CN113122501B

CN113122501B - Culture medium suitable for large-scale clinical grade virus vector preparation and application thereof

Info

Publication number: CN113122501B
Application number: CN201911398385.0A
Authority: CN
Inventors: 张巍; 唐文凤; 郑姣
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2023-06-16
Anticipated expiration: 2039-12-30
Also published as: CN113122501A

Abstract

The invention belongs to the field of biological viruses, and particularly relates to a culture method for preparing slow viruses by culturing a vaccine Xpress culture medium. The method can effectively reduce BSA residues, can be used for preparing clinical-grade lentiviruses, can obviously improve the titer of the lentiviruses, is suitable for large-scale clinical-grade lentiviruses production, and is simple, convenient and short in time consumption, and serum-free domestication of working cells is not required when the working cells are replaced to serum-free culture mediums.

Description

Culture medium suitable for large-scale clinical grade virus vector preparation and application thereof

Technical Field

The invention belongs to the field of biological viruses, and particularly relates to a method for culturing a lentiviral vector containing a target gene by using a glutamine-containing vaccine xpress culture medium.

Background

Currently, clinical gene therapy vectors are based on viral vectors, the major viral vectors comprising adenoviruses, oncolytic viruses and retroviruses. Among them, retroviral vectors, commonly used include gamma retrovirus and lentiviral vectors, have been used for clinical studies for stable gene transfer into mammalian cells for nearly 30 years. Retroviruses can be efficiently integrated into the target cell genome and stably and permanently express the exogenous gene.

Lentiviral vectors are one of the retroviruses, and have a broader host range than other retroviruses, and are capable of effectively infecting non-periodic and postmitotic cells. The lentiviral vector is a gene therapy vector modified based on human immunodeficiency virus type I (HIV), and is derived from HIV-1, and can infect non-dividing cells, stably integrate in vivo and have less gene silencing phenomenon because of being capable of bearing larger exogenous target gene segments. And for some cells which are difficult to infect, such as primary cells, stem cells, non-differentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene can be greatly improved, the probability of integrating the target gene into the genome of the host cell is increased, and the long-term and stable expression of the target gene can be conveniently and rapidly realized. The lentiviral vector has the advantages of small immune response, good safety and the like, is widely applied to clinical researches, and particularly has extremely high biological safety due to the 3 rd generation self-defective lentiviral vector.

With the development of research from the basic stage to the clinical stage, the demand for lentiviruses is greatly increased, the conventional lentivirus production method is difficult to meet, and a preparation scheme of lentivirus capable of meeting the clinical demand with high quality is urgently needed to be produced on a large scale. The conventional lentivirus preparation method needs to use DMEM culture medium containing FBS for culture, and liquid exchange is carried out before and after plasmid transfection, because the use of the FBS-containing culture medium leads to that the produced lentivirus feed liquid contains a large amount of BSA residues, but clinical lentivirus has definite limitation on the BSA residues, and the removal capacity of BSA by lentivirus purification is limited, so that the BSA residues after the lentivirus purification are still higher, and the clinical requirement cannot be met; on the other hand, although there are some serum-free media on the market at present, there is no media specially prepared for lentiviruses, and these serum-free media are not good for lentiviruses to prepare a large number of fruits, so there is an urgent need for a media and a culture scheme thereof that can be used for lentiviruses preparation.

Disclosure of Invention

In view of this, one of the objects of the present invention is a combination of two media that can act synergistically in lentiviral vector packaging and lentiviral preparation.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a combination of media suitable for large-scale clinical grade viral vector preparation, said media comprising conventional media and vaccine xpress media.

Further, the conventional medium in the medium is DMEM medium containing FBS, and the content of FBS is a conventional amount by those skilled in the art, such as DMEM medium containing 5% or 10% FBS.

Further, in the combination of media, glutamine is added to the vaccine xpress medium in an amount that is conventional to those skilled in the art, such as 1% glutamine.

Preferably, in the culture medium combination, the vaccine xpress culture medium is packaged independently.

Lentiviral vectors are viral vectors derived from human immunodeficiency virus-1, contain genetic information required for packaging, transfection, and stable integration, and are the main components of lentiviral vector systems. The lentiviral vector carrying the target gene (functional gene) is packaged into infectious viral particles by viruses with the aid of lentiviral packaging plasmids and cell lines, and the target gene is expressed in cells or tissues by infecting cells or living tissues. In the whole process, the invention adopts two culture mediums to jointly apply: the vaccine xprees culture medium can be used for preparing the lentivirus, and the infection capacity of the lentivirus can be obviously improved by using the culture medium, which is far higher than that of other types of serum-free culture medium and conventional DMEM culture medium with 5% FBS. Thus, the use of the vaccine xprees medium for lentivirus production enables higher viral yields than conventional production protocols.

The second purpose of the invention is to provide a virus with small bovine serum albumin residue and a preparation method thereof, and the product obtained by the method is suitable for clinical application scenes.

by using the method for reducing the residual amount of bovine serum albumin in the virus particles by using the culture medium combination, the cell line capable of producing the virus particles is cultured by using the vaccine xpress culture medium.

Further, the cell line is a HEK293T cell. HEK293T cells as packaging cells for lentiviruses are anchorage dependent epithelioid cells, and the traditional medium is DMEM medium containing FBS. The adherent cells are grown by culture to proliferate to form a monolayer of cells. Cells of equivalent function, such as E.coli strain DH5a, can also be used to amplify lentiviral vectors and helper packaging vector plasmids.

Further, the method specifically comprises the following steps:

(1) Pretreatment of

Culturing a cell line to be transfected which can generate virus particles by using the conventional culture medium, and preparing a packaging plasmid and a lentiviral plasmid carrying a target gene into a calcium phosphate-DNA complex; removal of

(2) Transfecting cells

Adding the calcium phosphate-DNA complex to the cell line capable of generating virus particles to be transfected, culturing the cell line by using the conventional culture medium, and obtaining the cell line capable of generating virus particles after the cell line is transfected, wherein the cell line capable of generating virus particles comprises a lentiviral vector of a target gene;

(3) Collection of viruses

The virus particle producing cell line was cultured with the vaccine xpress medium and virus was collected. Clinical grade viral vector particles carrying the gene of interest, which have only one-time infectivity and no replicative capacity, can be harvested in the upper part of the cell line.

Further, the time of culturing with the vaccine xpress medium is 40-48 hours after plasmid transfection.

Specifically, the lentiviral vector comprising the gene of interest comprises one of Human Immunodeficiency Virus (HIV), simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), equine Infectious Anemia Virus (EIAV), bovine Immunodeficiency Virus (BIV), and visna-medi virus (VMV) lentiviral plasmids.

The third object of the invention is to provide a new application of the vaccine xpress culture medium, which provides a new idea for mass production of lentiviral vectors.

use of said vaccine xpress medium as a medium for said virus particle producing cell line, such as transfected packaging cells.

Further, in the application, glutamine is added to the vaccine xpress medium.

The vaccine xpress medium is a serum-free medium developed by Hyclone company for vaccine production. Cell lines verified during development of the culture medium are mainly adherent cells and microcarriers thereof (such as Vero cells and Vero cell microcarriers) for expression of influenza (influenza), zika (Zika), ridge ash (Polio), dengue (Dengue) and respiratory syncytial virus (Respiratory syncytial virus, RSV) vaccine-related viruses for vaccine preparation. There is no information indicating that it was applied to lentivirus preparation, nor was it advertised in the website of Hyclone company for its application in lentivirus preparation. The conventional lentivirus preparation method needs to use DMEM culture medium containing FBS for culture, and liquid exchange is carried out before and after plasmid transfection, because the use of the FBS-containing culture medium leads to that the produced lentivirus feed liquid contains a large amount of BSA residues, but clinical lentivirus has definite limitation on the BSA residues, and the removal capacity of BSA by lentivirus purification is limited, so that the BSA residues after the lentivirus purification are still higher, and the clinical requirement cannot be met; the invention is innovatively applied to the preparation of the slow virus, can effectively reduce BSA residues, can obviously improve slow virus titer, and does not need to carry out serum-free domestication on working cells when being replaced to a serum-free culture medium.

It is therefore a fourth object of the present invention to provide a composition which can produce high titres of virus.

a composition suitable for large-scale clinical grade viral vector preparation, said composition comprising said vaccine xpress medium and said virus particle producing cell line. Equivalently, a composition suitable for large-scale clinical grade viral vector preparation, after a certain period of time, contains the viral particles produced by the vaccine xpress medium and the virus particle-producing cell line.

Further, the cell line in the composition is a HEK293T cell.

Further, the composition consists of the vaccine xpress medium, glutamine, and the virus particle-producing cell line.

The invention aims to provide a method for improving the interference efficiency of a lentiviral vector, which is simple to operate and suitable for large-scale production.

a method for improving the infection efficiency of a lentiviral vector, comprising transfecting a cell line capable of producing virus particles with a lentiviral vector containing a gene of interest under the condition of the conventional culture medium, culturing the cell line with the vaccine Xpress culture medium, and obtaining the supernatant as the virus particles produced by the cell line.

Further, in the method, the gene of interest comprises a chimeric antigen receptor nucleic acid sequence or a gene therapy-related nucleic acid sequence.

Further, in the method, the vaccine xpress medium contains 1-5% of the glutamine by volume fraction.

Further, the lentiviral vector comprising the gene of interest includes a viral vector, a vector comprising a viral accessory protein gene, a vector comprising a packaging protein gene, and a vector comprising the gene of interest. It is also understood to include packaging plasmids, envelope plasmids, helper protein plasmids and lentiviral plasmids carrying the gene of interest.

Further, the method comprises the steps of: (1) Culturing cells to be transfected with a DMEM medium containing FBS, the transfected cells not comprising a gene of interest; preparing a calcium phosphate-DNA complex from the packaging plasmid and the lentiviral plasmid carrying the target gene; (2) Adding the calcium phosphate-DNA complex into the cells to be transfected, and culturing the cells with the DMEM medium containing FBS to obtain a cell line capable of generating virus particles, wherein the cell line capable of generating the virus particles carries a lentiviral vector of a target gene; (3) The virus particle-producing cell line was removed from the supernatant and continued culture using glutamine-containing vaccine media.

The calcium phosphate-DNA complex is obtained by a calcium phosphate transfection method well known in the art; other known methods may be substituted.

Further, in the culture at the cell non-transfer stage, the following can be referred to: firstly, culturing target cells which do not contain lentiviral vectors containing target genes by using a DMEM medium containing 10-13% FBS, and then culturing by changing the target cells into a DMEM medium containing 5-8% FBS; preferably, it is: cells to be transfected (packaging cells) were first cultured in DMEM medium containing 10-13% fbs, and then cultured in DMEM medium containing 5-8% fbs for 3-8 hours. The two culture is preferably carried out under the conditions of 37-40deg.C and 5-8% CO ₂ Culturing in a carbon dioxide incubator.

Further, the lentiviral plasmid carrying the target gene in the step (2) comprises one of Human Immunodeficiency Virus (HIV), simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), equine Infectious Anemia Virus (EIAV), bovine Immunodeficiency Virus (BIV), visna-meydy virus (VMV) lentivirus plasmids.

Further, the calcium phosphate transfection method specifically comprises the following steps: adding sterilized ultrapure water into a mixture of the packaging plasmid, the lentiviral plasmid carrying the target gene and 2-3mol/L CaCl2 solution, uniformly mixing, slowly adding 2-x HBS solution with pH of 6.9-7.1 into the mixture, slowly dropwise adding the mixture while shaking and uniformly mixing, standing and incubating for 5-10 minutes after uniformly mixing, and obtaining the calcium phosphate-DNA compound.

Further, the time for culture using the vaccine xpress medium was: 40-48 hours after plasmid transfer.

Further, the vaccine xpress medium can be replaced immediately without serum-free acclimation of the virus particle-producing cell line in the medium.

The invention has the beneficial effects that:

(1) The culture medium and the culture method provided by the invention can effectively reduce BSA residues, and can be used for preparing clinical-grade slow virus vectors;

(2) The culture medium and the culture method provided by the invention can obviously improve the titer of the slow virus, and are suitable for large-scale clinical slow virus production;

(3) The culture and the culture method provided by the invention have the advantages that in the culture process, the serum-free culture medium is replaced without carrying out serum-free domestication on the working cells, and the method is simple, convenient and short in time consumption.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

For a better understanding of the concepts and relationships of the various bodies in the examples, the specific invention is directed to: (1) The transfer vector carrying the gene of interest is co-transfected with the packaging vector (e.g., HEK293T cells in this example) and cultured in conventional medium (e.g., DMEM medium with 10% FBS in this example). (2) Transfected packaging cells were not cultured with the conventional medium (as in the present embodiment, DMEM medium containing FBS), but with the vaccine Xpres medium containing 1% glutamine additive, and virus was collected. (3) The virus transduces target cells, such as T lymphocytes or other host cells (e.g., lymphocytes (PBMCs) in this example 4). (4) The gene of interest is integrated and expressed in target cells, such as lymphocytes (PBMCs) in this example 4.

EXAMPLE 1 HEK293T cell subculture

(1) Preparing DMEM medium containing 10% FBS (10% DMEM for short);

(2) Resuscitating HEK293T cells;

(3) Resuscitated HEK293T cells were added to 10ml of 10% DMEM medium, centrifuged at 200-300g for 5 min, the supernatant was discarded, the cells were resuspended in 10% DMEM and transferred to a cell flask, carbon dioxide incubator, 37℃C, 5% CO ₂ Culturing under the condition;

(4) When the cell density reaches a certain degree, the cells are passaged, and the good state of the cells is maintained.

EXAMPLE 2 lentiviral preparation

(1) 1 x 10 x 6HEK293T cells/hole are planted in a 6-hole plate, when the cell fusion degree in the hole plate reaches 60-80% in the next day, the original culture medium in the hole plate is removed, and the hole plate is replaced by a DMEM culture medium containing 5% FBS (hereinafter referred to as 5% DMEM), and the hole plate is cultured for about 2 hours;

(2) The packaging plasmid and the target plasmid, 2.5M CaCl were added to a 15ml centrifuge tube ₂ Adding sterilized ultrapure water into the solution mixture, uniformly mixing, slowly adding 2-x HBS solution with pH of 6.90-7.10 into the solution mixture, slowly dripping the solution while shaking and uniformly mixing, standing and incubating for 5-10 minutes after uniformly mixing to form a calcium phosphate-DNA compound;

(3) 200 μl of calcium phosphate-DNA complex was added to each well of a 6-well plate, and the mixture was placed in a carbon dioxide incubator at 37℃with 5% CO ₂ Culturing for 3-8 hours;

(4) Taking out the 6-well plate, removing the supernatant, and respectively adding 2ml of 5% DMEM, vaccine Xpres culture medium containing 1% glutamine additive, gibco FreeStyle 293 culture medium, gibco VP-SFM culture medium and OPTI PRO-SFM culture medium into different wells;

(5) The viral supernatants were harvested 40-48 hours after plasmid transfection.

Example 3 lentiviral titer detection

(1) HEK293T cells were digested, counted after resuspension, and the number of plated cells per well was 1 x 105.

(2) After 3-6 hours, after cell attachment in 24-well plates, 10. Mu.l of lentiviral supernatant produced in example 2 was added to the wells, and 1. Mu.l Polybrene,37℃and 5% CO were added ₂ The culture was carried out for 2 days.

(3) After 2 days, HEK293T cells in the 24-well plate are digested, and the positive rate of the HEK293T cells is detected in a flow mode; the detection reagent is Protein-L.

Protein-L can specifically recognize immunoglobulin light chain kappa chain, and the sequence of a single-chain antibody contained in a lentiviral vector can be recognized by the Protein-L; if 293T cells express the CAR structure in the viral vector, they will be detected by the tag. Pro-L positive rate indicates viral infectivity, and the stronger the infectivity of the same viral vector to the same cell, the higher the viral titer at the same volume of viral supernatant. Viral infection ability the conversion formula before viral infection ability and titer is:

the virus titer unit is TU/ml;

the viral volume unit is ml.

The experimental test results are shown in table 1:

TABLE 1 infection efficiency of cultured viruses of different cultures

Conclusion: the vaccine xprees culture medium can be used for preparing the lentivirus, and the infection capacity of the lentivirus can be obviously improved by using the culture medium, which is far higher than that of other types of serum-free culture medium and conventional DMEM culture medium with 5% FBS. Thus, the use of the vaccine xprees medium for lentivirus production enables higher viral yields than conventional production protocols.

Example 4 Virus transduction of target cells

The newly prepared mononuclear cell PBMC are cultured by RPMI 1640 complete culture medium containing 10% FBS, and lentiviral infection is carried out after the anti-CD 3 and anti-CD 28 monoclonal antibodies are activated; separately adding the prepared lentivirus containing the target gene, uninfected peripheral blood lymphocytes (PBMC) as a blank control; after 24h, the medium was changed to a complete RPMI 1640 medium containing 500IU/mL recombinant human IL-2, and the culture was continued for 10-20 days, and the expression of the target gene was detected by using Protein-L on the 10 th day of the culture.

Comparative example 1 single use test of vaccine Xpress Medium and DMEM Medium

According to the methods of example 2 and example 3, the vaccine xpress medium coupling protocol was changed to two changes of vaccine xpress medium and 2 changes of 5% dmem were performed for culture experiments and detection. The results of the cultured lentiviral vector assays are shown in Table 2 below.

TABLE 2 transfection efficiency of viruses cultured in Single Medium

Experimental group	VaccireXpress 2 times liquid change	5% DMEM 2 times liquid exchange
			1	9.70％	16.00％
2	9.40％	10.70％

Conclusion: the ability to prepare viruses using the vaccine xprees medium 293T cells was inferior to that of the conventional 5% DMEM medium, and the preparation scheme using dmem+vaccine xpress and the preparation scheme superior to DMEM alone was combined with the scheme of example 3, so that the preparation scheme of dmem+vaccine xpress was optimal, and thus the present invention used a slow virus in which the culture was performed by replacing the vaccine culture medium with the DMEM medium replacement solution containing FBS after 3-8 hours of the addition of the calcium phosphate-DNA complex.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

1. A method for improving the infection efficiency of a lentiviral vector, which is characterized in that a cell line capable of producing virus particles is transfected by the lentiviral vector containing a target gene under the condition of a conventional culture medium, the cell line is cultured by using a vaccine xpress culture medium, and the supernatant is the virus particles produced by the cell line; the method comprises the following steps: (1) culturing cells to be transfected with DMEM medium containing 10% fbs; preparing a calcium phosphate-DNA complex from the packaging plasmid and the lentiviral plasmid carrying the target gene; (2) Adding the calcium phosphate-DNA complex into the cells to be transfected, and culturing the cells with a DMEM medium containing 5% FBS to obtain a cell line capable of generating virus particles, wherein the cell line capable of generating virus particles carries a lentiviral vector of a target gene; (3) Removing the supernatant from the virus particle-producing cell line and continuing to culture the virus particle-producing cell line with glutamine-containing vaccine culture medium; the cell line is a HEK293T cell.

2. The method of claim 1, wherein the gene of interest comprises a chimeric antigen receptor nucleic acid sequence or a gene therapy-related nucleic acid sequence.

3. The method of claim 2, wherein said vaccine xpress medium comprises 1-5% by volume of said glutamine.