Purification method of bacteriophage preparation and application of bacteriophage preparation in industrial scale production

Abstract

The invention relates to the technical field of biology, in particular to a purification method of a bacteriophage preparation and application of the bacteriophage preparation in industrial scale production. The purification method of the bacteriophage preparation comprises the following steps: removing impurities such as bacteria and bacterial fragments in the bacteriophage preparation through grading membrane microfiltration; a hollow fiber ultrafiltration system is adopted to remove impurities including free lipopolysaccharide (LPS) and improve the titer of the bacteriophage at the same time; free LPS is further rapidly and accurately removed through a novel compound chromatography medium of molecular exclusion and charge adsorption, and a high-purity bacteriophage preparation can be obtained. According to the method, the endotoxin level in a phage preparation can be lower than 20 EU / 10 < 9 > PFU (the removal rate is greater than 99%), and the phage recovery rate is greater than 63.5%. The purification method provided by the invention has the advantages that the use of an organic solvent is reduced or avoided to the greatest extent, the technological process is green and environment-friendly, the endotoxin removal rate is high, and time and cost are saved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a method for purifying a bacteriophage preparation and its application in industrial-scale production. Background Art

[0002] Bacterial resistance to antibiotics is considered one of the biggest challenges facing humanity in the 21st century and a major threat to the global public health system. As a natural enemy of bacteria, bacteriophages have many advantages such as wide distribution, high bactericidal efficiency, self-replication and host specificity, and are recognized as potential alternatives to antibiotics. Humans have been studying bacteriophages for a long time, but with the emergence of a large number of antibiotics and chemical antibacterial drugs and their high efficiency in fighting bacterial infections, the research on bacteriophages in developed countries such as Europe and the United States was interrupted for a time. It was not until the 1980s that humans re-emphasized the research on bacteriophages due to the emergence of multi-drug resistant bacteria. With the development of modern biotechnology, bacteriophages have been widely used in human epidemic prevention and control, bacterial diseases of animals and plants, and food safety, becoming a weapon for humans to fight bacterial infections.

[0003] The purity of phage preparations, in other words, the level of impurities remaining, is directly related to its safety and is one of the important detection indicators for the quality control of phage preparations. For Gram-negative pathogens, phage lysis of host bacteria will cause a large amount of lipopolysaccharide (i.e., endotoxin) in the cell outer membrane layer to be released. The endotoxin content in a typical Gram-negative phage lysis solution can reach 10 5 ~10 6EU / mL. Bacterial endotoxin is a lipopolysaccharide (LPS) on the cell wall of Gram-negative bacteria. It is composed of core polysaccharides, lipid A and O-specific side chains. It has high thermal stability and will not be destroyed even if heated at 100°C for 1 hour. It can only be inactivated by heating at 160°C for 2 to 4 hours, or boiling with strong alkali, strong acid or strong oxidant for 30 minutes, so it is difficult to remove. Endotoxin units are generally measured in EU (Endotoxin Unit) or IU (International Unit), 1EU = 1IU, equivalent to 0.2ng lipopolysaccharide (FDA standard substance). Although endotoxin has low oral toxicity, there is a risk of entering the blood after the intestinal mucosal function is damaged. Overdose can cause fever, shock, multiple organ failure and even death. For the endotoxin limit in phage preparations, the European Pharmacopoeia stipulates that the endotoxin limit for intravenous injection and oral administration is 0.2IU or 5IU body weight, respectively. The requirements of the US FDA are the same as those of the European Pharmacopoeia. For clinical applications, the endotoxin limits for intravenous and oral preparations are 5EU / h / kg and 20EU / mL, respectively. Therefore, for phage preparations, effective strategies must be adopted to remove endotoxins from the source, the phage lysate, and detect its residual amount.

[0004] Scholars around the world have conducted extensive research on the detection and removal of endotoxins in phage preparations. The main methods include TritonX-100 dissociation and chloroform dialysis, cesium chloride density gradient centrifugation, and LPS affinity chromatography. The lipophilic solvent is used to reduce the content of lipopolysaccharide in phage preparations, and the endotoxin removal effect is significant. However, these methods generally have the disadvantages of many steps, complex processes, time-consuming and labor-intensive, high energy consumption, and environmental pollution. Chloroform can inhibit the central nervous system and may cause dizziness, headache, fatigue, and confusion; cesium chloride and TritionX-100 can cause irritation to the eyes, respiratory tract, and skin. In addition, although there is no clear evidence that TritionX-100 is carcinogenic, its potential toxicity and harm to the human body still need to be taken seriously. Some scholars have also tried to use polyethylene glycol precipitation, but this method has a small processing volume and is time-consuming and labor-intensive. Charge adsorption has also been used to purify phages in recent years, which has a high phage recovery rate but a low endotoxin removal rate.

[0005] Therefore, there is an urgent need to develop a new preparation method that can control the endotoxin content in the phage lysate to a low range without involving organic reagents and can purify phage preparations that meet safety requirements. Summary of the invention

[0006] The present invention aims to solve the problems of complex purification process, low endotoxin removal rate, harm to human body and pollution to environment of current phage preparations, and provide a method for purifying phage preparations without the involvement of organic reagents. The purification method provided by the present invention first uses a graded microfiltration membrane to remove impurities such as bacteria and bacterial fragments in the phage lysate, then uses a tangential flow filtration column to remove impurities including free endotoxins, and finally uses a new composite chromatographic medium to further remove free impurities including free endotoxins. All operations of the purification method provided by the present invention do not involve organic reagents, and separation is mainly achieved by using the difference in molecular weight between endotoxins and phage particles.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for purifying a phage preparation, comprising the following steps:

[0009] S1, taking a phage lysate, and sequentially microfiltering the phage lysate using graded microfiltration membranes with cutoff pore sizes of 0.45 μm and 0.22 μm to obtain a feed solution; the phage lysate contains endotoxins, and the endotoxins are derived from bacteria and / or bacterial fragments in the phage lysate;

[0010] S2. Ultrafiltration of the feed liquid is performed using a tangential flow filtration column with a molecular weight cutoff of 300 to 750 kDa to obtain a reflux end filtrate; the shear rate of the tangential flow filtration column is 4000 to 8000 s -1 , the system pressure of the tangential flow filtration column pipeline is set to a constant value, and the constant value ranges from 1 to 2 psi;

[0011] S3, using a "molecular exclusion-charge adsorption" composite chromatography column to purify the reflux end filtrate to obtain a high-purity phage preparation; the chromatography medium of the "molecular exclusion-charge adsorption" composite chromatography column is CaptoCore400 TM , the loading buffer was PBS buffer.

[0012] In the preferred embodiment, the molecular weight cut-off of the tangential flow filtration column in step S2 is 500 kDa, and the shear rate is 8000 s -1 , the system pressure of the tangential flow filtration column pipeline is set to 1 psi.

[0013] In a preferred embodiment, the phage lysis solution is selected from one or more of a long-tail phage lysis solution, a short-tail phage lysis solution and a muscle-tail phage lysis solution.

[0014] In a preferred embodiment, the bacteria and / or bacterial fragments in step S1 are Gram-negative bacteria, and the Gram-negative bacteria are selected from pathogenic Gram-negative bacteria in clinical, livestock and poultry, and aquaculture industries.

[0015] In a preferred embodiment, the pathogenic Gram-negative bacteria are selected from one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Salmonella.

[0016] In a preferred embodiment, before ultrafiltration, the tangential flow filtration column is rinsed with 0.1 M NaOH and PBS buffer in sequence.

[0017] In a preferred embodiment, the tangential flow filtration column is a hollow fiber column.

[0018] In the best embodiment, a method for purifying a phage preparation comprises the following steps:

[0019] S1, taking a phage lysate, and performing microfiltration on the phage lysate using a microfiltration membrane to remove impurities such as bacteria and bacterial fragments in the phage lysate to obtain a feed liquid;

[0020] S2, ultrafiltration of the feed liquid using a tangential flow filtration column, and collection of the filtrate at the reflux end;

[0021] S3. Use the "molecular exclusion-charge adsorption" composite chromatography column to continue purifying the reflux end filtrate. After pumping the reflux end filtrate into the "molecular exclusion-charge adsorption" composite chromatography column, observe the first penetration peak and collect the corresponding filtrate. The filtrate collected when the ultraviolet absorption detection value begins to rise and returns to the baseline is observed on the computer, which is the purified phage preparation.

[0022] The experimental conditions of the purification method of the phage preparation under the above optimal scheme are as follows:

[0023] In step S1, the phage lysate is filtered using microfiltration membranes with a cutoff pore size of 0.45 μm and 0.22 μm in sequence.

[0024] In step S2, the molecular cutoff of the tangential flow filtration column is 500 kDa, the system pressure should be kept constant at 1 system pressure during ultrafiltration, and the shear rate is 8000 s -1 When the molecular weight cut-off is too low, the amount of endotoxin that can be removed is limited; when the molecular weight cut-off is too high, a large amount of phage particles will be lost during the purification process. When the system pressure is too low, no liquid will flow out of the permeate end, and the separation of phage particles and impurities cannot be achieved. When the system pressure is too high, a large amount of phage particles will be lost. If the sample flow rate is too low, the entire separation time will be too long and the separation effect will be affected.

[0025] In step S3, the filler of the "molecular exclusion-charge adsorption" composite chromatography column is CaptoCore 400 TM The filler contains a large number of microspheres. Substances with a diameter less than 20nm will enter the microspheres through the small holes on the surface of the microspheres. The inside of the microspheres are functionalized ligands, which are both hydrophobic and positively charged, so that impurities entering the microspheres can be adsorbed in the core of the microspheres. Substances with a diameter greater than 20nm will flow out of the chromatography column, thereby achieving the separation of bacteriophages from impurities such as endotoxins in the phage fluid.

[0026] In step S3, the loading buffer selected is PBS buffer, which does not contain calcium and magnesium ions, and will not cause floccules to form during the subsequent elution of impurities from the chromatography column, thereby causing clogging of the chromatography column.

[0027] In a second aspect of the present invention, there is provided an application of any of the above purification methods in industrial-scale production, any one or more of the following:

[0028] (1) Used for preparing bacteriophage preparations;

[0029] (2) Used for the preparation of antibacterial products with bacteriophage preparations as active ingredients.

[0030] Beneficial effects of the present invention

[0031] 1. The endotoxin removal effect is significant, and the endotoxin content of phage preparations is less than 20EU / 10 9 PFU (removal rate> 99%), which is lower than the endotoxin limit of oral preparations for clinical application.

[0032] 2. The present invention has wide applicability and has a good endotoxin removal effect on the lysates of different types of phages of the Brachyura family, Myoura family and Doliura family.

[0033] 3. All steps of the present invention do not involve organic reagents, and compared with other endotoxin removal schemes, it is safer and more environmentally friendly.

[0034] 4. The purification method of the bacteriophage preparation provided by the present invention has a large processing capacity, a simple process, and is easy to realize industrialization.

[0035] 5. The endotoxin removal rate of the phage lysate purified by the purification scheme of the present invention is 99.20%, and the phage retention rate is 64.84%; the endotoxin removal rate of the phage lysate purified by the traditional dissociation scheme is 98.40%, and the phage retention rate is 25.02%. Compared with the traditional dissociation scheme, the purification scheme of the present invention not only significantly improves the endotoxin removal rate and phage recovery rate, but also avoids the participation of organic reagents. It is significantly superior to the traditional dissociation scheme in all aspects and is more suitable for industrial-scale application of phages. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a flow chart of phage purification;

[0037] Figure 2 This is the comparison result of the effect of different molecular weight cut-offs on the purification of phage lysate in cross-flow filtration in Example 1;

[0038] Figure 3 This is the comparison result of the effect of different shear rates on the purification of phage lysate in cross-flow filtration in Example 2;

[0039] Figure 4 This is a comparison result of the effect of different system pressures on the purification of phage lysate in cross-flow filtration in Example 3;

[0040] Figure 5 The changes in endotoxin content and phage titer before and after the purification of the long-tailed phage lysate in Example 4;

[0041] Figure 6 This is the comparison result of the effect of treating phage lysate in Example 5 and Comparative Example 1;

[0042] Figure 7 This is a comparison of the effects of Example 5 and Comparative Example 2 on treating phage lysate. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] The present invention provides a method suitable for industrial-scale purification of endotoxins in phage preparations and its application. The purification method of phage preparations includes the following steps: removing impurities such as bacteria and bacterial fragments in phage preparations by graded membrane microfiltration; using a hollow fiber ultrafiltration system to remove impurities including free lipopolysaccharide (LPS) while increasing the titer of phages; and further quickly and accurately removing free LPS through a new type of composite chromatography medium of "molecular exclusion + charge adsorption" to obtain a high-purity phage preparation. This method can reduce the endotoxin level in phage preparations to less than 20EU / 10 9 PFU (removal rate> 99%), phage recovery rate> 63.5%. The advantages of the invention are: the use of organic solvents is reduced or avoided to the greatest extent, the process is green and environmentally friendly, the endotoxin removal rate is high, and time and cost are saved.

[0045] The present invention is a comprehensive application based on the existing endotoxin removal scheme. It avoids the use of organic reagents on the basis of removing impurities such as endotoxins in phage preparations, thereby improving the safety of purified phage preparations. After purification by the present invention, the phage lysate can meet the clinical application standards. The invention is simple to operate, and the instruments and equipment used are common and easy to obtain, and can be industrialized.

[0046] The tangential flow filtration column and chromatography column fillers used in the present invention are purchased from Cytiva.

[0047] The endotoxin detection method described in the present invention is a horseshoe crab reagent dynamic colorimetric method, which is an endotoxin detection method provided in General Chapter 1143 of the Chinese Pharmacopoeia.

[0048] like Figure 1 As shown, a method for purifying a phage preparation comprises the following steps:

[0049] S1. Filter the phage lysate using microfiltration membranes with cutoff pore sizes of 0.45 μm and 0.22 μm through a suction pump to remove impurities such as bacteria and / or bacterial fragments in the phage lysate to obtain a feed solution.

[0050] The bacteria and / or bacterial fragments are Gram-negative bacteria, preferably Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and the like, which are representative Gram-negative bacteria.

[0051] S2. Use a tangential flow filtration column to ultrafilter the feed liquid to remove impurities including free endotoxins in the feed liquid, while increasing the titer of bacteriophages, and collect the filtrate at the reflux end.

[0052] Among them, the molecular weight cutoff of the tangential flow filtration column is 300-500 kDa, and the shear rate is 4000-8000 s -1 The system pressure of the tangential flow filtration column pipeline is set to a constant value, and the constant value ranges from 1 to 2 psi; more preferably, the molecular weight cutoff is 500 kDa and the system pressure is 1 psi.

[0053] In the preferred embodiment, the tangential flow filtration column uses a hollow fiber column (also known as a hollow fiber ultrafiltration system), the molecular weight cutoff of the hollow fiber column is 500 kDa, and the shear rate is 8000 s -1 , the system pressure is constant at 1psi;

[0054] Notes for step S2:

[0055] Before using the tangential flow filtration column, the pipeline should be cleaned with 0.5M NaOH solution. Before pumping in the phage lysis solution, the entire pipeline should be rinsed with PBS buffer. After draining the pipeline, pump in the phage lysis solution and maintain the pipeline system pressure at 1psi. After completion, the pipeline should be cleaned with 0.5M NaOH and the tangential flow filtration column should be preserved with 0.1M NaOH.

[0056] S3. Use a "molecular exclusion + charge adsorption" composite chromatography column to further remove free impurities including free endotoxin to obtain a pure phage preparation.

[0057] In the preferred embodiment, the filler in the "molecular exclusion + charge adsorption" composite chromatography column is a new composite chromatography medium CaptoCore 400 TM .

[0058] The "molecular exclusion + charge adsorption" composite chromatography column uses CaptoCore 400 TM The chromatographic column filled with fillers contains a large number of microspheres. Substances with a diameter less than 20nm will enter the microspheres through the small holes on the surface of the microspheres. The inside of the microspheres are functionalized ligands, which are both hydrophobic and positively charged, so that the impurities entering the microspheres are adsorbed in the core of the microspheres. Substances with a diameter greater than 20nm will flow out of the chromatographic column, thereby achieving the separation of bacteriophages and impurities such as endotoxins in the phage fluid.

[0059] Notes for step S3:

[0060] In step S3, the loading buffer is PBS buffer, and the phage is pumped in after the UV absorption curve is stable. When the UV absorption curve rises, the permeate is collected, and the collection is stopped after the UV absorption curve falls back. This is the purified phage. After collecting the phage, the chromatography column should be cleaned with 1M NaOH and 30% isopropanol. The UV absorption curve rises again, which is the impurity adsorbed inside the chromatography column. Stop cleaning after the UV curve falls back and stabilizes, and use 20% ethanol to preserve the chromatography column.

[0061] The above-mentioned method for purifying phage preparations is suitable for preparing phage preparations on an industrial scale.

[0062] Example 1 Effects of molecular weight cut-off on phage titer and endotoxin removal during ultrafiltration

[0063] A method for purifying a bacteriophage preparation comprises the following steps:

[0064] S1. In a sterile environment, filter the myoclonic Pseudomonas aeruginosa phage lysate using filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively, and store it in a 4°C refrigerator after filtration.

[0065] S2. Purify the same batch of phage lysate obtained in step S1 using hollow fiber columns with molecular weight cutoff of 300 kDa, 500 kDa, and 750 kDa, respectively, and maintain the shear rate at 8000 s -1 , the system pressure is 1psi. After alkaline washing, PBS rinsing, sample loading, top washing and pipeline cleaning operations, the phage lysate purified in this step is collected separately.

[0066] The endotoxin content and phage titer in the phage lysate purified by hollow fiber columns with different molecular weight cutoffs were measured using the limulus amebocyte lysate dynamic colorimetric method and the double-layer plate method. Figure 2 As shown in the figure, the purification effect of hollow fiber columns with different molecular weight cutoffs, the results show that when the molecular weight cutoff is 750kDa, the phage retention rate is too low, and when the molecular weight cutoff is 300kDa, the endotoxin removal rate is lower than that of the molecular weight cutoff of 500kDa. Therefore, the preferred molecular weight cutoff is 500kDa.

[0067] Example 2 Effect of shear rate on bacteriophage titer and endotoxin removal rate during ultrafiltration

[0068] A method for purifying a bacteriophage preparation comprises the following steps:

[0069] S1. In a sterile environment, filter the myoclonic Pseudomonas aeruginosa phage lysate using filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively, and store it in a 4°C refrigerator after filtration.

[0070] S2. Purify the same batch of phage lysate obtained in step S1 using a hollow fiber column with a molecular weight cutoff of 500 kDa, and select a shear rate of 2000 s -1 , 4000s -1 and 8000s -1 , keeping the system pressure at 1, after alkaline washing, buffer rinsing, sample loading, top washing and pipeline cleaning operations, the phage lysate purified in this step was collected separately.

[0071] The endotoxin content and phage titer in the phage lysate purified by hollow fiber column treated with different shear rates were measured by the lysate dynamic colorimetric method and the double-layer plate method. Figure 3 As shown, the purification effect of hollow fiber column at different shear rates. The results show that the shear rate is 2000s -1 The endotoxin removal rate was low when the shear rate was 4000s -1 and 8000s -1 When the shear rate was 8000s -1 The purification rate is faster when the shear rate is 8000s. -1.

[0072] Example 3 Effect of system pressure on bacteriophage titer and endotoxin removal rate during ultrafiltration

[0073] S1. In a sterile environment, filter the myoclonic Pseudomonas aeruginosa phage lysate using filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively, and store it in a 4°C refrigerator after filtration.

[0074] S2. Purify the same batch of phage lysate obtained in step S1 using a hollow fiber column with a molecular weight cutoff of 500 kDa, and select a shear rate of 8000 s -1 , maintaining the system pressure at 1psi, 1.5psi and 2psi respectively. After alkaline washing, buffer rinsing, sample loading, top washing and pipeline cleaning operations, the phage lysate purified in this step was collected respectively.

[0075] The endotoxin content and phage titer in the phage lysate purified by hollow fiber column treated with different system pressures were measured by the lysate dynamic colorimetric method and the double-layer plate method. Figure 4 As shown in the figure, the purification effect of the hollow fiber column with different system pressures, the results show that the phage retention rate is lower when the system pressure is 1.5 and 2, so the preferred system pressure is 1.

[0076] Example 4 Purification of long-tailed Pseudomonas aeruginosa phage lysate by the method of the present invention

[0077] A method for purifying a bacteriophage preparation comprises the following steps:

[0078] S1. In a sterile environment, filter the long-tailed Pseudomonas aeruginosa phage lysate using filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively, and store it in a 4°C refrigerator after filtration.

[0079] S2. Before starting this step, clean the 500kDa tangential flow filtration pipeline, circulate the entire pipeline with 0.5M NaOH solution for 5 minutes, empty the pipeline, and circulate it with pure water for 2 minutes before emptying it. Before pumping in the phage, circulate PBS buffer in the pipeline for 5 minutes and then empty it to prevent the phage particles from rupturing due to osmotic pressure imbalance. Then pump in the phage lysate to be processed, maintaining the shear rate at 8000s -1 , the system pressure is 1, place the reflux end at the sample loading end and circulate together until the volume is about one-fourth of the initial volume, end the cycle, and collect the filtrate at the reflux end. Pump in a system volume of PBS buffer to top wash out the phage particles retained in the pipeline, empty the system and store the phage liquid in a 4°C refrigerator. The tangential flow filtration column in this step should be recycled, so after the purification is completed, 0.5M NaOH should be used for circulation for 10 minutes, and 0.1M NaOH should be used to preserve the tangential flow filtration column.

[0080] S3. Perform the next purification operation on Akta Pure L1 (protein purification system). After rinsing the equipment with PBS buffer, inject the filtrate from the reflux end into the equipment and observe that the UV absorption curve shows an upward trend. This is the filtrate that has passed through the chromatography column. Collect the filtrate that has passed through until the UV absorption curve falls back. Store the collected filtrate in a 4°C refrigerator. This is the purified phage preparation. The chromatography column in this step can also be recycled repeatedly. Therefore, after the purification is completed, use 1M NaOH and 30% isoamyl alcohol to clean the pipeline. Observe the UV absorption curve for a peak. This is the impurity adsorbed inside the chromatography column. After the UV absorption curve falls back, use 20% ethanol to preserve the chromatography column. The chromatography column can be stored at room temperature.

[0081] In step S3, the chromatographic column is a "molecular exclusion + charge adsorption" composite chromatographic column, and the filler in the "molecular exclusion + charge adsorption" composite chromatographic column is CaptoCore 400 TM .

[0082] The endotoxin content and phage titer in the phage lysate retained in each link were measured using the horseshoe crab reagent dynamic colorimetric method and the double-layer plate method, respectively. Figure 5 As shown in the figure, the purification effect of the phage lysate of the present invention is shown. The results show that the toxin content of the purified phage is 18.8EU / 10 9 PFU, endotoxin removal rate was 99.29%, and phage retention rate was 64.72%.

[0083] Example 5 Purification of Pseudomonas aeruginosa phage lysate by the method of the present invention

[0084] A method for purifying a bacteriophage preparation comprises the following steps:

[0085] S1. In a sterile environment, filter 800 mL of Pseudomonas aeruginosa phage lysate using filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively, and store in a 4°C refrigerator after filtration.

[0086] S2. Purify the same batch of phage lysate using a hollow fiber column with a molecular weight cutoff of 500 kDa and a shear rate of 8000 s -1 , keeping the system pressure at 1, after alkaline washing, buffer rinsing, sample loading, top washing and pipeline cleaning operations, collect 200mL of phage lysate purified in this step.

[0087] S3. Perform the next purification operation on Akta Pure L1 (protein purification system). After rinsing the equipment with PBS buffer, inject the filtrate from the reflux end into the equipment and observe the rising trend of the UV absorption curve. This is the filtrate that passes through the chromatography column. Collect the filtrate until the UV absorption curve falls again. Store and collect 200 mL of the filtrate in a 4°C refrigerator. This is the purified phage preparation.

[0088] In step S3, the chromatographic column is a "molecular exclusion + charge adsorption" composite chromatographic column, and the filler in the "molecular exclusion + charge adsorption" composite chromatographic column is CaptoCore 400 TM .

[0089] Comparative Example 1 Traditional purification method using TritonX-100 dissociation

[0090] The traditional dissociation method for purifying phage preparations includes the following steps:

[0091] S1. Take 50 mL of the phage lysate of Pseudomonas aeruginosa from the same batch as in Example 5, inoculate chloroform at a ratio of 1%, shake evenly, and centrifuge at 8000 rpm for 10 min to obtain the supernatant.

[0092] S2. Inoculate TritonX-100 at a ratio of 5% and shake evenly, then place in a constant temperature shaker at 37°C and 220 rpm for shaking incubation for 2 to 3 hours. After the incubation is completed, centrifuge at 10,000 rpm for 15 minutes and take the supernatant.

[0093] S3, dialyze the supernatant using a dialysis bag with a molecular weight cutoff of 1000 kDa, place it in 100 volumes of normal saline, and dialyze it twice on a magnetic stirrer at 400 rpm, each time for 4 hours;

[0094] S4. Add chloroform at a ratio of 1:1, shake evenly, centrifuge at 5000 rpm for 5 min, and take 15 mL of the supernatant. This is the phage preparation purified by the traditional method.

[0095] The endotoxin content and phage titer in the phage lysate purified by two different methods in Example 5 and Comparative Example 1 were measured using the limulus reagent dynamic colorimetric method and the double-layer plate method, respectively. Figure 6As shown, the purification effect of the phage lysate of the scheme of the present invention (Example 5) and the traditional dissociation scheme (Comparative Example 1) is shown. The results show that the endotoxin removal rate of the phage lysate purified by the purification scheme of the present invention is 99.20%, and the phage retention rate is 64.84%; the endotoxin removal rate of the phage lysate purified by the traditional purification scheme is 98.40%, and the phage retention rate is 25.02%. Compared with the traditional dissociation scheme, the purification scheme of the present invention not only significantly improves the endotoxin removal rate and phage recovery rate, but also expands the single processing volume while avoiding the participation of organic reagents. It is significantly superior to the traditional dissociation scheme in all aspects and is more suitable for industrial-scale application of phages.

[0096] Comparative Example 2 Traditional purification method using ultrafiltration

[0097] The traditional ultrafiltration method for purifying phage preparations includes the following steps:

[0098] S1. Take 5 mL of the Pseudomonas aeruginosa phage preparation from the same batch as in Example 5 and place it in a Millpore ultrafiltration tube.

[0099] S2. Centrifuge at 2000 rpm for 15 min, then at 4000 rpm for 15 min.

[0100] S3. Use 5 mL of physiological saline to elute the phages attached to the ultrafiltration membrane and collect them.

[0101] The endotoxin content and phage titer in the phage lysate purified by different methods in Comparative Example 2 and Example 5 were measured using the limulus reagent dynamic colorimetric method and the double-layer plate method, respectively. Figure 7 As shown in Figure 5, the purification effect of phage lysate by the present invention (Example 5) and the traditional ultrafiltration scheme (Comparative Example 2) is shown. The results show that the endotoxin content of the phage lysate purified by the purification scheme of the present invention is 18.8EU / 10 9 PFU, endotoxin removal rate was 99.20%, phage retention rate was 64.84%; the endotoxin content of phage lysate purified by traditional purification scheme was 123EU / 10 9 PFU, endotoxin removal rate was 96.72%, and phage retention rate was 67.2%. Compared with the traditional ultrafiltration scheme, the endotoxin removal rate of the present invention is significantly improved and meets the requirement of less than 20EU / 10 9 The oral standard of PFU expands the single processing volume and is more suitable for large-scale application of bacteriophages.

[0102] The method of the present invention can effectively remove impurities including endotoxins remaining in the phage lysis solution, does not involve any organic reagents, and the purified phage preparation is safe and reliable, simple to operate, and the instrument is easily available. It is easy to achieve large-scale preparation of phage preparations, thus providing the possibility for the industrial application of phages.

[0103] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for purifying a bacteriophage preparation, characterized in that: The steps include: S1, taking a phage lysate, and sequentially microfiltering the phage lysate using graded microfiltration membranes with cutoff pore sizes of 0.45 μm and 0.22 μm to obtain a feed solution; the phage lysate contains endotoxins, and the endotoxins are derived from bacteria and / or bacterial fragments in the phage lysate; S2. Ultrafiltration of the feed liquid is performed using a tangential flow filtration column with a molecular weight cutoff of 300 to 750 kDa to obtain a reflux end filtrate; the shear rate of the tangential flow filtration column is 4000 to 8000 s -1 , the system pressure of the tangential flow filtration column pipeline is set to a constant value, and the constant value ranges from 1 to 2 psi; S3, using a "molecular exclusion-charge adsorption" composite chromatography column to purify the reflux end filtrate to obtain a high-purity phage preparation; the chromatographic medium of the "molecular exclusion-charge adsorption" composite chromatography column is CaptoCore 400 TM , the loading buffer was PBS buffer.

2. The method for purifying a phage preparation according to claim 1, characterized in that: The molecular weight cut-off of the tangential flow filtration column in step S2 is 500 kDa, and the shear rate is 8000 s -1 , the system pressure of the tangential flow filtration column pipeline is set to 1 psi.

3. The method for purifying a phage preparation according to claim 1, characterized in that: The phage lysis solution is selected from one or more of a long-tail phage lysis solution, a short-tail phage lysis solution and a muscle-tail phage lysis solution.

4. The method for purifying a phage preparation according to claim 1, characterized in that: In step S1, the bacteria and / or bacterial fragments are Gram-negative bacteria, and the Gram-negative bacteria are selected from pathogenic Gram-negative bacteria in clinical, livestock and poultry, and aquaculture industries.

5. The method for purifying a phage preparation according to claim 4, characterized in that: The pathogenic Gram-negative bacteria are selected from one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Salmonella.

6. The method for purifying a phage preparation according to claim 1, characterized in that: Before ultrafiltration, the tangential flow filtration column was rinsed with 0.1 M NaOH and PBS buffer in sequence.

7. The method for purifying a phage preparation according to claim 1, characterized in that: The tangential flow filtration column is a hollow fiber column.

8. Use of the purification method according to any one of claims 1 to 7 in industrial-scale production, characterized in that: Any one or more of the following: (1) Used for preparing bacteriophage preparations; (2) Used for the preparation of antibacterial products with bacteriophage preparations as active ingredients.

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