chloride 4～12g, glycerin 12～50g, potassium dihydrogen phosphate 2～10g, dipotassium hydrogen phosphate trihydrate 2～10g, sodium hydroxide 2g.

The supernatant was collected and applied to a hydrophobic medium chromatography column equilibrated by the equilibration solution A, Phenyl Sepharose 6 Fast Flow (low sub) packing, and the unbound protein was washed away by the equilibration solution A, and the heteroprotein eluent B was The hydrophobic protein is washed away, and finally the target protein is eluted with the target protein eluent C to obtain a protein of interest containing the CNB composition.

Each 1 kg of the balance liquid A contains: TRIS 1.5 to 3.5 g, sodium chloride 30 to 70 g, calcium chloride 0.2 to 1.0 g, and hydrochloric acid 1.0 to 1.5 g.

Each 1 kg of the heteroprotein eluent B contains: TRIS 1.5 to 3.5 g, calcium chloride 0.2 to 1.0 g, and hydrochloric acid 1.0 to 1.5 g.

Each 1 kg of the protein eluent C contained: TRIS 1.5 to 3.5 g, EGTA 0.1 to 0.5 g, and hydrochloric acid 1.0 to 1.5 g.

Example 2 Preparation of rhCNB dimer

In the pure liquid of rhCNB protein obtained in the preparation of Example 1, by using an oxidizing agent (hydrogen peroxide, the mass ratio of the rhCNB solution to the oxidizing agent is 20:1), in an environment having a pH of 6.6 and a temperature of 12 ° C Under the conditions, an oxidant with a concentration of 0.75% was added in sections every 1 h, oxidized for 12 h, and an oxidation stop solution (0.1% sodium hydrogen sulfite solution, the rhCNB solution and the oxidation) was added. The mass ratio of the terminating liquid was 90:1) to terminate the oxidation, to obtain a component solution in which the rhCNB dimer component was more than 75%, and the polymer was controlled to be within 1.5% (see Fig. 1). The rhCNB dimer protein was collected by eluting the oxidizing solution onto a size exclusion chromatography column (selecting a medium with a separation range of 3000 to 7000 Da), 10 mM sodium citrate-sodium citrate buffer pH 5.8-7.0. peak.

The SEC-HPLC liquid phase method detected a polymer content of <0.5% and a dimer content of >98% (Fig. 2).

Table 1 Figure 1 chromatogram data

Peak number	keep time	area	height		area%
1	10.093	23731	1014	1.164
2	10.684	1675350	76840	82.19
3	12.058	339296	10594	16.645
total		2038378	88448	100

Table 2 Figure 2 chromatogram data

Peak number	keep time	area	height		area%
1	10.096	2080	130	0.098
2	10.583	2113411	98751	99.156
3	11.896	15908	504	0.746
total		2131399	99385	100

Example 3

In the rhCNB protein solution prepared in Example 1, by using an oxidizing agent (oxidized glutathione, the mass ratio of the rhCNB solution to the oxidizing agent was 25:1), at a pH of 6.2 and a temperature of 10 ° C Under ambient conditions, 0.5% oxidant was added in sections every 45h, oxidized for 18h, and an oxidation stop solution (0.1% sodium hydrogen sulfite solution was added. The mass ratio of the rhCNB solution to the oxidation stop solution was 70:1. The oxidation was terminated to obtain a component solution in which the rhCNB dimer component was more than 75%, and the polymer was controlled within 1.5% (Fig. 3). The rhCNB dimer protein was collected by eluting the oxidizing solution onto a size exclusion chromatography column (selecting a medium with a separation range of 3000 to 7000 Da), 10 mM sodium citrate-sodium citrate buffer pH 5.8-7.0. peak. The SEC-HPLC liquid phase method detected a polymer content of <0.5% and a dimer content of >98% (Fig. 4).

Table 3 Figure 3 chromatogram data

Table 4 Figure 4 chromatogram data

Peak number	keep time	area	height		area%
1	10.096	1655	115	0.08
2	10.578	2052532	95328	99.176
3	11.813	15390	505	0.744
total		2069577	95947	100

Example 4

In the rhCNB protein solution obtained in the preparation of Example 1, by using an oxidizing agent (glutathione redox pair, the mass ratio of the rhCNB solution to the oxidizing agent is 28:1), at a pH of 7.0 and a temperature of 5 ° C Under the environmental conditions, the oxidant with a concentration of 0.1% was added in sections every 3 h, oxidized for 15 h, and an oxidation stop solution (0.1% sodium hydrogen sulfite solution was added, and the mass ratio of the rhCNB solution to the oxidation stop solution was 60: 1) The oxidation was terminated to obtain a component solution in which the rhCNB dimer component was more than 75%, and the polymer was controlled to be within 1.5% (Fig. 5). The rhCNB dimer protein was collected by eluting the oxidizing solution onto a size exclusion chromatography column (selecting a medium with a separation range of 3000 to 7000 Da), 10 mM sodium citrate-sodium citrate buffer pH 5.8-7.0. peak. The SEC-HPLC liquid phase method detected a polymer content of <0.5% and a dimer content of >98% (Fig. 6).

Table 5 Figure 5 chromatogram data

Peak number	keep time	area	height		area%
1	10.031	28897	1289	1.509
2	10.65	1443559	64406	75.383
3	11.948	442510	19743	23.108
total		1914966	85438	100

Table 6 Figure 6 chromatogram data

Peak number	keep time	area	height		area%
1	10.121	1969	132	0.091
2	10.578	2131922	99699	98.806
3	11.846	23797	565	1.103
total		2157688	100396	100

Example 5

In the rhCNB protein solution prepared in Example 1, by using an oxidizing agent (ozone, the mass ratio of the rhCNB solution to the oxidizing agent is 22:1), under an environmental condition of pH 6.8 and a temperature of 15 ° C, The oxidizing agent was added in a concentration of 1% every 2 h, oxidized for 20 h, and oxidation was terminated by adding an oxidation stop solution (0.1% sodium hydrogen sulfite solution, the mass ratio of the rhCNB solution to the oxidation stop solution was 100:1). A component solution in which the rhCNB dimer component was obtained to more than 75% and the polymer was controlled within 1.5% was obtained (Fig. 7). The rhCNB dimer protein was collected by eluting the oxidizing solution onto a size exclusion chromatography column (selecting a medium with a separation range of 3000 to 7000 Da), 10 mM sodium citrate-sodium citrate buffer pH 5.8-7.0. peak.

The SEC-HPLC liquid phase method detected a polymer content of <0.5% and a dimer content of >98% (Fig. 8).

Table 7 Figure 7 chromatogram data

Peak number	keep time	area	height		area%
1	9.961	27842	1243	1.445
2	10.581	1446695	64587	75.084
3	11.881	452231	20190	23.471
total		1926768	86020	100

Table 8 Figure 8 chromatogram data

Peak number	keep time	area	height		area%
1	10.121	1193	83	0.057
2	10.585	2062082	97126	99.166
3	11.813	16141	539	0.776
total		2079415	97748	100

Example 6

In the rhCNB protein solution prepared in Example 1, by using an oxidizing agent, high-purity oxygen was slowly introduced into the section at a pH of 6.0 and a temperature of 2 ° C every 5 hours, and each time the oxygen was introduced for 30 minutes, oxidation was performed. 22h, adding oxidation termination solution (0.1% sodium hydrogen sulfite solution, the mass ratio of the rhCNB solution to the oxidation stop solution is 50:1) to terminate the oxidation, to obtain the rhCNB dimer component to more than 75%, more The polymer solution was controlled within 1.5% of the component solution (Fig. 9). Then, the oxidizing solution was applied to a molecular exclusion chromatography column (selecting a medium with a separation range of 3,000 to 70,000 Da), and 10 mM sodium citrate-sodium citrate buffer pH 5.8 to 7.0 was eluted and collected. The rhCNB dimer protein peak is set. The SEC-HPLC liquid phase method detected a polymer content of <0.5% and a dimer content of >98% (Fig. 10).

Table 9 Figure 9 chromatogram data

Peak number	keep time	area	height		area%
1	10.102	19003	834	0.926
2	10.684	1580136	72634	77.026
3	12.058	452301	15502	22.048
total		2051440	88970	100

Table 10 chromatogram data of Figure 10

Peak number	keep time	area	height		area%
1	10.146	5842	308	0.261
2	10.581	2206694	103067	98.75
3	11.911	22096	677	0.989
total		2234632	104052	100

Example 7 screening test

1: hydrogen peroxide one-time addition method

0.96 kg of rhCNB protein solution collected in Example 1 (pH of the solution was 6.98), 4 g of 0.1% hydrogen peroxide solution was added, and the mixture was stirred for 24 hours. The ratio of each component of rhCNB was determined by SEC-HPLC. It is 3.3% for the polymer and 83.497% for the dimer (Fig. 11).

Table 11 Figure 11 chromatogram data

Peak number	keep time	area	height		area%
1	9.867	272806	10835	3.3
2	10.521	6902462	311800	83.497
3	11.872	1091408	33628	13.203
total		8266676	356263	100

2: Hydrogen peroxide segmentation method

The rhCNB protein solution collected in Example 1 was 0.97 kg (solution pH 7.01), and 0.1% hydrogen peroxide solution was added to the 0, 1, 3, 5, 7, 9, 11, 13 h, respectively, while stirring. g, and samples were taken at 0, 5, 9, 13, 24h, and the ratio of each component of rhCNB was detected by SEC-HPLC. The results are shown in Tables 12-17. The map is shown in Figures 12 to 16.

Table 12, chromatogram data of Figure 12

Table 13 Chromatogram data of Figure 13

Peak number	keep time	area	height		area%
1	10.217	6241	324	0.298
2	10.721	1018764	43661	48.571
3	12.081	1072488	44697	51.132
total		2097494	88682	100

Table 14 Figure 14 chromatogram data

Peak number	keep time	area	height		area%
1	10.152	15062	718	0.745
2	10.706	1408448	60895	69.65
3	12.096	598674	21105	29.605
total		2022184	82718	100

Table 15 chromatogram data of Figure 15

Peak number	keep time	area	height		area%
1	10.135	20694	844	1.001
2	10.703	1540674	62860	74.523
3	12.099	506012	20646	24.476
total		2067380	84350	100

Table 16 chromatogram data of Figure 16

Peak number	keep time	area	height		area%
1	10.118	25767	1092	1.264
2	10.702	1548751	67476	75.986
3	12.11	463675	14868	22.749
total		2038194	83435	100

Table 17 results of hydrogen peroxide addition method

3: Hydrogen peroxide addition method (solution pH changed from 7.0 to 6.5)

0.97 kg of the rhCNB protein solution collected in Example 1 (pH of the solution was 6.48), and 0.1% hydrogen peroxide solution was added to the 0, 1, 3, 5, 7, 9, 11 and 13 h, respectively, under stirring. g, and sampling at 0, 5, 9, 13, 24h, SEC-HPLC method to detect the proportion of rhCNB components, the results are shown in Tables 18-23. The map is shown in Figures 17-21.

Table 18 Figure 17 chromatogram data

Peak number	keep time	area	height		area%
1	10.158	533	twenty four	0.019
2	10.793	159250	5660	5.718
3	12.058	2625257	120276	94.263
total		2785040	125960	100

Table 19, Figure 18, chromatogram data

Peak number	keep time	area	height		area%
1	10.354	7937	379	0.288
2	10.724	1038688	43846	37.745
3	12.067	1705198	72055	61.966
total		2751823	116280	100

Table 20 Figure 19 chromatogram data

Peak number	keep time	area	height		area%
1	10.148	17883	814	0.662
2	10.698	1694768	74337	62.743
3	12.09	988466	37331	36.595
total		2701117	112482	100

Table 21, Figure 20, chromatogram data

Peak number	keep time	area	height		area%
1	10.123	28078	1230	1.041
2	10.696	1949314	85142	72.296
3	12.101	718920	24126	26.663
total		2696312	110498	100

Table 22 Figure 21 chromatogram data

Peak number	keep time	area	height		area%
1	10.119	34345	1475	1.249
2	10.692	2070056	89293	75.282
3	12.101	645346	20318	23.469
total		2749746	111086	100

Table 23 Hydrogen peroxide addition method (solution pH changed from 7.0 to 6.5)

4: Add oxidation stop solution

0.95kg of the rhCNB protein solution collected in the above process (pH of the solution is 6.49), and 0.5g of 0.1% hydrogen peroxide solution is added to the 0th, 1st, 3rd, 5th, 7th, 9th, 11th, and 13th, respectively. 0.1 g of sodium hydrogen sulfite 0.3 g was added at 14 h, 14.5 h, and 15 h, and samples were taken at 0, 13, 15 and 24 h, and the ratio of each component of rhCNB was detected by SEC-HPLC. The results are shown in Tables 24-28. The map is shown in Figures 22 to 25.

Table 24 Figure 22 chromatogram data

Peak number	keep time	area	height		area%
1	10.033	1172	49	0.056
2	10.663	239429	9906	11.518
3	11.888	1838084	89722	88.425
total		2078685	99677	100

Table 25 chromatogram data of Figure 23

Peak number	keep time	area	height		area%
1	9.962	27051	1123	1.366
2	10.589	1490504	70069	75.244
3	11.893	463350	18396	23.391
total		1980905	89588	100

Table 26, Figure 24, chromatogram data

Peak number	keep time	area	height		area%
1	9.957	28293	1174	1.465
2	10.588	1479647	69919	76.589
3	11.897	423996	16725	21.947
total		1931936	87818	100

Table 27, chromatogram data of Figure 25.

Peak number	keep time	area	height		area%
1	9.987	31769	1334	1.505
2	10.599	1629591	75032	77.217
3	11.891	449045	16568	21.278
total		2110405	92934	100

Table 28 Addition of Oxidation Stopper Results

Example 8 repeated test

Repeatability test:

Three batches of rhCNB stock solution were prepared according to the process, and the results are as follows (see Figure 26 to Figure 28 for the dimer and multimer content maps):

The results are shown in Tables 29 to 32:

Table 29 Figure 26 chromatogram data

Peak number	keep time	area	height		area%
1	10.071	3358	205	0.15
2	10.563	2219322	105659	99.289
3	11.894	12534	506	0.561
total		2235213	106370	100

Table 30, Figure 27, chromatogram data

Peak number	keep time	area	height		area%
1	9.983	4990	291	0.209
2	10.559	2358612	111762	98.952
3	11.754	19990	614	0.839
total		2383592	112668	100

Table 31, Figure 28, chromatogram data

Peak number	keep time	area	height		area%
1	10.071	4766	274	0.198
2	10.561	2384153	112798	99.159
3	11.884	15454	560	0.643
total		2404373	113632	100

Table 32 repeat test data

The method for preparing the rhCNB dimer provided by the present invention is described in detail above. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (10)

1. A method for preparing a rhCNB dimer, comprising the steps of:

   Step 1: Preparation of crude crude liquid of rhCNB;

   Step 2: Under the reaction conditions of pH 6.0-7.0, 2-15 ° C, the crude pure liquid of rhCNB is mixed with the oxidant, oxidized for 12-24 hours, and the oxidation is terminated, and then purified and separated.
2. The method according to claim 1, wherein the oxidizing agent is added in a stepwise manner every 1 to 5 hours.
3. The method according to claim 1 or 2, wherein the mass ratio of the rhCNB solution to the oxidizing agent is from 20:1 to 30:1.
4. The method according to any one of claims 1 to 3, wherein the oxidizing agent is one or two of hydrogen peroxide, iodine, oxidized glutathione, glutathione redox couple or oxygen The above mixture.
5. The method according to any one of claims 1 to 4, wherein the concentration of the oxidizing agent is 0.05% to 1% (w/w).
6. The method according to any one of claims 1 to 5, wherein the reagent for terminating oxidation is an oxidation stop solution, and the oxidation stop solution is sodium hydrogen sulfite, sodium sulfite, 0.1% DTT or reduced glutathione. One or a mixture of two or more of the glycopeptides.
7. The method according to any one of claims 1 to 6, wherein the concentration of the oxidation terminating liquid is 0.05% to 1% (w/w).
8. The method according to any one of claims 1 to 7, wherein the mass ratio of the rhCNB solution to the oxidation terminating liquid is from 50:1 to 100:1.
9. The method according to any one of claims 1 to 8, wherein the separation and purification are performed by size exclusion chromatography using 10 mM sodium citrate-citrate buffer The rhCNB dimer protein peak was collected by elution at a pH of 5.8 to 7.0.
10. The method according to any one of claims 1 to 9, wherein the size exclusion chromatography employs a medium having a separation range of 3,000 to 70,000 Da.

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