阅读说明：本技术 使用金属螯合色谱法去除重组人生长激素内毒素的方法 (Method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography ) 是由 邹青 岑选伉 胡一峰 刘海昌 朱复培 于 2021-11-08 设计创作，主要内容包括：本发明公开了使用金属螯合色谱法去除重组人生长激素内毒素的方法,包括如下步骤：菌体沉淀重悬、细胞破碎、破碎上清孵育、样品过滤、IMAC纯化、碱液预洗、平衡、样品装载、柱洗涤、去污剂洗脱内毒素、洗涤、目标蛋白洗脱。本发明提供一种简单有效的方法来纯化蛋白质样品,并且有效的去除其中的内毒素,从内毒素-蛋白质复合物中分离出内毒素,并在蛋白质浓度或者生物活性没有明显损失的情况下,使其内毒素含量下降到一定限度。(The invention discloses a method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography, which comprises the following steps: resuspending thallus precipitation, breaking cells, breaking supernatant for incubation, filtering a sample, carrying out IMAC purification, prewashing by alkali liquor, balancing, loading the sample, washing a column, eluting endotoxin by a detergent, washing and eluting target protein. The present invention provides a simple and efficient method for purifying a protein sample and efficiently removing endotoxin therefrom, separating endotoxin from an endotoxin-protein complex, and reducing the endotoxin content to a certain limit without significant loss of protein concentration or biological activity.)

1. A method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography, which is characterized by comprising the following steps:

step 1, bacterial precipitation and resuspension: resuspending the thallus precipitate with buffer solution containing 0.1-5% alcohol or nonionic surfactant at a ratio of thallus precipitate mass to buffer solution volume of 1: 5-20, and mixing;

step 2, cell disruption: carrying out cell disruption on the resuspended thalli;

and 3, crushing the supernatant for incubation: centrifuging the crushed cells, removing the precipitate, taking the supernatant, and stirring and incubating the supernatant;

and 4, filtering the sample: filtering the incubated recombinant human growth hormone fusion protein to obtain a recombinant human growth hormone fusion protein sample;

step 5, IMAC purification: taking an IMAC metal chelating chromatographic column, and cleaning the column by using purified water;

step 6, alkali liquor prewashing: washing the metal chelating chromatographic column with alkali liquor with certain concentration to remove residual endotoxin on the original column;

step 7, balancing: balancing the metal chelating chromatographic column by using an equilibrium buffer solution;

step 8, sample loading: loading the recombinant human growth hormone fusion protein sample on a metal chelating chromatographic column to ensure that the recombinant human growth hormone fusion protein sample is combined with the filler;

step 9, column washing: washing the column with an equilibration buffer;

step 10, detergent elution of endotoxin: eluting the endotoxin with a non-ionic detergent at a concentration;

step 11, washing: washing the column with an equilibration buffer;

step 12, eluting the target protein: eluting the target protein by using the elution mode of a conventional metal chelating chromatographic column.

2. The method for removing recombinant human growth hormone endotoxin according to claim 1, wherein the alcohol is ethanol, alkanediol or isopropanol.

3. The method for removing endotoxin in recombinant human growth hormone according to claim 1, wherein the non-ionic surfactant is one or more of TritonX-100, TritonX-114, Tween 80, Tween 40 and Tween 20.

4. The method for removing endotoxin in recombinant human growth hormone according to claim 1, wherein the step 2 is a method of cell disruption using mechanical stress, cavitation stress, turbulent stress or biological and chemical lysis.

5. The method for removing endotoxin in recombinant human growth hormone according to claim 1, wherein the incubation temperature in step 3 is 0-25 ℃ and the incubation time is 6-24 h.

6. The method for removing recombinant human growth hormone endotoxin according to claim 1, wherein the filtration pore size in step 4 is 0.2 to 2 μm.

7. The method for removing recombinant human growth hormone endotoxin by metal chelate chromatography as claimed in claim 1, wherein in the steps 7, 9 and 11, the equilibration buffer is prepared by ultrapure water or water for injection.

8. The method for removing recombinant human growth hormone endotoxin by metal chelate chromatography as claimed in claim 1, wherein the concentration of the nonionic surfactant in the nonionic detergent is 0.1 to 5%.

9. The method for removing recombinant human growth hormone endotoxin according to claim 1, wherein in the step 7, the equilibrium volume is 5 to 20 CV.

10. The method for removing recombinant human growth hormone endotoxin according to claim 1, wherein the elution volume in the step 10 is 5 to 20 CV.

Technical Field

The invention relates to a protein purification technology in the technical field of biotechnology pharmacy and bioengineering, in particular to a method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography.

Background

Recombinant Human Growth Hormone (Recombinant Human Growth Hormone) is a genetic engineering drug produced by Recombinant DNA technology, and comprises single-chain protein consisting of 191 amino acids, the relative molecular mass is about 22KDa, the isoelectric point is 4.9, two pairs of disulfide bonds exist in the molecule, the structure and the amino acid sequence of the disulfide bonds are consistent with those of natural Human Growth Hormone, most of the currently marketed Recombinant Human Growth Hormone drugs are escherichia coli secretory and intracellular soluble expression modes, and the bacterial endotoxin pollution can be inevitably caused in the research, development and production processes of the Recombinant Human Growth Hormone drugs.

Endotoxin, also known as Lipopolysaccharide (LPS), the cell wall constituent of gram-negative bacteria, is responsible for its organization and stability. In the pharmaceutical field, endotoxins may be present in various production stages or end products, and although endotoxins are associated with the bacterial cell wall, they are constantly released into the environment not only during cell death but also during cell growth and division, and are almost ubiquitous as bacteria can grow in nutrient-poor media, such as water, salts, buffers, etc., and a single E.coli contains about 200 or more tens of thousands of LPS molecules. Endotoxins are recognized by the immune system and can elicit a variety of pathophysiological responses. When the body is in excessive contact with lipopolysaccharide (e.g., when low concentrations of lipopolysaccharide enter the blood system), systemic inflammatory reactions can occur, which can lead to a variety of pathophysiological responses, such as endotoxic shock, tissue damage, and even death. (Minutoli L, 2008) however, endotoxin does not act directly on cells or organs, but rather releases a range of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), interleukin 6(IL-6) and interleukin 1 beta (IL-1 beta), by activating the immune system, particularly by leukocytes or macrophages (Anspach FB, 2001). Low endotoxin concentrations (1ng/ml) in mammals cause a fever response and even shock. All pharmacopoeias specify that the maximum amount of intravenous endotoxin for pharmaceutical or biological products is 5EU/(kg · h) (Daneshian M, 2006). EU is a unit representing the biological activity of endotoxin. For example, 100pg of the standard endotoxin EC-5 or 120pg of E.coli endotoxin O111: B4 have 1EU activity. Achieving this standard is always a challenge to the biological sciences or pharmaceutical industry.

In the biotechnological field, gram-negative bacteria are widely used for the production of recombinant DNA products, such as peptides and proteins, which are always contaminated with endotoxins, for which reason proteins produced by gram-negative bacteria must be as free as possible of endotoxins in order to avoid side effects in human and animal use, which, however, are very stable molecules that, in contrast to proteins, are resistant to extreme temperatures and pH values. Endotoxins are negatively charged substances, usually with slight hydrophobicity.

Based on the unique molecular characteristics of endotoxin molecules, a number of different methods have been developed to remove LPS from proteins, including LPS affinity chromatography (polymyxin B, immobilized L-histidine, poly L-lysine), biphasic extraction, ultrafiltration, hydrophobic interaction chromatography, ion exchange chromatography, and membrane adsorption. For the vessel in direct contact with the protein, 180-250 ℃ high temperature and 0.1-1mol/L acid or alkali concentration are usually used to remove endotoxin.

The conventional method has been a challenge to remove endotoxin from endotoxin-protein complexes because endotoxin can interact with many protein molecules, such as LPS-binding protein, bactericidal permeability-enhancing protein (BPI), amyloid P-component, cationic protein, enzyme used in biological endotoxin assay (anti-lipopolysaccharide factor (LAL) in limulus reagent), which directly participate in the reaction with endotoxin in various substances, and although some studies on the interaction mechanism of endotoxin with proteins (such as molecular recognition, electrostatic interaction, etc.) have been made, the interaction mechanism of many essential proteins with endotoxin is still unclear. Removal of endotoxin by a single process is not possible for most protein products and neither approach is universally applicable.

Therefore, the key to remove endotoxin from recombinant human growth hormone is 1, how to prevent recombinant human growth hormone from forming protein-endotoxin complex with endotoxin; 2. how endotoxin can be removed from the protein-endotoxin complex; 3. how to avoid the introduction of new endotoxins.

One of the most commonly used techniques for removing endotoxins from a protein sample of interest is anion exchange chromatography (Webber et al, 1995), which binds strongly to positively charged ligands of anion exchange materials due to the negative charge of endotoxins, however this method has several disadvantages: if the protein to be purified is also negatively charged, a great deal of effort must be expended to adjust the binding conditions to avoid co-efflux of endotoxins with the target protein; often with a large loss of the protein of interest.

The target protein and bacterial endotoxin are separated by ultrafiltration technology, the difference between the molecular sizes of protein molecules and endotoxin molecules is relied on, the difference between the molecular sizes of the target substance and the bacterial endotoxin molecules is generally required to be more than three times to be effective, and the basic unit size of lipopolysaccharide is 10-20KDa, so the method is suitable for small molecular substances.

Affinity chromatography (adsorption of endotoxins) (immobilized polymyxin B karp et al, 1987) (immobilized histidine Matsumae et al, 1990) (US3897309, US4276050, US4381239) is very effective in removing endotoxins from protein solutions and maintaining relatively high protein yields, but the disadvantages are also obvious, such as expensive equipment, high material consumption, complicated operation, and the need to specially tailor the chromatography medium, and the possible loss of affinity ligand, the introduction of new impurities to the protein solution, the increased risk of purification process, and the inability of these affinity media to be washed with standard chromatography media such as strong base sodium hydroxide or ethanol to remove heat sources.

Also known is the purification of polypeptides and removal of endotoxins by Immobilized Metal Affinity Chromatography (IMAC), as described in US6942802 for example in a method for removing endotoxin from the outer membrane protein of moraxella catarrhalis by immobilized metal ion affinity chromatography, which comprises loading a protein solution containing endotoxins onto IMAC packing, which packing has been pre-equilibrated to a buffer zone, followed by elution of endotoxin with a buffer, which binding of proteins to the packing is maintained, and finally elution and collection of proteins with a specific buffer, whereby the endotoxin concentration in the protein solution is effectively reduced by at least 200-fold and the protein recovery is greater than at least 60% after IMAC purification. The method has the disadvantages that the protein-endotoxin complex is not removed, the endotoxin reduction degree is not enough, and the requirement of intravenous injection is far from being met.

In view of the above, there is a need for a simple and efficient method or process for purifying a protein sample and efficiently removing endotoxin therefrom to isolate endotoxin from an endotoxin-protein complex, and it is also desired that such a protein purification method is effective in reducing the endotoxin content to a certain extent without significant loss of protein concentration or biological activity, so that it can be administered as a pharmaceutical or immunogenic composition. This is not only a critical point in recombinant human growth hormone production, but also a critical point in recombinant protein production.

Therefore, a method for removing endotoxin in the production process of expressing the recombinant human growth hormone by using the escherichia coli is developed, and the method can also be suitable for removing endotoxin in the production of other escherichia coli expression recombinant proteins by changing parameters.

Disclosure of Invention

The invention aims to provide a method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography, which can effectively remove endotoxin in escherichia coli expression recombinant human growth hormone and reduce protein-endotoxin complex formed by endotoxin and protein, thereby achieving the purpose of removing endotoxin in large quantity and even completely removing endotoxin.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for removing recombinant human growth hormone endotoxin by using metal chelate chromatography, comprising the following steps:

step 1, bacterial precipitation and resuspension: resuspending the thallus precipitate with buffer solution containing 0.1-5% alcohol or nonionic surfactant at a ratio of thallus precipitate mass to buffer solution volume of 1: 5-20, and mixing;

step 2, cell disruption: carrying out cell disruption on the resuspended thalli;

and 3, crushing the supernatant for incubation: centrifuging the crushed cells, removing the precipitate, taking the supernatant, and stirring and incubating the supernatant;

and 4, filtering the sample: filtering the incubated recombinant human growth hormone fusion protein to obtain a recombinant human growth hormone fusion protein sample;

step 5, IMAC purification: taking an IMAC metal chelating chromatographic column, and cleaning the column by using purified water;

step 6, alkali liquor prewashing: washing the metal chelating chromatographic column with alkali liquor with certain concentration to remove residual endotoxin on the original column;

step 7, balancing: balancing the metal chelating chromatographic column by using an equilibrium buffer solution;

step 8, sample loading: loading the recombinant human growth hormone fusion protein sample on a metal chelating chromatographic column to ensure that the recombinant human growth hormone fusion protein sample is combined with the filler;

step 9, column washing: washing the column with an equilibrium buffer solution to remove impurities which are not hung on the column;

step 10, detergent elution of endotoxin: eluting the endotoxin with a non-ionic detergent at a concentration;

step 11, washing: washing the column with an equilibrium buffer solution to remove the residual non-ionic detergent and endotoxin on the column;

step 12, eluting the target protein: eluting the target protein by using the elution mode of a conventional metal chelating chromatographic column.

Preferably, the alcohol is ethanol, alkylene glycol or isopropanol.

Preferably, the nonionic surfactant is one or more of TritonX-100, TritonX-114, Tween 80, Tween 40 and Tween 20.

Preferably, in step 2, the cell disruption method is mechanical stress, cavitation stress, turbulent stress or biological and chemical lysis method.

Preferably, in the step 3, the incubation temperature is 0-25 ℃ and the incubation time is 6-24 h.

Preferably, in the step 4, the filtration pore size is 0.2 to 2 μm.

Preferably, in the steps 7, 9 and 11, the equilibration buffer is a buffer prepared using ultrapure water or water for injection.

Preferably, the concentration of the nonionic surfactant in the nonionic detergent is 0.1-5%.

Preferably, in the step 7, the equilibrium volume is 5 to 20 CV.

Preferably, in the step 10, the elution volume is 5 to 20 CV.

Compared with the prior art, the invention has the advantages that:

the present invention provides a simple and efficient method for purifying a protein sample and efficiently removing endotoxin therefrom, separating endotoxin from an endotoxin-protein complex, and reducing the endotoxin content to a certain limit without significant loss of protein concentration or biological activity.

Furthermore, the invention adopts the alcohol or non-ionic surfactant to compete for endotoxin with protein through hydrophobic interaction competition, so as to reduce the level of the combination body of protein and lipopolysaccharide, and prevent the protein and endotoxin from forming a complex at the beginning of purification; and loading the target protein to an IMAC metal chelating chromatographic column and eluting endotoxin by using a non-ionic surfactant so as to achieve the aim of removing a large amount of endotoxin and even completely removing the endotoxin.

Drawings

FIG. 1 is a process flow diagram of the present invention.

FIG. 2 is an IMAC purification chromatogram of an embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further specifically described below by way of embodiments in combination with the accompanying drawings.

Example (b): referring to fig. 1, the present example provides a method for removing endotoxin from recombinant human growth hormone by metal chelate chromatography, which is performed by constructing fused recombinant human growth hormone containing polyhistidine tag (6 × His,8 × His or 10 × His) in molecular biology, culturing seed solution, inoculating in a fermentation tank, centrifuging in a lower tank to obtain thallus, adding a certain amount of alcohol (such as ethanol, alkylene glycol, isopropanol, etc.) or nonionic surfactant (such as triton x-100, triton x-114, tween 80, tween 40, tween 20, etc.) before or during disruption of escherichia coli cells, competing for endotoxin with protein by hydrophobic interaction to reduce the level of protein and lipopolysaccharide conjugate, and preventing the protein and endotoxin from forming a complex at the beginning of purification. Loading the target protein onto an IMAC metal chelating chromatography column (metal chelating agent such as Cu)²⁺,Ni²⁺,Co²⁺,Zn²⁺,Fe³⁺And (3) performing complexation with filler by using transition metal with d-layer empty valence electron orbits, including IDA, TED, NTA and the like, eluting endotoxin by using a non-ionic surfactant (such as TritonX-100, TritonX-114, Tween 80, Tween 40, Tween 20 and the like), and finally eluting the target protein by using a conventional elution mode of a metal chelating chromatographic column (such as a buffer solution containing Gly, His, imidazole and ammonia and a mode of reducing the pH of the buffer solution).

As a preferred embodiment of the invention, the specific operation steps are as follows:

step 1: the pellet was resuspended in Tris-HCl buffer (50mM Tris-HCl,300mM NaCl, pH8.00) containing 1% Tween 80 at a ratio of 1:10(W (g)/V (mL)) by mass to volume of buffer for 30min until well mixed (no visible particles).

Step 2: and (3) cell disruption, namely homogenizing the resuspended thalli for 3 times by using a high-pressure homogenizer at the temperature of 4 ℃ and the pressure of 600-800 bar.

And step 3: and (3) crushing and incubating the supernatant, centrifuging the crushed cells, discarding the precipitate, taking the supernatant, stirring and incubating the supernatant, wherein the incubation temperature is 20 ℃, and the incubation time is 12 h.

And 4, step 4: and (3) filtering the sample, and filtering the incubated recombinant human growth hormone fusion protein, wherein the filtering aperture is 0.45 mu m.

And 5: IMAC purification, namely taking an IMAC metal chelating chromatographic column, using Cytiva Ni Sepharose FF metal chelating chromatographic packing, and washing the column by using purified water.

Step 6: pre-washing with alkali liquor, and washing the metal chelating chromatographic column with alkali liquor (0.1-1mol/L NaOH solution, preferably 0.5mol/L NaOH solution) with a certain concentration to remove residual endotoxin on the original column, wherein the washing time is 3h, and the specific time is determined according to the alkali liquor tolerance of the filler.

And 7: equilibration, the metal chelate column is equilibrated with the same buffer as used for resuspension of the cell pellet but prepared with ultrapure water or water for injection without alcohol or nonionic surfactant, preferably 50mM Tris-HCl,300mM NaCl, pH8.00, prepared with ultrapure water. The equilibrium volume is 5-20CV (CV is one column volume).

And 8: and (3) loading the sample, namely loading the recombinant human growth hormone fusion protein sample subjected to the incubation and filtration after the crushing into a metal chelating chromatographic column, and ensuring that the recombinant human growth hormone fusion protein sample is combined with the filler.

And step 9: the column is washed, and the impurities not attached to the column are removed by washing the column with an equilibration buffer, preferably 50mM Tris-HCl,300mM NaCl, pH8.00, prepared with ultrapure water.

Step 10: eluting endotoxin with detergent, eluting endotoxin with certain concentration (0.1-5%) of nonionic detergent (such as Triton X-100, Triton X-114, Tween 80, Tween 40, Tween 20, etc.) at a volume of 5-20CV, preferably 50mM Tris-HCl prepared with ultrapure water, 300mM NaCl, PH8.00. containing 1% Triton X-114 nonionic detergent.

Step 11: washing, using balance buffer solution column washing, removing the column residual non ionic detergent and endotoxin, balance buffer solution preferably using ultrapure water prepared 50mM Tris-HCl,300mM NaCl, pH8.00.

Step 12: eluting the target protein by using a conventional elution mode of a metal chelating chromatographic column, preferably 50mM Tris-HCl,300mM NaCl,300mM imidazole and pH8.00.

By adopting the preferred scheme of the embodiment, the recombinant human growth hormone fusion protein containing a polyhistidine (6 × His tag) tag is subjected to metal chelate chromatographic column purification, compared with a method which does not adopt the scheme and only uses a non-ionic surfactant to elute endotoxin at the column purification stage, the eluted target protein is subjected to endotoxin detection by a limulus reagent method, and the protein yield is detected by SDS-PAGE and Lowry, and the result shows that (the comparison result is shown in a table 1, and an IMAC purification chromatogram is shown in a figure 2), the preferred scheme of the embodiment of the invention has the endotoxin content of less than 9.92EU/mg and the yield of 81.82%; the endotoxin content of 166-333EU/mg eluted with the nonionic surfactant in the column purification stage was 61%, while the yield was 61% (the lower yield may be due to co-elution of the endotoxin-protein complex with the nonionic surfactant), and the endotoxin content of 25000EU/mg was 83% without this protocol. Therefore, the endotoxin content is reduced by about 2000 times by only one-step purification, and the endotoxin content is lower by matching with the subsequent purification steps, thereby reaching the pharmacopoeia standard of intravenous injection.

TABLE 1 comparison of endotoxin-removing effects

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.