CN116410295A

CN116410295A - Purification method of escherichia coli expression

Info

Publication number: CN116410295A
Application number: CN202111649587.5A
Authority: CN
Inventors: 陈坤; 郭帅利; 田赵源; 季荣钰; 梅菲; 谭小钉
Original assignee: Jiangsu Maiweikang New Drug Research And Development Co ltd
Current assignee: Jiangsu Maiweikang New Drug Research And Development Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-07-11

Abstract

The invention discloses a purification method of an escherichia coli expression, which comprises the following steps: subjecting the E.coli expression sample to cationic chromatography to obtain a first chromatographic solution, wherein the cationic chromatography is equilibrated with a buffer solution having a pH of 4.0, and the chromatographic column is rinsed; subjecting the first chromatographic liquid to anion chromatography to obtain a second chromatographic liquid; filtering the second chromatographic liquid to obtain filtrate; the filtrate was subjected to ultrafiltration concentration to obtain a purified expression product. The purification process can prepare the G-CSF protein expressed by escherichia coli, so that the protein product with higher purity can be prepared, the process is simpler, the amplification can be realized, and the cost is saved.

Description

Purification method of escherichia coli expression

Technical Field

The invention relates to the field of biological medicines, in particular to a purification method of an antibody medicine, and particularly relates to a method for removing endotoxin in G-CSF. The invention also relates to therapeutic and diagnostic uses of these granulocyte colony stimulating factor proteins, in particular in the treatment, prevention and/or diagnosis of related diseases, such as autoimmune related diseases.

Background

Endotoxin is a generic term for toxic substances present in cells of gram-negative bacteria, and is a component of the cell wall of various gram-negative bacteria, and is a toxin released after cleavage of the cells, also called "pyrogen". The chemical component of the composition is a phospholipid polysaccharide-protein complex, and the toxic component of the composition is mainly lipid A. Endotoxin is located on the outermost layer of the cell wall and covers the cell wall's mucin. The endotoxin of various bacteria has weak toxic effects, and can cause fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation and the like. Endotoxin is thermostable and stable, and has weak antigenicity. Can stimulate organism to produce antibody without neutralization to form antitoxin, which can not become toxoid after formaldehyde treatment. Although endotoxins are tightly embedded in the cell wall, they are constantly released into the surrounding environment, not only due to cell disintegration and death, but also during normal cell growth and division.

In the existing biological product purification process, endotoxin pollution of a sample can be removed by a chromatography method and a TFF tangential flow membrane filtration method. In addition, additives such as non-ionic detergents Triton X-114 and arginine can be used to facilitate dissociation of endotoxin interactions with sample molecules and reduce endotoxin content of the final product. However, there is no general scheme for endotoxin removal in biopharmaceutical technology, and there are many chromatographic products, and the operating process has many influencing factors and low removal efficiency, so when endotoxin pollution is serious, a method with higher optimization efficiency needs to be explored to obtain a protein product with higher purity.

Disclosure of Invention

The invention aims to solve the problem of high impurity content of biological products in the prior art, and particularly solves the technical problem that an escherichia coli expression system has high endotoxin content. To this end, an object of the present invention is to prepare granulocyte colony stimulating factor (G-CSF) with a low endotoxin content and a higher purity.

The present invention has been completed based on the following work of the inventors:

the G-CSF has very wide clinical application and very low toxic and side effects, is mainly used for preventing and treating bone marrow suppression and the like caused by chemotherapy and radiotherapy at present, and can not meet the clinical requirements due to very limited natural human G-CSF sources, and successfully constructs the expression clone with very high expression level by means of genetic engineering, thereby having higher expression in escherichia coli.

However, the E.coli expression system is particularly high endotoxin. The G-CSF inclusion body expressed by colibacillus is denatured and renatured, SP Sepharose Fast Flow cation chromatography, Q Sepharose Fastflow anion chromatography and ion exchange filter (Kleenpak Posidyne 0.2 μm) to obtain the protein with higher purity and lower endotoxin.

Thus, according to one aspect of embodiments of the present invention, there is provided a method of purifying an E.coli expression comprising:

carrying out cationic chromatography on the escherichia coli expression sample so as to obtain a first chromatographic solution, wherein the cationic chromatography adopts buffer solution with the pH of 4.0 for balancing, and washing a chromatographic column;

subjecting the first chromatographic liquid to anion chromatography so as to obtain a second chromatographic liquid;

filtering the second chromatographic liquid to obtain filtrate;

and carrying out ultrafiltration concentration treatment on the filtrate so as to obtain the purified escherichia coli expression.

According to the method for reducing the endotoxin content in the biological protein, provided by the embodiment of the invention, the purification process of escherichia coli expression can be used for preparing a product with higher purity, and the process is simpler, easy to amplify and cost-saving.

In addition, the antibodies or antigen-binding fragments according to the above embodiments of the present invention may have the following additional technical features

According to an embodiment of the invention, the cationic chromatography is performed with a buffer solution having a pH of 6.5.

According to an embodiment of the invention, the cationic chromatography loading is less than or equal to 30mg/ml.

According to an embodiment of the invention, the anion chromatography is pre-equilibrated with a buffer solution of pH 4.0, and the column is rinsed.

According to an embodiment of the invention, sodium chloride is added to the pre-equilibrated buffer.

According to an embodiment of the invention, the anion chromatography uses a flow-through mode to collect the eluate.

According to an embodiment of the invention, the buffer is an acetic acid/sodium acetate buffer.

According to an embodiment of the invention, the E.coli expression is subjected to colony fermentation and washing with a buffer at pH 7.5 and colony solubilization and renaturation with a buffer at pH 8.5 prior to the cationic chromatography.

According to an embodiment of the present invention, the filter is a nylon filter.

According to another aspect of the invention there is provided a method of removing endotoxin from a G-CSF protein. According to an embodiment of the invention, the E.coli expression sequence is as set forth in SEQ ID NO: 1.

According to the above embodiments of the present invention, the inventors have found that, through two-step chromatography, including combined elution and flow-through elution, and further through a simple filtration manner, the endotoxin content can be further reduced while the sterilization can be performed, so that the endotoxin content can be successfully reduced to the injection standard specified in pharmacopoeia.

For a better understanding of the invention, some terms are first defined. Other definitions are set forth throughout the detailed description.

The term "affinity chromatography", also known as "affinity chromatography", is a chromatographic method that utilizes the binding properties of a stationary phase to separate molecules. Affinity chromatography involves attaching molecules with a certain binding capacity to the substance to be separated to a gel filtration column, and their binding is reversible, allowing the two to separate from each other when the mobile phase conditions are changed. Affinity chromatography can be used to purify or concentrate a molecule from a mixture, or to remove or reduce the content of a molecule in a mixture.

The term "anion chromatography" is one type of ion exchange chromatography. Ion exchange chromatography is one of the most widely used methods in the purification of biological macromolecules. Separation methods are performed under certain pH conditions according to the difference of charges of proteins. Ion exchange chromatography using a weak base diethylaminoethyl cellulose (DEAE cellulose) as an ion exchanger for protein separation is anion chromatography.

The term "cation chromatography" is also one type of ion exchange chromatography. Ion exchange chromatography using weak acid carboxymethyl cellulose (CM cellulose) as ion exchanger for protein separation is cation chromatography.

The term "ultrafiltration" is one of membrane separation techniques that uses pressure as the driving force. The purpose of separating large molecules from small molecules is that the pore diameter of the membrane is between 20 and 1000A degrees. The hollow fiber ultrafilter (membrane) has the advantages of high packing density in unit container, small occupied area, etc.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:

FIG. 1 shows an electrophoretogram after G-CSF inclusion body renaturation according to an embodiment of the invention;

FIG. 2 shows a cation exchange chromatography according to an embodiment of the invention;

FIG. 3 shows a G-CSF non-reducing electrophoresis pattern in accordance with an embodiment of the invention;

FIG. 4 shows an anion exchange chromatography according to an embodiment of the invention;

FIG. 5 shows a diagram of G-CSF non-reducing electrophoresis after purification according to an embodiment of the invention;

FIG. 6 shows a SEC-HPLC profile of a purified G-CSF protein according to an embodiment of the invention.

Detailed Description

The invention establishes a G-CSF purification route expressed by escherichia coli, and can obtain the G-CSF protein with higher purity and lower endotoxin after the inclusion body is separated.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are conventional products available commercially, such as those available from Illumina corporation, without the manufacturer's knowledge.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Example 1: coli expression

And (5) colony fermentation. E.coli colony of the encoding G-CSF gene is inoculated in LB culture medium, cultured for 6 hours at 37 ℃ under 220rmp shaking, inoculated in a fermentation tank for culture, fed with 1mmol/L IPTG for induction at 37 ℃ under the rotating speed of 450rpm, and after 4 hours of induction culture, the bacteria are collected by centrifugation.

G-CSF inclusion body denaturation. Re-suspending the bacterial mud, homogenizing, centrifuging, re-suspending the bacterial buffer, homogenizing twice at 800bar, centrifuging at 10000rpm for 30min, and collecting the precipitate.

G-CSF inclusion body washing. Re-suspending the sediment after bacteria breaking by using a washing buffer solution, centrifuging at 10000rpm for 30min, and taking the sediment; re-suspending the precipitate with pure water, centrifuging at 10000rpm for 30min, and collecting the precipitate; re-suspending the precipitate with pure water, centrifuging at 10000rpm for 30min, and collecting the precipitate.

Solubilization and renaturation of G-CSF inclusion bodies. Adding the washed precipitate into a solubilization buffer solution, controlling the temperature to be 10+/-2 ℃, stirring and solubilizing for 6 hours, diluting and renaturating the solubilization solution and the dilution solution according to the proportion of 1:50, rapidly adding the solubilization solution into the dilution solution, controlling the temperature to be 10+/-2 ℃, and stirring for 22 hours.

The composition ratios and the pH of the bacteria-destroying buffer solution, the washing buffer solution, the solubilization buffer solution and the dilution renaturation solution are shown in the following table 1.

Concentrating the sample by using a 5KD ultrafiltration membrane bag for standby to obtain a sample SPF0, wherein the G-CSF protein sequence is shown as SEQ ID NO: 1. SPF0 electrophoresis pattern is shown in FIG. 1, and it can be seen that G-CSF protein has better expression.

TABLE 1 composition of colony fermentation buffer

Species of type	Composition ratio	pH
			Bacteria-destroying buffer solution	50mM Tris-HCl+0.1M NaCl+1mM EDTA-2Na	7.5±0.1
Washing buffer	20mM Tris-HCl+2M Urea+5mM EDTA-2Na	7.5±0.1
			Solubilization buffer	20mM Tris-HCl+9M Urea+5mM EDTA-2Na+50mM beta-mercaptoethanol	8.5±0.1
Dilution renaturation liquid	20mM Tris-HCl	8.5±0.1

Example 2: SP Sepharose Fast Flow cationic chromatography

The pH of the SPF0 sample solution is regulated to 4.0+/-0.1, the electric conductance is less than or equal to 5mS/cm, and the sample is loaded after 3-4CV washing by using an equilibrium buffer solution. The chromatographic column packing is SP Sepharose Fast Flow, the loading retention time is less than or equal to 5min, and the loading capacity is less than or equal to 30mg/ml. After the loading is finished, the chromatography column is rebalanced by 2-3CV by using a balancing buffer, washed by 2-3CV by using a leaching buffer, and eluted by using an eluting buffer. And the absorption of the ultraviolet 280nm of the instrument rises to 50mAU, the sample starts to be collected, and falls to 50mAU, and the sample is stopped to be collected, so that the sample SPF2 is obtained.

The composition ratios and pH of the equilibration buffer, the elution buffer and the elution buffer are shown in table 2 below.

TABLE 2 composition ratio of cationic chromatography buffers

Species of type	Composition ratio	pH
			Balanced buffer	20mmol/L NaAc-HAc	4.0±0.1
Eluting buffer	100mmol/L NaAc-HAc	4.0±0.1
			Elution buffer	100mmol/L NaAc-HAc	6.5±0.1

Endotoxin in SPF2 was detected. SPF2 chromatographic patterns are shown in FIG. 2, non-reducing electrophoresis patterns are shown in FIG. 3, detection data are shown in Table 3, more polymer impurities are found to be removed, and the endotoxin content of the sample is partially reduced.

The purity measurement method is as follows:

content analysis by SEC-HPLC, high performance liquid chromatograph: agilent 1260; chromatographic column: TSK _ge lG300SW _XL The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase: 0.1mol/L PB+0.1mol/LNa ₂ SO ₄ ，pH6.8。

Sample preparation: samples were diluted to 1.0mg/ml with mobile phase, centrifuged at 12000rpm for 10min, and supernatants were taken for analysis conditions: flow rate: 0.6ml/min; column temperature: 25 ℃; detection wavelength: 214nm; analysis time: 25min.

And (3) testing system applicability: taking 20 microliters of working reference sample and carrying out HPLC analysis according to analysis conditions, wherein the theoretical plate number of the monomer peak is as follows: 7711; retention time 13.954; the degree of separation between monomer and polymer was 3.4.

Table 3: results of cationic chromatography endotoxin content

Sample name	Endotoxin (EU/ml)
		SPF0	＞200
SPF2	98

Example 3: q Sepharose Fast flow anion chromatography

And (3) eluting the sample SPF2 obtained by cation chromatography, and regulating the pH value to 4.0+/-0.1, wherein the electric conductivity is less than or equal to 5mS/cm. The sample was loaded after pre-equilibration with 15-20CV using a equilibration buffer, and rinsing with equilibration buffer until pH and conductance were no longer changing. And the chromatographic column packing is Q Sepharose Fast Flow, the loading retention time is less than or equal to 5min, the loading capacity is less than or equal to 120mg/ml, the sample is collected and flowed through in the loading process, the absorption of the ultraviolet 280nm of the instrument is increased to 50mAU, the sample is collected, the sample is reduced to 50mAU, and the sample QF1 is obtained.

The composition ratios and the pH of the equilibration buffer and the regeneration buffer are shown in Table 4 below.

TABLE 4 anionic chromatographic buffer composition ratios

Species of type	Composition ratio	pH
			Balanced buffer	20mmol/L NaAc-HAc	4.0±0.1
Regeneration buffer	20mmol/L NaAc-HAc+1mol/L NaCl	4.0±0.1

Endotoxin in QF1 was detected. QF1 chromatographic chart is shown in figure 4, and endotoxin content of sample is measured in table 5 below. After anion chromatography, the endotoxin content in QF1 is further reduced.

Table 5: results of anion chromatography endotoxin content

Sample name	Endotoxin (EU/ml)
		SPF2	98
QF1	3.4

Example 4: kleenpak Posidyne 0.2.2 mu m nylon material filter

Using nylon filter (Kleenpak Posidyne; model KA1NFZP1; area: 200 cm) ² The method comprises the steps of carrying out a first treatment on the surface of the Pore diameter: 0.2 μm), the filter buffer (20 mmol/L NaAc-HAc, pH 4.0), the sample was applied and buffered by filtrationWashing the solution to obtain a sample NF1.

Endotoxin in NF1 was tested and endotoxin content data is shown in table 6 below.

TABLE 6 endotoxin removal assay results

Sample name	Endotoxin (EU/ml)
		QF1	3.4
NF1	＜0.2

After nylon filtration, the endotoxin content is reduced by more than one order of magnitude, and the standard of the endotoxin content of the injection-grade medicine is reached.

Example 5: ultrafiltration concentration and displacement

NF1 was replaced into the formulation recipe by ultrafiltration, keeping the sample stable. Ultrafiltration membrane bag ultrafiltration of 5KD is used to replace NF1 into the prescription buffer solution, and then the concentration is carried out firstly and then the replacement concentration is carried out. The sample was concentrated to 1mg/ml and replaced with 6-8 CV of replacement buffer. Then, the sample was concentrated to 2mg/ml to give an ultrafiltration sample DS. The ultrafiltration buffer was 50mmol PB (pH 6.0). TMP was set at 1.0bar, inlet flux at 150LMH, membrane loading at 200g/m ² The film wrapping area is 88cm ² 。

The endotoxin in the sample DS is detected, the non-reduction electrophoresis diagram of the endotoxin in the purified G-CSF protein is shown in figure 5, the SEC-HPLC diagram of the content of the G-CSF protein is shown in figure 6, and the detection result is shown in table 7 below.

TABLE 7 endotoxin removal assay results

Sample name	Endotoxin (EU/ml)
		DS	＜0.2

The G-CSF protein expressed by colibacillus is denatured and renatured to form high-expression correctly folded protein, then part of incorrectly folded protein, most of polymer impurities and part of endotoxin are removed by SP Sepharose Fast Flow chromatography, most of endotoxin is removed by Q Sepharose Fastflow anion chromatography collecting and flow-through mode, finally the endotoxin content is further reduced by nylon material filter (Kleenpak Posidyne 0.2.2 mu m), and finally the G-CSF protein with higher purity and low endotoxin content is obtained.

Sequence listing

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Claims

1. A method for purifying an escherichia coli expression, comprising:

filtering the second chromatographic liquid to obtain filtrate;

2. The method of claim 1, wherein the cationic chromatography is performed using a buffer at pH 6.5.

3. The method of claim 2, wherein the cationic chromatography loading is 30mg/ml or less.

4. The method of claim 3, wherein the anion chromatography is pre-equilibrated with a buffer at pH 4.0 and the column is flushed.

5. The method of claim 4, wherein sodium chloride is added to the pre-equilibrated buffer.

6. The method of claim 5, wherein the anion chromatography uses a flow-through mode to collect the eluate.

7. The method of any one of claims 1-6, wherein the buffer is an acetic acid/sodium acetate buffer.

8. The method of claim 7, wherein said E.coli expression is colony fermented and washed with a buffer at pH 7.5 and colony solubilized and renatured with a buffer at pH 8.5 prior to said cationic chromatography.

9. The method of claim 8, wherein the filter is a nylon filter.

10. The method of any one of claims 1-9, wherein the escherichia coli expression sequence is set forth in SEQ ID NO: 1.