CN113980917A - Lentiviral lysis buffer solution and application thereof - Google Patents

Abstract

The invention discloses a slow virus dissolving buffer solution and application thereof, wherein the slow virus dissolving buffer solution comprises 3-10mM Tris, 15-35mM proline and 1-5mM MgCl₂5-20% of lactose, 0.005-0.02% of Tween80 and the balance of water, wherein the percentages are mass percentages, and the pH value of the slow virus dissolving buffer solution is 7-9. The components of the slow virus dissolving buffer solution are all medicinal auxiliary materials, so that the influence on the activity of the slow virus can be avoided, and proline is added into the slow virus dissolving buffer solution to stabilize a virus structure and enhance the activity of the virus; tween80 was able to prevent protein or virus aggregation; MgCl₂Has the function of maintaining the activity of protein or virus, and can be comprehensively improved through the combined action of a plurality of components in the formulaActivity and recovery when lentivirus was purified and increased the transduction efficiency of lentivirus and the positive rate of transduced CAR.

Description

Lentiviral lysis buffer solution and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a slow virus dissolving buffer solution and application thereof.

Background

Lentiviruses are commonly used in Chimeric Antigen Receptor-T Cell therapy (CAR-T), which has the advantages of high transduction efficiency, integration of T Cell genome and sustained expression of target proteins. Today CAR-T production is mostly based on lentiviral vector mediated CAR gene transfer. However, lentivirus is an extremely fragile viral vector, which is greatly affected by shear force, extrusion, temperature, pH, ionic strength, and handling time during production; in addition, lentiviruses also aggregate relatively easily, which poses a major challenge for lentivirus production and storage.

In the prior art, the buffer solution generally used for lentivirus dissolution is PBS buffer solution, 50 mM Tris buffer solution, PBS +4% lactose buffer solution, DMEM +2% HSA buffer solution, HATM buffer solution or HSTM buffer solution, however, the existing buffer solution has poor use effect and cannot provide a proper dissolution environment for lentiviruses.

Disclosure of Invention

The invention aims to provide a slow virus dissolving buffer solution capable of solving the problems that the existing slow virus dissolving buffer solution in the background art has poor use effect and cannot provide a proper dissolving environment for slow viruses.

In order to achieve the purpose, the invention is realized by the following technical scheme: a lentivirus lysis buffer comprising 3-10mM Tris, 15-35mM proline, 1-5mM MgCl₂5-20% of lactose, 0.005-0.02% of Tween80 and the balance of water, wherein the percentages are mass percentages, and the pH value of the slow virus dissolving buffer solution is 7-9.

As a further improvement of the slow virus dissolving buffer solution, the slow virus dissolving buffer solution also comprises 0.05% -0.2% of poloxamer. The reduction of activity of cells or lentiviruses due to mechanical compression can be prevented by the addition of poloxamers.

As a further improvement of the above-mentioned slow virus dissolving buffer, the Poloxamer is Poloxamer 188, P407 or F108. By selecting three poloxamers, namely Poloxamer 188, P407 and F108, the activity of cells or lentiviruses can be further prevented from being reduced due to mechanical extrusion.

The second object of the present invention is to provide the use of the lentiviral lysis buffer described in the above protocol, which can be used for the purification of lentiviruses for cell therapy.

A third object of the present invention is to provide a kit comprising the lentiviral lysis buffer of any one of claims 1 to 3.

The fourth object of the present invention is to provide a method for purifying a lentivirus using the lentivirus lysis buffer described in the above scheme, which comprises: the method comprises the following steps:

s1, slow virus packaging is carried out by using suspension cells;

s2, centrifuging to obtain supernatant containing lentivirus;

s3, pre-filtering by using a 0.45 mu m membrane;

s4, concentrating by 5-10 times by using 500 KD hollow fibers;

s5, washing and filtering with a lentivirus dissolving buffer solution and a hollow fiber with the density of 500 KD, and washing and filtering 10 concentrated volumes;

and S6, after the washing and filtering are finished, continuously concentrating to enable the total concentration multiple to be about 15-25 times, and sampling to send a sample to test the virus infection titer (FACS).

S7, setting MOI to 1 or 3 transductions activates CD3+ T cells;

s8, after transduction is carried out for 48 hours, the culture medium is supplemented to 8 mL;

and S9, continuing culturing for 2-3 days, and then detecting the CAR positive rate.

As a further improvement of the above lentivirus purification method, in step S7, the transduced cells are at a density of 1X 10⁶cells/mL。

As a further improvement of the above-described lentivirus purification method, in step S8, the medium added is X-Vivo15+2% SR +200IU/mL IL-2 medium. The culture medium is produced by Lonza company, Thermo company and Beijing tetracyclic biopharmaceutical company, wherein X-Vivo and SR are CAR-T production raw materials meeting GMP regulations in the field of cell therapy, and IL-2 is medicinal grade.

The invention has the beneficial effects that: 1) the formula of the slow virus dissolving buffer solution not only comprises Tris and lactose, but also comprises proline and MgCl₂Tween80, PBS and DMEM +2% HSA in the buffer solution formula in the prior art are removed, and the use amount of Tris is greatly reduced; the components of the invention are all pharmaceutic adjuvants, which can avoid the influence on the activity of the lentivirus, 2) the inventor finds that the addition of proline in the lentivirus buffer solution can stabilize the virus structure and enhance the virus activity; tween80 also called polysorbate 80 is a pharmaceutical adjuvant, and can prevent protein or virus aggregation; MgCl₂The slow virus purification agent is a pharmaceutic adjuvant, has the function of maintaining protein or virus activity, can comprehensively improve the activity and recovery rate of slow virus during purification and improve the transduction efficiency of the slow virus through the combined action of a plurality of components in the formula, and has obviously higher CAR positive rate during CAR-T transduction; 3) poloxamers such as Poloxamer 188, P407 or F108 in the formula of the slow virus lysis buffer are high molecular compounds, so that damage to cells or viruses due to physical shearing force can be prevented, combination of the viruses and the cells can be promoted, and the capability of the viruses in infecting the cells can be enhanced.

Drawings

FIG. 1 is a graph showing the FACS titers measured in comparative example 1 for four lentivirus-containing buffers stored at 4 ℃ for 0h, 4h, 24h, and 48 h.

FIG. 2 is a graph showing the FACS titers measured in comparative example 1 after storage of four lentivirus-containing buffers at room temperature (25 ℃. + -. 5 ℃) for 0h, 4h, 24h and 48h, which are not shown because the FACS titer values are below the limit of detection after storage for 24h and 48 h.

FIG. 3 is a graph showing the FACS titers measured in comparative example 1 after storage of the four lentivirus-containing buffers at 37 ℃ for 0h, 2h, 4h and 24h, which are not shown because the FACS titer values are below the detection limit after storage for 24 h.

FIG. 4 is a graph showing FACS titers measured in comparative example 1 after repeated freezing and thawing of four lentivirus-containing buffers at-80 deg.C/37 deg.C for 0-3 times.

FIG. 5 is a graph showing the positive rate of CAR in comparative example 2, which was detected after activation of CD3+ T cells by lentivirus transduction using PBS buffer, HSTM buffer, HATM buffer and TLTM buffer at a multiplicity of infection of 3.

FIG. 6 is a graph of the data obtained for titer and recovery of lentiviruses in comparative example 3 using four lentivirus lysis buffers TLTM, TLTM1, TLTM7 and TLTM 8; in the figure, the bar graph represents the FACS infectious titer of lentivirus and the triangular line represents the recovery of lentivirus.

FIG. 7 is a graph of data showing the positive rate of CAR after transduction of activated CD3+ T cells in comparative example 4 at multiplicity of infection of 1 and 3 using four lentiviral lysis buffers TLTM, TLTM1, TLTM7, and TLTM 8.

Detailed Description

The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A lentivirus lysis buffer comprising 3mM Tris, 5% lactose, 35mM proline, 5mM MgCl₂And 0.005% Tween80, the remainder being water, the pH of the lentivirus lysis buffer being 7, this formulation being designated TLTM buffer.

Tris in the formulation of this example is a buffer salt, widely used in the biomedical field, and can be used to maintain pH stability. Lactose is a pharmaceutical adjuvant, has the function of maintaining the integrity of viruses, and is a protective agent. Proline stabilizes viral structure and enhances viral activity. MgCl₂Is a pharmaceutical adjuvant, and has effect in maintaining protein or virus activity. Tween80, also known as polysorbate 80, is a pharmaceutical excipient and can prevent aggregation of proteins or viruses.

Example 2

Lentiviral lysisBuffer comprising 10mM Tris, 20% lactose, 15mM proline, 1mM MgCl₂0.02% Tween80 and 0.05% Poloxamer 188, the remainder being water, the pH of the lentiviral lysis buffer being 8, this formulation being designated TLTM1 buffer.

Example 3

A lentivirus lysis buffer comprising 5mM Tris, 10% lactose, 25mM proline, 2mM MgCl₂0.01% Tween80 and 0.1% P407, the remainder being water, the pH of the lentiviral lysis buffer was 7.5, and this formulation was designated TLTM7 buffer.

Example 4

A lentivirus lysis buffer comprising 7mM Tris, 15% lactose, 20mM proline, 3mM MgCl₂0.015% Tween80 and 0.2% F108, the remainder being water, the pH of the lentivirus lysis buffer being 9, this formulation being designated TLTM8 buffer.

The Poloxamer 188, P407 and F108 in the above examples 2-4 are three high molecular compounds of Poloxamer, which can enhance the activity of virus, promote the combination of virus and cell, and enhance the ability of virus to infect cell.

Application example 1

The method for purifying and recovering the lentivirus by adopting the lentivirus dissolving buffer solution of the embodiment comprises the following specific steps:

s1, slow virus packaging is carried out by using suspension cells;

s2, centrifuging to obtain supernatant containing lentivirus;

s3, pre-filtering by using a 0.45 mu m membrane;

s4, concentrating by 5-10 times by using 500 KD hollow fibers;

s5, washing and filtering the lentivirus dissolving buffer solution in the above examples 1-4 by using a hollow fiber with 500 KD, and washing and filtering 10 concentrated volumes; then, the samples were dispensed, and 1 of each of the dispensed samples was left at room temperature for 2 hours (defined as room temperature for 2 hours), and the remaining samples were immediately frozen at-80 ℃ for 0 hour (defined as room temperature).

And S6, after the washing and filtering are finished, continuously concentrating to enable the total concentration multiple to be about 15-25 times, and sampling to send a sample to test the virus infection titer (FACS).

S7, setting MOI to 1 or 3 transductions activates CD3+ T cells;

s8, after transduction is carried out for 48 hours, the culture medium is supplemented to 8 mL;

and S9, continuing culturing for 2-3 days, and then detecting the CAR positive rate.

Comparative example 1

The effect of using the TLTM buffer of the present invention with the existing HATM buffer and HSTM buffer was compared as follows:

1) four groups of lentivirus lysis buffers were prepared, wherein the first group was a PBS buffer consisting of 10mM (disodium hydrogen phosphate + sodium dihydrogen phosphate) +150 mM NaCl, pH 7.4; the second group was HATM buffer consisting of 10mM HEPES +100mM Arg +4% sucrose +0.01% Tween80+2mM MgCl₂pH7.5, the third group is HSTM buffer solution, which comprises 10mM HEPES +100mM NaCl +4% sucrose +0.01% Tween80+2mM MgCl2, pH7.5, and the fourth group is TLTM buffer solution of the present invention, which comprises 5mM Tris +10% lactose +25mM proline +0.01% Tween80+2mM MgCl 80₂，pH7.5。

2) Suspending cells packaging lentivirus after harvest centrifugal removal of cells and debris, 0.45 m prefiltration using 500 KD hollow fiber for concentration, then divided into four equal parts, respectively using the 4 buffer solution washing filter 10 volumes for buffer solution replacement, i.e. different buffer solution containing lentivirus, different buffer solution can make the active FACS retention degree different because of the very weak lentivirus.

3) Several buffer comparisons containing lentivirus: 4 ℃ stability, Room temperature stability, 37 ℃ stability, -80 ℃/37 ℃ repeated freeze thaw stability, and FACS titer changes were compared for the 4 lentivirus-containing buffers described above at different temperatures for different time periods.

FIG. 1 is a graph showing FACS titers measured after 0h, 4h, 24h and 48h storage at 4 ℃ in PBS buffer, HATM buffer, HSTM buffer and TLTM buffer containing lentiviruses; FIG. 2 is a graph showing FACS titers measured in the four kinds of buffers containing lentivirus after storage at room temperature (25 ℃. + -. 5 ℃) for 0h, 4h, 24h and 48 h; FIG. 3 is a graph showing FACS titers measured in the four lentivirus-containing buffers after storage at 37 ℃ for 0h, 2h, 4h and 24 h; FIG. 4 is a graph showing FACS titer measurements after repeated freezing and thawing at-80 deg.C/37 deg.C for 0, 1, 2, and 3 cycles in the four buffers containing lentivirus. As can be seen from the data graphs of FIGS. 1 to 4, the TLTM buffer of the present invention has a higher FACS titer than the lentiviruses of the conventional PBS buffer, HATM buffer and HSTM buffer. Experimental detection shows that the PBS buffer solution widely used in the prior art easily makes lentivirus inactive, and because DMEM is not a culture medium with pharmaceutical excipient grade, the requirement of pharmaceutical regulations is not met during subsequent CAR-T production, and the removal of residual DMEM is very complicated.

Comparative example 2

After the filtered samples were washed with the four lentivirus lysis buffers in comparative example 1, the CD3+ T cells were transduced and activated at the multiplicity of infection of 3, and the CAR positivity was determined, as shown in FIG. 5, and the comparison of the data in FIG. 5 revealed that the CAR positivity after the samples were washed with the TLTM buffer was 52.1% after the CD3+ T cells were transduced and 25.7%, 33.2% and 25.9% after the CD3+ T cells were transduced with the PBS buffer, the HATM buffer and the HSTM buffer, respectively, and the performance of the TLTM buffer was much higher than that of the existing PBS buffer, HATM buffer and HSTM buffer.

Comparative example 3

As shown in FIG. 6, the TLTM buffer, TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer of examples 1-4 were used for lentivirus lysis and the recovery rate and FACS infectious titer of the lentivirus-containing buffer were measured according to the method of comparative example 1, and the graphs of the experimental data obtained are shown in FIG. 6, where TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer were different buffers prepared by adding different poloxamers to the TLTM buffer, where 0h at room temperature represents the results of the samples frozen at-80 ℃ and 2h at room temperature represents the results of the samples left at room temperature for 2h, and it can be seen from the graph of data in FIG. 6 that the FACS infectious titer of lentiviruses infected with TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer was higher than the FACS infectious titer of lentiviruses infected with TLTM buffer regardless of the control group at room temperature of 0h or the control group at room temperature of 2h, and wherein the FACS infection titer of lentivirus using TLTM7 lentivirus lysis buffer was much higher than that of each other group, the application effect was optimal. Furthermore, the recovery rates of lentiviruses using TLTM1 buffer, TLTM7 buffer and TLTM8 buffer were all higher than those using TLTM buffer in the control group at room temperature of 0h, and those using TLTM1 buffer and TLTM7 buffer were all higher than those using TLTM buffer in the control group at room temperature of 2 h. In addition, in the control groups at room temperature of 0h and room temperature of 2h, the recovery rate of the lentivirus using the TLTM7 buffer solution is far higher than that of other groups, and the application effect is optimal.

Comparative example 4

As shown in FIG. 7, a graph of the data for the positive rate of CAR after transduction of activated CD3+ T cells using the TLTM buffer, TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer of examples 1-4 for lentivirus lysis at a multiplicity of infection of 1 and 3. As can be seen from the data graphs, after the samples washed and filtered by using the lentivirus lysis buffer solutions of the above examples 1-4 are infected with 3 transduction-activated CD3+ T cells, the detected CAR positive rates are all above 40%, and are obviously improved compared with the CAR positive rates after the lentivirus transduction using the PBS buffer solution, the HATM buffer solution and the HSTM buffer solution in the comparative example 2, wherein the CAR positive rate after the lentivirus transduction using the TLTM7 buffer solution is the highest and can reach above 60%, and the most preferred example is shown.

As can be seen from the above comparative examples 1-4, the TLTM buffer, TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer disclosed in examples 1-4 of the present invention were used for lentivirus solubilization, and the FACS titer, recovery rate, and positive rate after transduction of the harvested lentivirus were all higher than those of the conventional common buffers such as PBS buffer, HATM buffer, and HSTM buffer, and the performance of TLTM1 buffer, TLTM7 buffer, and TLTM8 buffer after the addition of poloxamer was improved to various degrees, with the best performance of TLTM7 buffer after the addition of P407.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (8)

1. A lentiviral lysis buffer comprising: it comprises 3-10mM Tris, 15-35mM proline, 1-5mM MgCl₂5-20% of lactose, 0.005-0.02% of Tween80 and the balance of water, wherein the percentages are mass percentages, and the pH value of the slow virus dissolving buffer solution is 7-9.

2. The lentiviral lysis buffer of claim 1, wherein: also comprises 0.05 to 0.2 percent of poloxamer.

3. The lentiviral lysis buffer of claim 2, wherein: the Poloxamer is Poloxamer 188, P407 or F108.

4. Use of a lentiviral lysis buffer according to any one of claims 1 to 3, wherein: purification of lentiviruses for cell therapy.

5. A kit, characterized in that: comprising a lentiviral lysis buffer according to any one of claims 1 to 3.

6. A method for lentivirus purification using a lentivirus lysis buffer according to any one of claims 1 to 3, wherein: the method comprises the following steps:

s1, slow virus packaging is carried out by using suspension cells;

s2, centrifuging to obtain supernatant containing lentivirus;

s3, pre-filtering by using a 0.45 mu m membrane;

s4, concentrating by 5-10 times by using 500 KD hollow fibers;

s5, washing and filtering with a lentivirus dissolving buffer solution and a hollow fiber with the density of 500 KD, and washing and filtering 10 concentrated volumes;

s6, after the washing and filtering are finished, continuously concentrating to enable the total concentration multiple to be about 15-25 times, and sampling and sending to a virus infection titer FACS;

s7, setting MOI to 1 or 3 transductions activates CD3+ T cells;

s8, after transduction is carried out for 48 hours, the culture medium is supplemented to 8 mL;

and S9, continuing culturing for 2-3 days, and then detecting the CAR positive rate.

7. The lentivirus purification method of claim 6, wherein: in step S7, the transduced cell density was 1X 10⁶ cells/mL。

8. The lentivirus purification method of claim 6, wherein: in step S8, the medium added was X-Vivo15+2% SR +200IU/mL IL-2 medium.

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