CN118931968B

CN118931968B - A method for purifying BaEV envelope pseudotype lentivirus

Info

Publication number: CN118931968B
Application number: CN202411439852.0A
Authority: CN
Inventors: 赵义林; 余鼎; 梁洪; 刘�东; 曾金杰
Original assignee: Chengdu Rongsheng Pharmaceuticals Co Ltd
Current assignee: Chengdu Rongsheng Pharmaceuticals Co Ltd
Priority date: 2024-10-15
Filing date: 2024-10-15
Publication date: 2025-01-24
Anticipated expiration: 2044-10-15
Also published as: CN118931968A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a purification method of BaEV envelope pseudotyped slow virus. The purification method comprises the steps of (1) concentrating a BaEV enveloped pseudotyped lentivirus clarified liquid, centrifuging, discarding supernatant to obtain lentivirus sediment, (2) re-suspending the lentivirus sediment by using a buffer solution, adding a omnipotent nuclease for enzyme digestion to obtain a crude and pure sample, (3) purifying the crude and pure sample by chromatography to obtain a refined and pure sample, and (4) carrying out ultrafiltration concentration and sterilization on the refined and pure sample to obtain the purified BaEV enveloped pseudotyped lentivirus. The BaEV envelope pseudotyped slow virus purified by the method has obviously improved infection titer and titer recovery rate, and can effectively reduce host cell protein, host cell DNA and endotoxin residues. The purification method provided by the invention can effectively remove impurities without affecting the activity of the purified lentivirus, and can be used for preparing a BaEV pseudotype lentivirus product with high titer and high purity.

Description

Purification method of BaEV envelope pseudotyped slow virus

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a purification method of BaEV envelope pseudotyped slow virus.

Background

Lentiviral vectors are a genetic vector derived from human immunodeficiency virus and are widely used for gene editing in eukaryotic cells such as hematopoietic stem cells and neural cells. With the long-term intensive research into gene delivery tools, lentiviruses have been widely used as gene vectors in basic research and clinical practice. With the advent of the post-genomic era, gene therapy has gained an increasing popularity in medical research. Some traditional diseases which are difficult to radically cure or alleviate can be relieved or even cured through gene therapy, and lentiviruses provide new tools and methods for the development of future gene therapy. Among them, vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviruses have been widely used in gene therapy. However, the pseudotyped lentivirus has relatively low transduction efficiency when it transduces hematopoietic stem cells and part of lymphocytes.

Patent application number 201280053749.1 discloses a method for producing a pseudotyped lentiviral vector of mutant baboon endogenous retrovirus (BaEV) glycoprotein, and the BaEV glycoprotein pseudotyped lentiviral vector obtained by the method can effectively improve the transfection efficiency of hematopoietic stem cells and natural killer cells and is used for preparing medicines for treating hematopoietic dysfunction or autoimmune diseases, but in the preparation process of the pseudotyped lentiviral vector, baEV glycoprotein is extremely easy to lead adherent cells to form syncytia and to generate shedding death, so that the prepared lentiviral titer is lower, and more host cell impurities in virus liquid are remained. In order to solve the above problems, there is a need to develop a method for obtaining BaEV pseudotyped lentiviruses with high titer and high purity.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a purification method of BaEV envelope pseudotyped slow virus.

The invention provides a purification method of BaEV envelope pseudotyped slow virus, which is characterized by comprising the following steps:

Step 1, concentrating BaEV envelope pseudotyped lentivirus clarified liquid, centrifuging, and discarding supernatant to obtain lentivirus sediment;

Step 2, re-suspending slow virus precipitation by using buffer solution, and adding omnipotent nuclease for enzyme digestion to obtain a crude and pure sample;

Step 3, purifying the crude and pure sample by chromatography to obtain a refined and pure sample, wherein the chromatography is Mustang Q XT membrane chromatography capsule filter chromatography or DEAE cellulose chromatography;

and 4, washing, filtering, concentrating and sterilizing the refined sample to obtain the purified BaEV envelope pseudotyped slow virus.

In step 1, the BaEV enveloped pseudotyped lentivirus clarified solution is obtained by centrifuging a BaEV enveloped pseudotyped lentivirus harvest solution and filtering.

In the step 1, the method for concentrating the BaEV envelope pseudotyped lentivirus clarified liquid comprises the steps of adding polyethylene glycol solution into the BaEV envelope pseudotyped lentivirus clarified liquid, and uniformly mixing, wherein the volume ratio of the BaEV envelope pseudotyped lentivirus clarified liquid to the polyethylene glycol solution is (2-8): 1, the polyethylene glycol solution comprises 20-50% g/L polyethylene glycol and 0.6-1.2M NaCl, and the pH value of the polyethylene glycol solution is 7.5-8.5.

Further, in the step 1, the polyethylene glycol is PEG8000, the volume ratio of the BaEV envelope pseudotyped lentivirus clarified liquid to the polyethylene glycol solution is 4:1, and the preparation method of the polyethylene glycol solution comprises the following steps of mixing the polyethylene glycol and NaCl in a buffer solution, adjusting pH, sterilizing, wherein the polyethylene glycol solution comprises 25% -45% g/L polyethylene glycol and 0.5% -0.7M NaCl, and the pH of the polyethylene glycol solution is 7.5% -8.0.

Further, the polyethylene glycol solution contains 40% g/L polyethylene glycol and 0.6M NaCl, the pH of the polyethylene glycol solution is 8.0, and the sterilization mode is that the sterilization is carried out through a 0.45 μm filter.

Further, in the step 2, the buffer solution is a buffer solution containing 40-60 mM Tris-HCl and 1-5 mM MgCl ₂, the pH value of the buffer solution is 7.5-8.5, and the activity of the omnipotent nuclease is 5-50U/ml.

Further, in the step 2, the buffer solution is a buffer solution containing 50mM Tris-HCl and 2mM MgCl ₂, the pH value of the buffer solution is 8.0, and the activity of the omnipotent nuclease is 40U/ml.

Further, in step3, the chromatography is Mustang Q XT membrane chromatography capsule filter chromatography, and the Mustang Q XT membrane chromatography capsule filter chromatography comprises the following steps:

(1) Balancing, namely balancing a Mustang Q XT membrane by using a balancing buffer;

(2) Sampling, namely loading a crude and pure sample, and continuously balancing by using a balancing buffer solution until the UV ₂₈₀ value is stable after loading is finished;

(3) The method comprises the steps of eluting by using an eluent, collecting the eluent, wherein the eluent consists of an eluting buffer A and an eluting buffer B, the eluting buffer A comprises 20-50 mM Tris-HCl and 0-0.3M NaCl, the pH value is 7.5-8.0, the eluting buffer B comprises 20-50 mM Tris-HCl and 1-2M NaCl, the pH value is 7.5-8.0, the eluting procedure comprises the steps of firstly performing linear elution, increasing the volume ratio of the eluting buffer B in the eluent from 0% to 60%, then performing isocratic elution, starting to collect the eluent when UV ₂₈₀ is more than 50 mAU, and ending to collect the eluent when UV ₂₈₀ is less than 50 mAU.

Further, in the step (1), the balance buffer comprises 50 mM Tris-HCl and 0.3M NaCl, and the pH value is 7.5-8.0;

The eluting buffer solution A comprises 50mM Tris-HCl and 0.15~0.3M NaCl,pH with the value of 7.5-8.0, and the eluting buffer solution B comprises 50mM Tris-HCl and 1-1.5M NaCl with the pH value of 7.5-8.0;

The elution process is that the linear elution is carried out at the flow rate of 5 ml/min, the volume proportion of the eluting buffer B in the eluent is increased from 0% to 60%, then the isocratic elution is carried out, the elution is started when the eluting solution UV ₂₈₀ is more than 50 mAU, and the collection is ended when the eluting solution UV ₂₈₀ is less than 50 mAU.

Further, in the step 4, the washing, filtering and concentrating comprises the steps of concentrating by using a centrifugal concentrating pipe, washing and filtering by using PBS, wherein the molecular retention of the centrifugal concentrating pipe is 100-500 KDa, and the sterilization mode is that the sterilization is carried out by filtering by using a 0.22 mu m filter.

Further, the molecular cut-off amount of the centrifugal concentration tube is 300 KDa.

In the present invention, a BaEV pseudotyped lentivirus refers to a lentiviral vector pseudotyped with a mutant BaEV glycoprotein, the viral envelope of which has a unique structure and surface markers of the BaEV envelope.

Compared with VSVG pseudotyped lentivirus, the plasmid transfection of the mutant BaEV envelope glycoprotein is extremely easy to cause adherent cells to form syncytia, so that the cells fall off and die after being transfected for 48 hours, not only the packaging titer of the lentivirus is reduced, but also a large amount of host DNA residues and host protein residues are introduced, and the quality of the lentivirus is seriously influenced.

The purification method of the BaEV envelope pseudotyped slow virus adopts a specific chromatographic column, optimizes the PEG solution formula used in concentrating the slow virus and the buffer solution formula used in chromatography, obviously improves the infection titer and the titer recovery rate of the BaEV envelope pseudotyped slow virus obtained by purification by the method, and can effectively reduce HCP, HCD and endotoxin residues. The purification method provided by the invention can effectively remove impurities without affecting the activity of the purified lentivirus, and can be used for preparing a BaEV pseudotype lentivirus product with high titer and high purity.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows fluorescence imaging of 293T cells infected with BaEV envelope pseudotyped lentivirus before purification (indicated as clear solution) and after purification (indicated as purified solution).

Detailed Description

In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available.

The omnipotent nuclease used in the examples was PANNARASE omnipotent nuclease from Shanghai Sedan Biotechnology Co., ltd, product number CYG002F0010.

UV ₂₈₀ values refer to the UV absorbance at a wavelength of 280 nm.

EXAMPLE 1 purification method of BaEV envelope pseudotyped lentivirus

1. Preparation of lentivirus clear solution

Preparation of BaEV envelope pseudotyped lentiviruses using 293T suspension cells:

(1) Plasmid transfection was performed with a 293T suspension cell density of 3.0X10 ⁶/ml, plasmid DNA transfection ratio of 0.8. Mu.g/10 ⁶ cells,

(2) Taking shuttle plasmid (pCDH-GFP), packaging plasmid (pSpax) and envelope plasmid (pCMV-BaEV-Rless) according to the proportion of 1.2:1.2:1, mixing the above 3 plasmids, adding Opti-MEM culture medium, carefully mixing, standing for 5min, and recording as A solution;

(3) PEIpro (the ratio of PEIpro to the total mass of 3 plasmids is 1:1) was diluted with Opti-MEM medium, carefully mixed, left to stand for 5 minutes and recorded as solution B;

(4) Slowly adding the solution B into the solution A, slowly and uniformly mixing, and standing for 10 minutes to obtain a mixed solution;

(5) Slowly adding the mixed solution into cells, gently mixing, and starting transfection;

(6) One volume of fresh medium is added after 24 hours of transfection;

(7) The cell culture fluid is harvested after 48 transfection to obtain BaEV envelope pseudotyped lentivirus harvest fluid, and the BaEV envelope pseudotyped lentivirus harvest fluid is filtered by a 0.45 mu m filter after low-speed centrifugation to obtain lentivirus clarified fluid.

2. Optimized preparation of PEG8000 solution

The PEG8000 solution was prepared using PBS at 40% (w/v, unit g/L), then NaCl at 0.6M was added, dissolved in a 50℃water bath, and then pH was adjusted to 8.0, and filtered through a 0.45 μm filter to give a 40% optimized PEG8000 solution.

3. Optimized PEG8000 solution concentration lentivirus

50 Adding 36 ml lentiviral clarified solution and 9 ml 40% optimized PEG8000 solution into a ml centrifuge tube, uniformly mixing once every 20min, standing at 4 ℃ for 6h, centrifuging at 3000 Xg and 4 ℃ for 30 min, and discarding the supernatant to obtain precipitate.

4. Adding 0.5 ml buffer (50 mM Tris-HCl, mgCl ₂ of 2mM, pH 8.0) to resuspend the precipitate, mixing well, adding omnipotent nuclease with activity of 40U/ml, and treating 1 h at 37 ℃ to obtain crude pure sample.

5. Mustang Q XT5 membrane chromatography capsule filter chromatography purification

(1) Balancing, wherein a balancing Buffer is 5 CV (column volume) and balances Mustang Q XT5 (cytiva, XT5MSTGQPM 6) to the stable conductivity and pH value at the flow rate of 10ml/min, and the balancing Buffer is 50 mM Tris-HCl, 0.3M NaCl,pH 8.0;

(2) Feeding a 10CV crude pure sample at a flow rate of 5 ml/min through an infusion valve, and continuously pumping a 10CV balance buffer at a flow rate of 5 ml/min until the UV ₂₈₀ value is stable after the sample feeding is finished;

(3) The elution is carried out by switching an infusion valve to an eluent consisting of eluent bufffer-A and bufffer-B, carrying out linear elution at a flow rate of 5ml/min, increasing the volume ratio of bufffer-B in the eluent from 0% to 60%, and eluting the eluent by 6 CV to ensure that the concentration of the eluting salt is increased from 0.3M to 1.32M, then keeping stable and continuing to elute for 5 CV, beginning to collect the eluent when UV ₂₈₀ is more than 50 mAU, ending the collection when UV ₂₈₀ is less than 50 mAU, adding PBS into the eluent immediately after the collection of the eluent, and diluting the NaCl concentration to be lower than 0.3M. Wherein the elution Buffer A liquid is 50 mM Tris-HCl, 0.3M NaCl,pH 8.0, and the elution Buffer B liquid is 50 mMTris-HCl,1.5M NaCl,pH 8.0;

(4) CIP is that an infusion valve is switched to CIP-bufffer, and 6 CV solution is pumped into the infusion valve at the flow rate of 10 ml/min for cleaning and regeneration in situ, wherein CIP-bufffer is 1M NaOH aqueous solution.

6. Washing, filtering and concentrating

Immediately transferring the diluted eluent into 300 KDa centrifugal concentration tube (sartorius, VS 2052), centrifugally concentrating at 3000 Xg and 4 ℃, washing and filtering with PBS for 2 times (namely sequentially removing the supernatant, adding PBS for resuspension, centrifugally concentrating, removing the supernatant, adding PBS for resuspension and centrifugally concentrating), finally adding X-VIVO 15 culture medium for resuspension to 20ml, filtering and sterilizing the concentrated lentivirus by a 0.22 mu m filter, and obtaining the purified BaEV envelope pseudotyped lentivirus.

EXAMPLE 2 purification method of BaEV envelope pseudotyped lentivirus

1. Preparation of lentivirus clear solution

(1) 293T cells were seeded in 15cm dishes and incubated in 10% foetal calf serum (Gibco) medium to a confluency of 70-80% at transfection.

(2) 1 Hour prior to transfection, the cell culture medium was replaced with Opti-MEM medium;

(3) Taking shuttle plasmid (pCDH-GFP), packaging plasmid (pSpax 2), coating plasmid (pCMV-BaEV-Rless) with the proportion of 1.2:1.2:1, mixing 25 mug of the 3 plasmids, adding Opti-MEM culture medium to 500 mug, carefully mixing, standing for 5 minutes, and marking as A solution;

(4) PEIpro to 500 μl (PEIpro to 3 plasmids total mass ratio of 1:1) was diluted with Opti-MEM medium, carefully mixed, left to stand for 5min, and recorded as solution B;

(5) Slowly adding the solution B into the solution A, slowly and uniformly mixing, and standing for 10 minutes to obtain a mixed solution;

(6) Slowly adding the mixed solution into a cell culture medium, gently mixing, and starting transfection;

(7) The culture supernatant was collected at 48 hours after transfection by changing the culture medium to 10% fetal bovine serum (Gibco) at 6 hours after transfection, and the supernatant was filtered using a 0.45 μm filter after low-speed centrifugation.

2. Optimized preparation of PEG8000 solution

3. Optimized PEG8000 solution concentration lentivirus

50 Adding 36 ml lentiviral clarified solution and 9 ml 40% optimized PEG8000 solution into a ml centrifuge tube, uniformly mixing every 20min for 5 times, standing for 6h in a4 ℃ refrigerator, centrifuging at 4000 Xg and 4 ℃ for 50 min, and discarding the supernatant to obtain precipitate.

4. Adding 1ml PBS to resuspend and precipitate, fully mixing the heavy suspension, adding totipotent nuclease with the activity of 20U/ml, and treating 1 h at 37 ℃ to obtain a crude pure sample.

4. DEAE chromatographic purification

(1) Balancing 5CV balancing buffer with flow rate of 3 ml/min until conductivity and pH value are stable, wherein the balancing buffer is 50 mM Tris-HCl and 0.15M NaCl,pH 8.0;

(2) Sampling, namely sampling a 10CV crude pure sample through an infusion valve at a flow rate of 3ml/min, and continuously pumping the 10CV crude pure sample into a 10CV balance buffer at a flow rate of 3ml/min after the sampling is finished until the UV ₂₈₀ value is stable;

(3) The method comprises the steps of (1) switching an infusion valve to an eluent consisting of eluent bufffer-A and bufffer-B, performing linear elution at a flow rate of 3 ml/min, wherein the volume ratio of bufffer-B in the eluent is increased from 0% to 60%, the elution volume is 6 CV, the concentration of an eluting salt is increased from 0.15M to 0.66M, then, keeping stable and continuous elution for 5 CV, starting to collect the eluent with UV ₂₈₀ of >50 mAU, ending the collection with UV ₂₈₀ of <50 mAU, immediately adding PBS into the eluent after collecting the eluent, diluting the concentration of NaCl in the eluent to be lower than 0.3M, eluting bufffer-A into 50 mM Tris-HCl and 0.15M NaCl,pH 8.0, and eluting bufffer-B into 50 mMTris-HCl and 1M NaCl, wherein the pH is 8.0;

(4) CIP is that a transfusion valve is switched to CIP-bufffer, 6 CV solution is pumped into the transfusion valve at the flow rate of 3 ml/min for in-situ cleaning and regeneration, and CIP buffer is 1M NaOH aqueous solution;

(5) Washing, namely pumping 10 CV balance buffer into a chromatographic column at a flow rate of 3 ml/min to wash the chromatographic column until the conductivity and the pH value are stable;

(6) Column preservation, namely switching an infusion valve, pumping 8 CV20% ethanol at a flow rate of 3 ml/min for balancing, and taking down the column for preservation to 4 ℃.

5. Washing, filtering and concentrating

Immediately after elution, transferring the diluted eluent into a 300 KDa centrifugal concentration tube, carrying out centrifugal concentration at 3000 Xg and 4 ℃, washing and filtering 3 times by using PBS, concentrating to 3ml, and filtering and sterilizing the concentrated lentivirus by using a 0.22 mu m filter head to obtain the purified BaEV envelope pseudotyped lentivirus.

EXAMPLE 3 purification method of BaEV envelope pseudotyped lentivirus

1. Preparation of lentivirus clear solution

Step 1 is the same as in example 2.

2. Optimized preparation of PEG8000 solution

The PEG8000 solution was prepared using PBS at 25% (w/v, unit g/L), then NaCl at 0.6M was added, dissolved in a 50℃water bath, and then pH was adjusted to 8.0, and filtered through a 0.45 μm filter to give a 25% optimized PEG8000 solution.

3. Optimized PEG8000 solution concentration lentivirus

50 Adding 36 ml lentivirus clarified solution and 9 ml 25% optimized PEG8000 solution into a ml centrifuge tube, uniformly mixing every 20min for 4 times, standing overnight at 4 ℃ in a refrigerator, centrifuging at 3000 Xg at 4 ℃ for 30min the next day, and discarding the supernatant to obtain precipitate.

4. Adding 1ml PBS to resuspend the precipitate, fully and uniformly mixing, adding omnipotent nuclease with the activity of 20U/ml, and treating 1 h at 37 ℃ to obtain a crude and pure sample.

5. DEAE chromatographic purification

(1) Balancing 5 CV balancing buffer at 3 ml/min until conductivity and pH value are stable, wherein the balancing buffer is 50 mM Tris-HCl and 0.15M NaCl,pH 8.0;

(2) Sampling, namely sampling a 10CV crude pure sample at a flow rate of 3ml/min through an infusion valve, and continuously pumping the 10CV crude pure sample into a 10CV balance buffer at a flow rate of 3ml/min after the sampling is finished until the UV ₂₈₀ value is stable;

(3) The elution is carried out by switching an infusion valve to an eluent consisting of an eluent bufffer-A and a bufffer-B, carrying out linear elution at a flow rate of 3 ml/min, wherein the volume ratio of the liquid bufffer-B in the eluent is increased from 0% to 60%, the elution volume is 6 CV, the concentration of the eluting salt is increased from 0.1M to 0.66M, then the stable elution is kept for 5 CV, the collection of the eluent is started by UV ₂₈₀ >50 mAU, the collection of the eluent is ended by UV ₂₈₀ <50 mAU, PBS is added into the eluent immediately after the collection of the eluent, the NaCl concentration in the eluent is diluted to be lower than 0.3M, wherein the elution bufffer-A is 50mM Tris-HCl and 0.15M NaCl,pH 8.0, the elution bufffer-B is 50 mMTris-HCl and 1M NaCl, and the pH is 8.0;

(4) CIP is that a transfusion valve is switched to CIP-bufffer, and 6 CV solution is pumped into the transfusion valve at the flow rate of 3 ml/min for in-situ cleaning and regeneration, wherein CIP buffer is 1M NaOH aqueous solution;

(6) Column preservation by switching the infusion valve to pump 8 CV 20% ethanol at a flow rate of 3 ml/min for equilibration and removing the column for preservation to 4 ℃.

5. Washing and concentrating, namely immediately transferring the diluted eluent into a 100 KDa ultrafiltration centrifuge tube, centrifugally concentrating at 3000 Xg and 4 ℃, washing and filtering 3 times by PBS, and finally concentrating to 3ml. Filtering and sterilizing the concentrated lentivirus by a 0.22 mu m filter head to obtain the purified BaEV envelope pseudotype lentivirus.

The technical effects of the present invention are demonstrated by experimental examples below.

Experimental example 1 PEG8000 concentration Condition recovery of infectious titer before and after optimization

1. Experimental sample

A pre-concentrate sample, example 1 lentivirus clarified solution;

optimized PEG8000 concentrated sample, concentrated lentiviral pellet obtained according to example 1, step 3;

The non-optimized PEG8000 solution was prepared by preparing a PEG8000 solution with ultra pure water at a concentration of 25% (w/v, unit g/L), adding NaCl at a concentration of 0.8M, and autoclaving at 121℃for 30min to obtain a non-optimized PEG8000 solution.

2. Experimental method

Count after digestion of 293T cells, adjust the density to 2.0X10 ⁵/ml, inoculate to 12 well plates, 1ml in each well.

2. The virus solution was diluted 10-fold in a 1.5ml centrifuge tube. The dilution method comprises preparing 5 1.5ml tubes for each virus, adding 135 μl culture medium into each tube, adding 15 μl stock solution of virus into the first tube, mixing, sucking 15 μl, and adding into the second tube. And so on, 5 dilutions were made. After the cells are attached, the original culture solution in the 12-well plate is discarded, 0.9ml of new complete culture medium is replaced, 100 μl of diluted virus solution is added into each well, and the labeling is performed. 3 wells were repeated for each dilution and fresh medium was replaced after 48h of incubation.

3. Lentiviruses themselves express green fluorescent protein, and after 72h of infection, the digested cells were subjected to flow detection to calculate the lentivirus titer using the percentage of cells expressing green fluorescent protein.

Lentivirus virus titer calculation formula:

virus infection titer (TU/ml) =cell number×percent fluorescence×10 ³/virus stock volume (μl);

total infectious titer = viral infectious titer x viral volume;

Recovery of infectious titer = total infectious titer after concentration/total infectious titer before concentration x 100%.

3. Experimental results

The results are shown in Table 1, with the optimized PEG8000 group, the recovery of infectious titer was significantly improved compared to the non-optimized PEG8000 group. Experimental results show that the BaEV envelope pseudotyped slow virus can obviously improve the total infection titer and titer recovery rate of the virus by utilizing the optimized PEG8000 solution.

TABLE 1 recovery of infectious titer before and after optimization of PEG8000 concentration conditions

Experimental example 2 evaluation of recovery rate and quality of lentivirus infection titer

1. Experimental sample

Clear solutions and purified solutions prepared in examples 1-3. Wherein the clarified liquid refers to the lentivirus clarified liquid before purification, and the purified liquid refers to the BaEV envelope pseudotype lentivirus after purification.

2. Experimental method

The virus infection titer test method is described in experimental example 1;

Recovery of infectious titer = (purified solution volume×purified solution infectious titer)/(clarified solution volume×clarified solution infectious titer) ×100%;

The endotoxin residue in the purified solution was detected by a dynamic color development method known in the art, the Host Cell Protein (HCP) content in the purified solution was detected by an ELISA method known in the art, and the Host Cell DNA (HCD) content in the purified solution was detected by a qPCR method known in the art.

3. Experimental results

As shown in Table 2, it can be seen that the BaEV envelope pseudotyped lentivirus obtained by the purification method of the invention has significantly improved infectious titer compared with the lentivirus clarified liquid before purification, and the purification method of the invention can effectively reduce HCP, HCD and endotoxin residues.

Further, the BaEV-enveloped pseudotyped lentiviruses obtained in example 1 were found to have the highest infectious titer, highest infectious titer recovery rate, and the lowest HCP, HCD and endotoxin residues compared with examples 2 and 3.

TABLE 2 evaluation of recovery and quality of lentiviral infection titres

Experimental example 3 Activity of BaEV envelope pseudotyped lentivirus

1. Experimental sample

Example 1 lentiviruses before and after purification, wherein the lentiviruses before purification refer to lentivirus clarified solution and the lentiviruses after purification refer to BaEV envelope pseudotyped lentivirus purified solution after purification.

2. Experimental method

Count after digestion of 293T cells, adjust the density to 2.0X10 ⁵/ml, inoculate to 12 well plates, and inoculate 1ml of cell suspension in each well.

2. 100 Mu l of lentivirus clarified liquid and 0.5 mu l of lentivirus purified liquid are respectively taken and added into 12-well plate cells to make marks, and fresh culture medium is replaced after continuous culture for 48 hours.

3. The lentivirus expresses the green fluorescent protein, and after 72 h infection, the green fluorescent expression of the cells is observed under a fluorescent microscope and photographed.

3. Experimental results

As shown in FIG. 1, the green fluorescence of the purified liquid group is obviously increased compared with that of the clarified liquid group, which proves that the infection titer of the BaEV envelope pseudotyped lentivirus cells purified by the method is obviously improved.

Claims

1. A method for purifying BaEV envelope pseudotyped lentivirus, characterized in that the method comprises the steps of:

The method for preparing the BaEV film-coated pseudotyped lentivirus clarified liquid comprises the steps of concentrating the BaEV film-coated pseudotyped lentivirus clarified liquid, centrifuging, discarding supernatant to obtain lentivirus precipitate, wherein the method for concentrating the BaEV film-coated pseudotyped lentivirus clarified liquid comprises the steps of adding polyethylene glycol solution into the BaEV film-coated pseudotyped lentivirus clarified liquid, uniformly mixing, wherein the polyethylene glycol is PEG8000, the volume ratio of the BaEV film-coated pseudotyped lentivirus clarified liquid to the polyethylene glycol solution is 4:1, the preparation method of the polyethylene glycol solution comprises the steps of mixing polyethylene glycol and NaCl in a buffer solution, adjusting pH, sterilizing, wherein the polyethylene glycol solution comprises 25-45% g/L polyethylene glycol and 0.5-0.7MNaCl, and the pH of the polyethylene glycol solution is 7.5-8.0;

step 3, purifying the crude and pure sample by chromatography to obtain a refined and pure sample, wherein the chromatography is Mustang QXT membrane chromatography capsule filter chromatography or DEAE cellulose chromatography;

2. The method according to claim 1, wherein in step 1, the clear solution of the BaEV-enveloped pseudotyped lentivirus is obtained by centrifuging a BaEV-enveloped pseudotyped lentivirus harvest solution and filtering the same.

3. The method according to claim 1, wherein in step 1, the polyethylene glycol solution contains 40% g/L polyethylene glycol and 0.6M NaCl, the pH of the polyethylene glycol solution is 8.0, and the sterilization is performed by filtration through a 0.45 μm filter.

4. The purification method according to claim 1, wherein in the step 2, the buffer solution is a buffer solution containing 40-60 mM Tris-HCl and 1-5 mM MgCl ₂, the pH value of the buffer solution is 7.5-8.5, and the activity of the omnipotent nuclease is 5-50U/ml.

5. The method according to claim 4, wherein in the step 2, the buffer is a buffer containing 50mM Tris-HCl and 2mM MgCl ₂, the pH of the buffer is 8.0, and the activity of the omnipotent nuclease is 40U/ml.

6. The purification method of claim 1, wherein in step 3, the chromatography is Mustang Q XT membrane-chromatography capsule-filter chromatography, comprising the steps of:

(3) The method comprises the steps of eluting by using an eluent, collecting the eluent, wherein the eluent consists of an eluting buffer A and an eluting buffer B, the eluting buffer A comprises 20-50 mM Tris-HCl and 0-0.3M NaCl, the pH value is 7.5-8.0, the eluting buffer B comprises 20-50 mM Tris-HCl and 1-2M NaCl, the pH value is 7.5-8.0, the eluting procedure comprises the steps of firstly performing linear elution, increasing the volume ratio of the eluting buffer B in the eluent from 0% to 60%, then performing isocratic elution, starting to collect the eluent when the UV ₂₈₀ is more than 50mAU, and ending to collect the eluent when the UV ₂₈₀ is less than 50 mAU.

7. The method of claim 6, wherein in step (1), the equilibration buffer comprises 50mM Tris-HCl and 0.3M NaCl, pH 7.5-8.0;

The elution process comprises the steps of firstly carrying out linear elution at a flow rate of 5ml/min, increasing the volume proportion of the eluting buffer B in the eluent from 0% to 60%, then carrying out isocratic elution, starting to collect the eluting solution when the UV ₂₈₀ is more than 50mAU, and ending to collect the eluting solution when the UV ₂₈₀ is less than 50 mAU.

8. The method according to any one of claims 1 to 7, wherein in the step 4, the washing and concentrating comprises the steps of concentrating by a centrifugal concentrating tube, washing and filtering by PBS, wherein the molecular cut-off amount of the centrifugal concentrating tube is 100-500kDa, and the sterilization is carried out by filtration through a 0.22 μm filter.