CN112175915A - Method for removing endotoxin in phage preparation and application - Google Patents

Abstract

The invention relates to a method for removing endotoxin (LPS) in a phage preparation and application thereof, wherein the method comprises the following steps: sterilizing with chloroform, removing impurities such as bacterial fragments in a phage preparation by a centrifugal mode → an active agent TritonX-100 dissociates LPS combined with the phage, degrades macromolecular LPS into small molecular LPS → dialyzes to remove free small molecular LPS → PEG8000 concentrates to improve the titer of the phage and remove the macromolecular LPS → removes introduced impurities by chloroform → dialyzes to remove organic solvents. Its advantages are: (1) the cost is low, the used equipment is simple, and the requirements of conventional laboratories can be met. (2) On the basis of not influencing the activity of the bacteriophage, the removal of the content of the LPS in the bacteriophage preparation to be below 5EU/ml is realized. (3) After recovery, the titer of the active phage can reach 120 percent of that before recovery. (4) Has universal applicability to endotoxin removal from bacteriophage or protein preparations.

Description

Method for removing endotoxin in phage preparation and application

Technical Field

The invention relates to the technical field of biology, in particular to a method for removing endotoxin in a phage preparation and application thereof.

Background

(one) phage module:

bacterial resistance has become a great challenge in the anti-infective field and is one of the problems of widespread concern in the medical field worldwide. According to data issued by the Chinese information drug-resistant network in 2019, about 70% of pathogenic bacteria of the caused diseases are gram-negative bacteria. The first five strains of the main clinical isolates in 2019 are respectively Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa. Many of these pathogenic bacteria develop varying degrees of resistance to different classes of antibiotics. So that the clinical treatment effect is increasingly poor, the side effect of the medicine is increasingly large, and the treatment cost is increased year by year. According to the world health organization, 70 million people die worldwide each year from "superbacterial" infections, including 23 million neonates. In order to solve the problem of bacterial drug resistance, although the world countries pay attention to the research and development of antibiotics, in recent years, the development speed of novel antibiotics is remarkably slowed down, and the difficulty is increased gradually. For some multi-drug resistant bacteria, the treatment effect of the existing antibiotics is more and more limited. Therefore, there is a need to find other methods for treating clinically intractable bacterial infections, and the research focus at home and abroad is mainly on phage anti-infection treatment.

The bacteriophage is a prokaryotic cell virus, specifically infects bacteria, is a natural antibacterial drug, can crack the bacteria, has no change of treatment effect due to drug resistance of the bacteria, and provides a new idea for treating drug-resistant bacterial infection. Phage are typically obtained by amplification in host bacteria, and in the case of gram-negative bacteria, large amounts of endotoxin are released simultaneously during the release of phage-lysed bacteria. The excessive endotoxin level becomes a great obstacle to the safety of the phage preparation, and the removal of endotoxin to obtain pure and safe phage is a great problem which needs to be solved in the clinical application of the phage.

(II) endotoxin module:

endotoxin (endotoxin), also called Lipopolysaccharide (LPS), is a cell wall component of gram-negative bacteria with a basic unit size of 10kDa to 20kDa, but free endotoxin can aggregate into different sized micelles.

Endotoxin is very heat resistant and its biological activity is not destroyed even when heated at a high temperature of 100 ℃ for 1 hour. Endotoxins cause a wide range of biological effects in humans. At moderate doses, endotoxins produce moderate fever and stimulate the immune system. At high doses, high fever, hypotension, disseminated coagulation and fatal shock occur. Thus, the european and us pharmacopoeias set endotoxin thresholds (≦ 5EU/kg body weight × hr) for human therapeutic preparations, but currently, there is no solution in phage preparations that is widely applicable and capable of industrial production of low endotoxin.

(III) removal of endotoxin:

currently, there are two main types of methods for removing endotoxin:

1, non-selective removal:

1. removing LPS according to molecular weight by physical method

1) And (3) ultrafiltration: the selection of a proper ultrafiltration membrane can remove free small molecular LPS in the solution, is mainly suitable for the solution with a large molecular weight of a target product, but has poor effect on LPS combined with phage and large molecular LPS and low recovery rate.

2) Density gradient ultracentrifugation: LPS is separated and removed by using a sucrose density gradient or cesium chloride density gradient ultracentrifugation method. The method has the advantages that: the separation effect is good, and pure particles can be obtained at one time; the method has wide application range, can separate particles with a sedimentation coefficient difference like a high-speed centrifugation method, and can separate particles with a certain buoyancy density difference; the particles are not crushed and deformed, the activity of the particles is maintained, and the formed zones are prevented from causing mixing due to convection.

The disadvantages of this method are: due to different existence forms of LPS and different molecular sizes, densities and sedimentation coefficients, the method is limited in application. The requirements on equipment and personnel are high, an ultracentrifuge and professional personnel are required to operate, the operation is strict and difficult to master, and the requirement is difficult to achieve in a common laboratory. The method has high requirements on samples, and for phage, the virus titer needs to reach high titer to generate a more obvious target band, but most phage (viruses) have difficulty in reaching high titer, so that the method is difficult to remove endotoxin in a phage preparation. Another reason for this is that the amount of sample per treatment is small, the centrifugation time is long, the process is complicated, and it is difficult to industrially produce, especially for a large variety of bacteriophages.

3) Activated carbon adsorption: the method is suitable for removing LPS in a solution with simple components or water, has poor selectivity, is easy to absorb effective components, and ensures that residual activated carbon is difficult to remove. Molecular sieve: the space network structure of the gel molecules is utilized for filtration chromatography, the treatment capacity is small, the time consumption is long, and the method is suitable for the solution with small protein molecular weight.

2. Removal was performed according to the negative charge characteristics of LPS in solution.

1) Charged microfiltration: the method selects the positively charged membrane material such as the microporous filter membrane of the polyaluminium sulfate, the polyacrylonitrile and the like to carry out charged microfiltration, and screens a proper PH condition, so that the removal effect of LPS molecules can be improved, but the optimal condition of each phage needs to be searched in the early stage, the operation is complex, the sample recovery rate is low, and the industrial production is difficult to carry out.

2) Ion exchange: is suitable for removing LPS from alkaline protein, and has low cost and large adsorption capacity. But not in the presence of other negatively charged species (e.g., phage) in the solution.

3. The removal of LPS is carried out according to the characteristics of lipid A (which is the bioactive center of LPS and has hydrophilic and hydrophobic amphipathy). Two-phase extraction method: TritonX-114 extraction: TritonX-114 is a hydrophobic surfactant, and has good effect in removing cytochrome C, catalase and LPS in serum albumin by phase separation method. However, Triton X-114 has high toxicity and is easy to remain, and the performance of the product is influenced.

And (2) selectively removing:

1. immune ligand: according to the mechanism of antigen-antibody high-specificity binding, researchers use an adsorption column using an anti-rHu-TNFa monoclonal antibody as a ligand to eliminate LPS in LPS blood disease. The immunoaffinity has high specificity, but the ligand is difficult to prepare, has narrow application range, and has the problems of high price and difficult elution.

2. Polymyxin B (PMX-B): PMX-B degrades the cell wall of gram-negative bacteria by virtue of its cyclic peptide structure, and the electrostatic and hydrophobic forces between its cation and phosphate groups of lipid A make it able to recognize LPS molecules of different origins. The LPS is removed by adopting a cellulose (or agarose) carrier for fixing the PMX-B, and the method has good effect and stability. However, PMX-B is expensive, nephrotoxic and neurotoxic, and should be used with caution.

3. Deoxycholate (DOC): DOC is a surfactant, can dissociate and destroy micelle structures formed by hydrophobic interaction of LPS, and prevents combination of LPS to realize separation. Thus having a certain effect of removing endotoxin in the solution, but the single use of the endotoxin-removing agent has a general effect of removing endotoxin in the solution.

TritonX-100: triton X-100 (polyethylene glycol octyl phenyl ether), a hydrophilic nonionic surfactant (or detergent). It can dissolve lipid to increase the permeability of cell membrane to antibody, has better dissociation property to virus and LPS in protein, dissociates LPS combined with protein, and degrades large molecular LPS into small molecular LPS, but has no ability to eliminate LPS. Therefore, the effect is not ideal when used alone.

Polycation: the polycation can be stably combined with LPS molecules by electrostatic action, van der Waals force and hydrogen bond, and the adsorption medium taking the polycation as a ligand can adsorb the LPS molecules in a similar way to a flocculation process, so that the adsorption of the polycation with protein molecules is very little. The ligands such as Polyethyleneimine (PEI), poly-L-lysine (PLL), poly-L-histidine (PLH) and the like have biocompatibility superior to that of PMX-B and adsorption capacity superior to that of histidine. However, the LPS removal effect is general, the operation is complex, the cost is high, and the method cannot be used for industrial production.

(IV) application market of low endotoxin phage preparation

Multiple-drug resistant (MDR) bacterial infections caused by "superbacteria" result in approximately 25000 patients dying from multiple-drug resistant bacterial infections each year in europe with a mortality rate of over 50%. Medical costs incurred by drug-resistant bacterial infections in the united states are as high as $ 200 billion per year. Methicillin-resistant staphylococcus aureus (MARS) first appears in the united states to result in about 19000 deaths per year and a property loss of $ 30-40 million. The discovery of new antibiotics has become difficult, and the problem of bacterial resistance has become more acute. According to the forecast of rand corporation (RNAD corporation), 250 million people die of bacterial resistance (AMR) in the coming 2020, rising to 590 million people after ten years and reaching 1400 million people in the coming 2050, according to the current bacterial resistance rate. And this figure is as high as 4.44 billion in 2050 if by extreme cases worsened to 100% drug resistance. On the other hand, AMR in both cases represents a worldwide economic loss of between 0.06% and 3.1% of GDP per year, resulting in cumulative GDP losses between $ 2.1 trillion and $ 124.5 trillion.

In the end of antibiotic therapy, it is imperative that phage therapy maintain or even reduce the rate of progression of bacterial resistance and the associated losses in the battle field. The bacteriophage shows excellent natural endowment in resisting drug-resistant bacteria due to a unique sterilization mechanism. Therefore, the attention and investment of phage in countries and pharmaceutical companies around the world are increasing. A plurality of phage treatment, research and development organizations and companies are established at home and abroad in turn. In recent years, only two companies, Boehringer Invehehold (Boehringer Ingelheim) and Roivant, invested in 20 more billion dollars in the field of phage infection resistance; locus Biosciences authorized Producer to develop commercial engineering phage technology platforms and pipelines at US $ 8.18 million; bioharmony Therapeutics signed a phage lytic enzyme-under-the-product authorization agreement with Boringer Vargahan for a total value of about $ 5 million; IntRon Biotechnology and Roivant have agreed a total of $ 6.67 million for a phage lytic enzyme drug. Some potential phage companies are also present in China. For example, the feijileke, which has acquired investments including pharmacon, zaar, boyuan capital, yuan grass origin, etc., has two phage cocktail lines and two phage lyase lines, and phage products for plants and animals have been marketed. The phage and phage protein drugs for animal and aquaculture have been on the market or are under development by companies such as Qingdao Nuobeite, Qingdao Rui union, Dalian Saim, Dalian Hanxin, Nanjing Jubao, and the like. Although there are many phage related products at home and abroad, there is a distance according to the real clinical application. The major bottleneck problem is whether the endotoxin content in the phage biological preparation can meet the international standard.

The complexity of drug-resistant bacterial infection can make phage therapy difficult. Whether the phage can be directly contacted with the pathogenic bacteria or not is directly related to the treatment effect of the phage. The difficulty of treatment of phages without their bleeding stems to some extent from the choice of the route of administration of the phage. If the phage is treated by bleeding, the phage is required to reach the standard of drug bleeding under the relevant national regulation, which is also the problem to be solved by the current phage treatment.

In a word, the endotoxin index in the phage preparation prepared by the current method is far higher than 5EU/ml, the endotoxin removing process of the phage preparation needs to be improved, and the batch removal of the endotoxin in the phage preparation is imperative and is one of the major problems which need to be overcome when the phage is developed to clinical treatment.

The invention aims to provide a method for removing bacterial endotoxin in phage lysate aiming at the defects in the prior art. There are no reports on the present invention.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for removing endotoxin in a phage preparation.

In order to achieve the purpose, the invention adopts the technical scheme that:

in one aspect, the present invention provides a method for removing endotoxin from a phage preparation, comprising the steps of:

(1) adding chloroform into the mother liquid for preparing bacteriophage to crack bacteria and release all bacteriophage;

(2) adding 5% TritonX-100 to dissociate LPS combined with phage preparation, and degrading free macromolecular LPS in solution into small molecular LPS;

(3) dialyzing to remove free small molecular LPS in the solution;

(4) concentrating PEG8000 to remove macromolecular LPS and TritonX-100, and increasing bacteriophage titer;

(5) adding chloroform to remove residual TritonX-100 and PEG 8000;

(6) and (4) dialyzing to remove residual chloroform, TritonX-100, PEG8000 and other impurities.

Preferably, the concentration of TritonX-100 in step (2) is 5%.

Preferably, the specific steps of step (2) are: adding 5% TritonX-100, performing vortex oscillation, placing in a constant temperature shaking table at 37 deg.C and 220rpm, performing oscillation incubation for 2-3h, centrifuging at 10000rpm/min for 15min, and collecting supernatant.

Preferably, the proportion of PEG8000 added in step (4) is 10%.

Preferably, the resulting phage preparation has an endotoxin content of 5EU/ml or less.

Preferably, the titer of active phage after recovery is up to 50% -120% of that before recovery.

In another aspect, the invention also provides the use of a method as described above for the preparation of a medicament using bacteria.

Preferably, the bacteria include gram-negative bacteria producing LPS such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the like.

The invention has the advantages that:

1. low cost, simple equipment, simple and convenient operation procedure, stable effect and easy large-scale industrial production.

2. The endotoxin removal effect is good and is lower than the international requirement standard for the endotoxin content of the blood preparation, the influence on the activity of the phage is small, and the titer of the active phage after recovery can reach 120 percent of that before recovery.

3. The method has wide applicability, has good endotoxin removing effect on lysates of bacteriophage of brachyury, Myoviridae and longtail, and has universal applicability to other bacteria fermentation preparation products.

4. The economic effect is as follows: low cost and wide market:

the cost is low: only 2800 yuan, on average 2.8 yuan/ml, was required to make an ultra low endotoxin phage preparation per liter.

Name of Material	Price/specification	Dosage of	Cost of
				Diamond Tryptone (Tryptone)	1238 yuan/500 g	10g	24.76 yuan
Diamond Yeast extract (Yeast)	675 yuan/500 g	5g	6.75 yuan
				Diamond sodium chloride (NaCl)	118 yuan/500 g	68.5g	16.16 yuan
Chloroform	850 yuan/500 ml	30ml	51 yuan
				TritonX-100	198 yuan/500 ml	50ml	19.8 yuan
Dialysis membrane	5700 yuan/10 m	3.1m	1767 yuan
				Physiological saline	20 yuan/3000 ml	220000ml	800 Yuan
PEG8000
		100 yuan/500G	100g	20 Yuan
50ml centrifuge tube					1.76 yuan/piece	10 are provided with	18 yuan
	EP tube, lance tip, etc	0.545/one	18 are provided with	10 Yuan
Total up to							2733.47≈2800

The market is wide:

the method can remove the endotoxin content in the phage preparation in batches, so that the blood entry standard can be reached, and the application step of the phage is greatly accelerated. Thus, a whole set of phage therapy industry chain can be formed. The economic value is immeasurable, and the social effect is huge.

In particular, the advantages of the present invention over the prior art are specified in the following table:

drawings

FIG. 1 shows the technical route for removing endotoxin (LPS) from the phage preparation of the present invention.

FIG. 2 shows the results of the safety verification experiment of the bacteriophage with TritonX-100 at different concentrations in example 4.

FIG. 3 shows the effect of different concentrations of Tritonx-100 on the change in phage titer after Tritonx-100 (including multiple concentrations) dialysis-concentration-dialysis treatment.

Fig. 4 shows the results of the effect of TritonX-100 on endotoxin changes after TritonX-100 (including multiple concentrations) dialysis-concentration-dialysis treatment.

FIG. 5 shows the results of titer changes before and after the short, myo, and long-tailed phage preparation treatment.

FIG. 6 shows the results of endotoxin changes before and after the treatment with the preparation of short, myo-and long-tailed phage.

FIG. 7 shows the results of the variation of endotoxin to titer ratio before and after the short, myo, and long-tailed phage preparation treatment.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

The method is comprehensively applied and optimized by summarizing and exploring the reported and researched method for effectively removing the LPS in the solution, can reach the standard of clinical application through common instruments and equipment and simple operation, and can be used for industrial production. The method of the invention is divided into three parts: the first part is mainly to remove free LPS and micromolecule LPS in the solution; the second part is mainly to remove large molecular LPS and increase the titer of phage; the third part is to remove the residue of the organic reagent. The details can be found in the examples below.

The reagents and equipment involved in the following examples are as follows:

desk type centrifuge (eppendorf Centrifuge 5810R)

Magnetic stirrer MS-H380-Pro

LB Broth, LB agar, agar powder-Purchase Beijing Solebao science and technology Co

Chloroform-purchased from Changsheng biotechnology Limited of Beijing Ding Guo

TritonX-100 (TritonX-100) -Purchase Biotechnology engineering (Shanghai) Ltd

Biotech CE Tubin MWCO:1000 kD-American Spectrapor dialysis Membrane

0.9% physiological saline-purchased from Shandongdu Dudu pharmaceutical Co Ltd

Sodium chloride-purchased from Biotechnology (Shanghai) Ltd

Polyethylene glycol 8000-purchased from Biotechnology (Shanghai) Ltd

Gram-negative bacteria lipopolysaccharide detection kit (color development method) -Tianjin Xinuo biological medicine GmbH is purchased.

Example 1 removal of free and small LPS from solution

1. Phage amplification

The host bacteria are streaked on an LB solid culture medium by a flat plate, the obtained product is placed in a constant temperature incubator at 37 ℃ for about 12 hours of culture, a single bacterial colony which accords with the bacterial morphology is selected and placed in 5ml of LB liquid culture medium, and the obtained product is subjected to shaking culture at 37 ℃ and 220rpm for about 8 hours. The host bacteria are transferred according to the proportion of 1 percent and cultured to the logarithmic phase.

Host bacteria in logarithmic growth phase are transferred according to the proportion of 1 percent, simultaneously target phage in equal proportion is added, and then the mixture is placed at 37 ℃ and is cultured for about 2 hours in a shaking way at 220 rpm. In addition, 2 groups of control experiments are set, wherein one group only inoculates bacteria, and the other group neither inoculates host bacteria nor bacteriophage. Compared with three groups of changes, the solution of the control group 1 (only bacteria is added) should be turbid, the solution of the control group 2 (no bacteriophage and no bacteria is added) should be clear and unchanged, the amplification group should be clear and bright under normal conditions, and if the solution is turbid, the amplification fails. Adding appropriate amount of host bacteria in logarithmic phase into the clarified amplification group, performing shake culture at 37 deg.C and 220rpm for about 30min, observing solution change, clarifying the solution, culturing for 30min, and adding appropriate amount of host bacteria liquid in logarithmic phase if the solution is still clarified. Repeating the above steps 3 to 5 times, and finally clarifying the mother liquor (possibly some bacterial fragments), namely completing the amplification, wherein the obtained clarified liquor is the phage mother liquor.

2. Chloroform sterilization

Adding chloroform into the phage mother liquor according to the proportion of 1%, vortexing and shaking for 1min, mixing well, and then ice-bathing for 20min, wherein the time is reversed from top to bottom for 2-3 times. After the ice bath was completed, centrifugation was carried out at 8000rpm/min for 10 min. And taking the supernatant, wherein the supernatant is the bacteriophage amplification sterilization liquid.

The operation purpose and the function of the step are as follows:

(1) the lytic bacteria release all phage.

(2) Impurities such as bacterial debris generated by bacteriophage lysis bacteria in the phage preparation are removed by chloroform.

3. On the basis of early preliminary experiments, TritonX-100 is used for dissociating and degrading LPS

Adding TritonX-100 into bacteriophage amplification and sterilization liquid according to the proportion of 5%, and performing vortex oscillation to completely dissolve the TritonX-100 in the solution. Then placing the mixture in a constant-temperature shaking table with the temperature of 37 ℃ and the rpm of 220 to shake and incubate for 2 to 3 hours, after the incubation is finished, centrifuging the mixture for 15 minutes at the speed of 10000rpm/min, and taking supernatant (possibly without obvious layering phenomenon, but discarding a few milliliters of solution at the bottom) for next treatment.

The purpose and function of this step are:

(1) the LPS bound to the phage is dissociated by using the characteristics of TritonX-100, and the two are separated.

(2) Free macromolecular LPS in the solution is degraded into micromolecular LPS, so that the subsequent removal is convenient.

4. Dialysis

The supernatant treated in step 3 was put into a dialysis membrane of Biotech CE piping MWCO:1000kD, placed in physiological saline of 100 times the volume of the sample solution, and dialyzed twice for 4 hours each time on a magnetic stirrer at 400 rpm/min. The solution turns into a colorless transparent liquid after dialysis with a pale yellow liquid (culture medium color).

The purpose and function of this step are:

(1) and (4) dialyzing to remove free small molecular LPS in the solution, thereby achieving a certain LPS removal effect.

(2) Medium components and impurities in the sample solution are replaced, so that the sample solution does not contain the medium components and the impurities, and safety guarantee is provided for subsequent clinical application.

Example 2 removal of macromolecular LPS and increase of phage titer

5. PEG concentration

The dialyzed solution of step 4 of example 1 was placed in an appropriate centrifuge tube (15ml or 50ml), and solid NaCl was added at 5.84g/100ml to sufficiently dissolve the solution. PEG8000 was added at a ratio of 10% (W/V) to dissolve the mixture, thereby making the solution homogeneous. Standing at 4 deg.C overnight or standing for more than 8 hr, centrifuging at 12000rpm/min for 20min, discarding supernatant, and inverting the centrifuge tube for 2-3min to remove supernatant as much as possible. Washing solution (0.9% physiological saline, PBS solution, SM buffer, etc.) is added to 10% of the original solution for resuspension, and the tube wall is washed thoroughly. The resulting liquid was the phage concentrate.

The purpose of this step is:

(1) removing the LPS of the macromolecules in the phage preparation.

(2) Removing TritonX-100 reagent to remove the introduced reagent as much as possible.

(3) The titer of the phage is increased, and the follow-up clinical application is guaranteed (the clinical application generally has higher requirement on the titer of the phage).

Example 3 removal of organic reagent residue

6. Chloroform extraction

The resuspended solution in step 5 of example 2 was resuspended in 1: 1, adding a chloroform solution, performing vortex oscillation for 1min, fully and uniformly mixing, then performing centrifugation at 5000rpm/min for 5min, obviously layering the solution, and taking supernatant for repeated extraction once.

The purpose of this step is:

(1) the residual TritonX-100 reagent was further removed.

(2) Remove PEG8000 and other impurities in the solution.

7. Dialysis

Diluting the solution obtained in step 6 according to clinical requirement, transferring into Biotech CE tubular MWCO:1000kD dialysis membrane, placing in 100 times volume of physiological saline, dialyzing for 2 times, and removing chloroform solution.

The purpose of this step is:

(1) the main function is to remove the various reagents introduced in the solution.

(2) The residual chloroform solution was removed from the solution.

(3) The TritonX-100 reagent was further removed.

(4) The PEG8000 reagent was further removed.

(5) Removing other ions and impurities in the solution.

Example 4 safety verification experiment

In the embodiment, phage preparations are treated by using only TritonX-100, and the groups are different in the concentration of TritonX-100, so that the influence of the concentration of TritonX-100 on the phage preparations is verified.

The experimental results are shown in FIG. 2.

As can be seen from fig. 2: TritonX-100 concentrations from 0.5% to 10% do not adversely affect phage titer, infectivity.

EXAMPLE 5 control test

In the embodiment, phage preparations are treated according to the TritonX-100-dialysis-concentration-dialysis method, the concentration of TritonX-10 is different in each group, the influence of the TritonX-100 concentration on the phage preparations is verified, and the results are recorded.

The experimental results are shown in figures 3-4, which show that the treatment effect of the 5% TritonX-10 is best.

EXAMPLE 6 Effect test

This example is directed to the testing of phage preparations prepared in examples 1-3, including short, myocaudate and long tail strains.

1. Experimental methods

The endotoxin detection in the test process adopts the following steps: a gram-negative lipopolysaccharide detection kit (a color development method) EKT-25M:50 persons/box of Tianjin Xinuo biomedicine GmbH can accurately quantify the content of endotoxin in a solution. The standard curve range is as follows: 0.005-5 EU.

2. Results of the experiment

2.1 Change in titer, endotoxin to titer ratio before and after treatment with phage preparations of the short, myo-and long-tailed strains the results are shown in FIGS. 5-7, respectively.

3 conclusion

The method solves the problem that endotoxin exceeds the standard in the prior art, which is a difficulty in restricting the development of phage preparations, realizes the large-scale and mass removal of endotoxin in the phage preparations, keeps higher recovery rate, does not damage the infectivity of the phage, can reach the American FDA standard in endotoxin removal, and provides guarantee for the clinical application of the phage.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims (7)

1. A method for removing endotoxin from a phage preparation, comprising the steps of:

(1) adding chloroform into the phage mother liquor to crack bacteria and release all phages;

(2) adding 5% TritonX-100 to dissociate LPS combined with phage preparation, and degrading free macromolecular LPS in solution into small molecular LPS;

(3) dialyzing to remove free small molecular LPS in the solution;

(4) concentrating PEG8000 to remove macromolecular LPS and TritonX-100, and increasing bacteriophage titer;

(5) adding chloroform to remove residual TritonX-100 and PEG 8000;

(6) dialyzing to remove residual impurities to obtain the phage preparation with ultralow endotoxin level.

2. The method according to claim 1, wherein the specific steps of step (2) are as follows: adding 5% TritonX-100, performing vortex oscillation, placing in a constant temperature shaking table at 37 deg.C and 220rpm, performing oscillation incubation for 2-3h, centrifuging at 10000rpm/min for 15min after incubation is completed, and collecting supernatant.

3. The method according to claim 1, wherein the concentration of PEG8000 added in step (4) is 10%.

4. The method of claim 1, wherein the titer of active phage after recovery is up to 50% -120% of that before recovery.

5. The method according to claim 1, comprising the steps of:

(1) preparing a phage mother solution;

(2) and (3) chloroform sterilization: adding chloroform into the phage mother liquor according to the proportion of 1%, performing vortex oscillation for 1min, fully mixing, performing ice bath for 20min, reversing the ice bath for 2-3 times, centrifuging at 8000rpm/min for 10min after the ice bath is finished, and taking supernatant, wherein the supernatant is phage amplification sterilization liquid;

(3) TritonX-100 dissociates and degrades LPS: adding TritonX-100 into bacteriophage amplification sterilization liquid according to the proportion of 5%, performing vortex oscillation to completely dissolve TritonX-100 in the solution, placing in a constant temperature shaking table at 37 ℃ and 220rpm, performing oscillation incubation for 2-3h, centrifuging at 10000rpm/min for 15min after incubation is completed, and taking supernatant;

(4) and (3) dialysis: placing the supernatant in a Biotech CE tubular MWCO 1000kD dialysis membrane, placing in 100 times volume of physiological saline, and dialyzing twice on a magnetic stirrer at 400rpm/min for 4h each time;

(5) PEG8000 concentration: placing the dialyzed solution in a centrifuge tube, adding solid NaCl according to the proportion of 5.84g/100ml, fully dissolving, adding PEG8000 according to the proportion of 10% to dissolve the solution to obtain a homogeneous solution, then placing the solution at 4 ℃ overnight or standing for 8h, then centrifuging at 12000rpm/min for 20min, discarding the supernatant after centrifugation, inverting the centrifuge tube for 2-3min, adding a washing solution according to the proportion of 10% of the original solution to carry out heavy suspension, and fully cleaning the tube wall to obtain a liquid, namely a phage concentrate;

(6) chloroform extraction: the resuspended solution was mixed according to 1: 1, adding a chloroform solution, performing vortex oscillation for 1min, fully and uniformly mixing, then performing centrifugation at 5000rpm/min for 5min, and taking the supernatant to perform repeated extraction once;

(7) and (3) dialysis: diluting the solution obtained in step 6, transferring into a Biotech CE tubular MWCO:1000kD dialysis membrane, placing into 100 times volume of physiological saline for dialysis, dialyzing for 2 times, and removing chloroform solution from the solution.

6. Use of a method according to any one of claims 1 to 5 for the preparation of a medicament using bacteria.

7. The use according to claim 6, wherein said bacteria comprise gram-negative LPS-producing bacteria selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

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