Method and system for totally-enclosed production of lentiviral vector
阅读说明:本技术 一种慢病毒载体全封闭生产的方法及系统 (Method and system for totally-enclosed production of lentiviral vector ) 是由 姚树元 刘丹 于开鹏 徐燕 曹鹏亮 张会 陈星星 杨吕杰 于 2019-11-06 设计创作,主要内容包括:本发明公开了一种慢病毒载体全封闭生产的方法及系统,方法包括步骤1:慢病毒载体宿主细胞的培养增殖;步骤2:慢病毒载体宿主细胞的包装,将含有慢病毒载体基因组序列的质粒通过转染试剂转染入慢病毒载体宿主细胞,制得慢病毒载体培养细胞上清液并收集;步骤3:慢病毒载体上清液的纯化,将慢病毒载体上清液依次进行澄清、酶切消化、超滤、层析柱纯化后制得慢病毒载体。系统包括宿主细胞增殖培养装置、慢病毒载体包装装置、慢病毒载体纯化装置,各装置使用一次性无菌耗材,有效避免交叉污染,实现多种产品的共线生产。(The invention discloses a method and a system for totally-enclosed production of a lentiviral vector, wherein the method comprises the following steps of 1: culturing and propagating the lentivirus vector host cells; step 2: packaging a lentivirus vector host cell, namely transfecting a plasmid containing a lentivirus vector genome sequence into the lentivirus vector host cell through a transfection reagent to prepare and collect a lentivirus vector culture cell supernatant; and step 3: and (3) purifying the supernatant of the lentiviral vector, namely sequentially clarifying the supernatant of the lentiviral vector, digesting by enzyme digestion, ultrafiltering and purifying by a chromatographic column to obtain the lentiviral vector. The system comprises a host cell proliferation culture device, a lentivirus vector packaging device and a lentivirus vector purification device, wherein each device uses disposable sterile consumables, cross contamination is effectively avoided, and collinear production of various products is realized.)
1. A method for totally-enclosed production of lentiviral vectors, comprising the steps of:
step 1: culturing and propagating the lentivirus vector host cells; step 2: packaging a lentivirus vector host cell, namely transfecting a plasmid containing a lentivirus vector genome sequence into the lentivirus vector host cell through a transfection reagent to prepare and collect a lentivirus vector culture cell supernatant;
and step 3: and (3) purifying the supernatant of the lentiviral vector, namely sequentially clarifying the supernatant of the lentiviral vector, digesting by enzyme digestion, ultrafiltering and purifying by a chromatographic column to obtain the lentiviral vector.
2. The method for totally-enclosed production of lentiviral vectors according to claim 1, wherein the lentiviral vector host cells in step 1 are cultured in a multilayer cell culture flask in a pre-closed manner, and the pre-closed culture comprises resuscitating, culturing, digesting and passaging the host cells under closed conditions.
3. The method of claim 2, wherein the lentiviral vector host cell is a 293T cell.
4. The method of claim 3, wherein the transfection reagent is calcium chloride.
5. The method for whole-plant production of lentiviral vector of claim 4, wherein step 3 comprises clarification with a capsule filter, digestion with nuclease, and ultrafiltration with hollow fiber column.
6. A system for totally-enclosed production of lentiviral vectors is characterized by comprising a host cell proliferation culture device, a lentiviral vector packaging device and a lentiviral vector purification device which are sequentially connected.
7. The system for the whole-plant production of lentiviral vectors according to claim 6, wherein the host cell proliferation culture device comprises a first collection roller bottle and a digestive juice culture conveying component, a culture medium feed liquid conveying component and a host cell culture component which are respectively connected with the first collection roller bottle;
the digestive juice culturing and conveying assembly comprises a digestive juice feed bag, a digestive juice conveying pipeline, a first digestive juice sterile pipe connecting machine, a first digestive juice sterile pipe sealing machine, a digestive juice multilayer cell culture bottle, a second digestive juice sterile pipe connecting machine and a second digestive juice sterile pipe sealing machine which are connected to the digestive juice conveying pipeline, wherein two ends of the digestive juice conveying pipeline are respectively communicated with the digestive juice feed bag and the first collecting roller bottle;
the culture medium liquid conveying assembly comprises a culture medium liquid bag and a culture medium liquid conveying pipeline, two ends of the culture medium liquid conveying pipeline are respectively communicated with the culture medium liquid bag and the first collecting roller bottle, and a culture medium liquid sterile pipe sealing machine and a culture medium liquid sterile pipe connecting machine are sequentially installed on the culture medium liquid conveying pipeline from the culture medium liquid bag to the direction of communication of the first collecting roller bottle;
the host cell culture assembly comprises a host cell conveying pipeline, a first host cell multilayer cell culture bottle and a second host cell multilayer cell culture bottle, two ends of the host cell conveying pipeline are respectively communicated with the first host cell multilayer cell culture bottle and the first collecting roller bottle, a host cell sterile pipe connecting machine is installed on the host cell conveying pipeline, a host cell conveying branch pipe connected with the second host cell multilayer cell culture bottle is installed on the host cell conveying pipeline between the host cell sterile pipe connecting machine and the first host cell multilayer cell culture bottle, and a host cell sterile pipe sealing machine is connected with the joint of the host cell conveying branch pipe and the host cell conveying pipeline.
8. The system for totally-enclosed production of lentiviral vectors as claimed in claim 6 or 7, wherein the lentiviral vector packaging device comprises a second collection roller bottle and a transfection reagent feed liquid bag, wherein the transfection reagent feed liquid bag is provided with a transfection reagent feed liquid conveying pipeline communicated with the second collection roller bottle, the transfection reagent feed liquid conveying pipeline is sequentially provided with a transfection reagent sterile tube sealing machine and a transfection reagent sterile tube connecting machine in the direction of the second collection roller bottle from the transfection reagent feed liquid bag, the second collection roller bottle is provided with a supernatant conveying pipeline communicated with the first host cell multilayer cell culture bottle or the second host cell multilayer cell culture bottle, and the supernatant conveying pipeline is sequentially provided with a supernatant sterile tube sealing machine in the direction of the second collection roller bottle, The supernatant fluid is connected to a tube machine in an aseptic mode.
9. The system according to claim 6 or 7, wherein the lentiviral vector purification device comprises a lentiviral vector conveying pipeline, and a viral supernatant fluid bag, a lentiviral vector first sterile tube connector, a lentiviral vector first sterile tube sealing machine, a bag filter, a lentiviral vector second sterile tube connector, a lentiviral vector second sterile tube sealing machine, a clarified sample fluid bag, a hollow fiber column, an ultrafiltration sample fluid bag, a lentiviral vector third sterile tube connector, a lentiviral vector third sterile tube sealing machine, a chromatography column and a purified fluid bag which are sequentially arranged on the lentiviral vector conveying pipeline, wherein the viral supernatant fluid bag is provided with a supernatant fluid extraction pipeline communicated with the host cell first multilayer cell culture bottle or the host cell second multilayer cell culture bottle, and a supernatant sterile pipe connecting machine is installed on the supernatant extracting pipeline, and a clarification pipeline communicated with the lentivirus carrier conveying pipeline is arranged at one end of the liquid inlet of the bag filter.
10. The system for totally enclosed production of lentiviral vectors according to claim 8, wherein the lentiviral vector purification device comprises a lentiviral vector conveying pipeline, and a viral supernatant fluid bag, a lentiviral vector first sterile tube connector, a bag filter, a lentiviral vector second sterile tube connector, a clarified sample fluid bag, a hollow fiber column, an ultrafiltration sample fluid bag, a lentiviral vector third sterile tube connector, a chromatography column, and a purified fluid bag which are sequentially arranged on the lentiviral vector conveying pipeline, wherein the viral supernatant fluid bag is provided with a supernatant fluid extraction pipeline communicated with the host cell first multilayer cell culture bottle or the host cell second multilayer cell culture bottle, and a supernatant sterile pipe connecting machine is installed on the supernatant extracting pipeline, and a clarification pipeline communicated with the lentivirus carrier conveying pipeline is arranged at one end of the liquid inlet of the bag filter.
Technical Field
The invention relates to the field of lentivirus vector production, in particular to a method and a system for totally-enclosed production of lentivirus vectors.
Background
The lentivirus vector is a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency virus type 1), belongs to the family of retroviridae, is an RNA virus, has the capacity of infecting both split cells and non-split cells, and can effectively integrate foreign genes onto a host chromosome so as to achieve persistent expression;
further intensive in recent years research into gene therapy and transgenic animals, lentiviral vectors have been spotlighted as one of the research vectors, which have proven to be very effective in a variety of different applications including cancer gene therapy;
because the lentivirus vector can be applied to in vitro cell modification and is returned to a patient body, the preparation process of the lentivirus vector has very high requirements on the aseptic control of the environment, personnel and equipment, and the conventional production process can easily cause cross contamination to the equipment, the environment and the personnel in the process flow, so that the multi-variety collinear production of products can not be realized; the production characteristics of the product are small batch, high product quality requirement and multi-variety collinear production, so that a set of totally-enclosed production process is developed, and the totally-enclosed production process has profound influence on the quality and effect of the product and the development of the whole gene therapy industry.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method and a system for totally-enclosed production of a lentiviral vector, wherein disposable sterile consumables are used in the whole process, cross contamination is effectively avoided, collinear production of various products can be realized, used equipment cannot contact the used materials, pipelines through which sample liquid and balance liquid flow are totally enclosed, and a sterile tube sealing machine and a sterile tube connecting machine are adopted for disconnection and connection, so that pollution and cross contamination can be effectively avoided in the production process, the influence of environment, equipment, personnel and other external factors on cell products is reduced, the quality and the stability of the products are improved, and the defects caused by the prior art are overcome.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for the totally enclosed production of lentiviral vectors, comprising the steps of:
step 1: culturing and propagating the lentivirus vector host cells;
step 2: packaging a lentivirus vector host cell, namely transfecting a plasmid containing a lentivirus vector genome sequence into the lentivirus vector host cell through a transfection reagent to prepare and collect a lentivirus vector culture cell supernatant;
and step 3: and (3) purifying the supernatant of the lentiviral vector, namely sequentially clarifying the supernatant of the lentiviral vector, digesting by enzyme digestion, ultrafiltering and purifying by a chromatographic column to obtain the lentiviral vector. In the method for totally-enclosed production of the lentiviral vector, the lentiviral vector host cell in the step 1 needs to be cultured in a multilayer cell culture flask in a closed manner in advance, and the closed culture in advance comprises the steps of recovering, culturing, digesting and passaging the host cell under a closed condition.
The method for totally-enclosed production of the lentiviral vector, wherein the host cell of the lentiviral vector is 293T cell.
The method for totally-enclosed production of the lentiviral vector is characterized in that the transfection reagent is calcium chloride.
In the above method for totally-enclosed production of the lentiviral vector, in the step 3, a bag filter is used for clarification, nuclease is added for digestion, and hollow fiber column is used for ultrafiltration.
A system for totally-enclosed production of lentiviral vectors comprises a host cell proliferation culture device, a lentiviral vector packaging device and a lentiviral vector purification device which are sequentially connected.
The system for totally-enclosed production of the lentiviral vector is characterized in that the host cell proliferation culture device comprises a first collection roller bottle, and a digestion solution culture conveying assembly, a culture medium liquid conveying assembly and a host cell culture assembly which are respectively connected with the first collection roller bottle;
the digestive juice culturing and conveying assembly comprises a digestive juice feed bag, a digestive juice conveying pipeline, a first digestive juice sterile pipe connecting machine, a first digestive juice sterile pipe sealing machine, a digestive juice multilayer cell culture bottle, a second digestive juice sterile pipe connecting machine and a second digestive juice sterile pipe sealing machine which are connected to the digestive juice conveying pipeline, wherein two ends of the digestive juice conveying pipeline are respectively communicated with the digestive juice feed bag and the first collecting roller bottle;
the culture medium liquid conveying assembly comprises a culture medium liquid bag and a culture medium liquid conveying pipeline, two ends of the culture medium liquid conveying pipeline are respectively communicated with the culture medium liquid bag and the first collecting roller bottle, and a culture medium liquid sterile pipe sealing machine and a culture medium liquid sterile pipe connecting machine are sequentially installed on the culture medium liquid conveying pipeline from the culture medium liquid bag to the direction of communication of the first collecting roller bottle;
the host cell culture assembly comprises a host cell conveying pipeline, a first host cell multilayer cell culture bottle and a second host cell multilayer cell culture bottle, two ends of the host cell conveying pipeline are respectively communicated with the first host cell multilayer cell culture bottle and the first collecting roller bottle, a host cell sterile pipe connecting machine is installed on the host cell conveying pipeline, a host cell conveying branch pipe connected with the second host cell multilayer cell culture bottle is installed on the host cell conveying pipeline between the host cell sterile pipe connecting machine and the first host cell multilayer cell culture bottle, and a host cell sterile pipe sealing machine is connected with the joint of the host cell conveying branch pipe and the host cell conveying pipeline.
The system for the totally-enclosed production of the lentiviral vector comprises a second collection roller bottle and a transfection reagent material liquid bag, wherein the transfection reagent material liquid bag is provided with a transfection reagent material liquid conveying pipeline communicated with the second collection roller bottle, the transfection reagent material liquid conveying pipeline is formed by sequentially mounting a transfection reagent sterile tube sealing machine and a transfection reagent sterile tube connecting machine on the transfection reagent material liquid bag in the direction of the second collection roller bottle, the second collection roller bottle is provided with a supernatant conveying pipeline communicated with the first multilayer cell culture bottle of the host cells or the second multilayer cell culture bottle of the host cells, and the supernatant conveying pipeline is formed by sequentially mounting a supernatant sterile tube sealing machine on the second collection roller bottle in the direction of the first multilayer cell culture bottle of the host cells or the second multilayer cell culture bottle of the host cells, The supernatant fluid is connected to a tube machine in an aseptic mode.
The system for the totally-enclosed production of the lentiviral vector comprises a lentiviral vector conveying pipeline, and a viral supernatant liquid bag, a lentiviral vector first sterile tube connecting machine, a lentiviral vector first sterile tube sealing machine, a bag filter, a lentiviral vector second sterile tube connecting machine, a lentiviral vector second sterile tube sealing machine, a clarified sample liquid bag, a hollow fiber column, an ultrafiltration sample liquid bag, a lentiviral vector third sterile tube connecting machine, a lentiviral vector third sterile tube sealing machine, a chromatography column and a purification liquid bag which are sequentially arranged on the lentiviral vector conveying pipeline, and a supernatant extracting pipeline communicated with the first host cell multilayer cell culture bottle or the second host cell multilayer cell culture bottle is arranged on the virus supernatant feed liquid bag, and a supernatant sterile pipe connecting machine is arranged on the supernatant extracting pipeline.
The technical scheme provided by the method and the system for totally-enclosed production of the lentiviral vector has the following technical effects:
the whole process uses disposable aseptic consumables, cross contamination is effectively avoided, collinear production of various products can be realized, used equipment cannot contact used materials, pipelines through which sample liquid and balance liquid flow are totally closed, an aseptic tube sealing machine and an aseptic tube connecting machine are adopted for disconnection and connection, contamination and cross contamination can be effectively avoided in the production process, the influence of environment, equipment, personnel and other external factors on cell products is reduced, and the quality and the stability of the products are improved.
Drawings
FIG. 1 is a flow chart of a process for the whole-enclosure production of lentiviral vectors of the present invention;
FIG. 2 is a schematic structural diagram of a host cell proliferation culture device in a system for totally-enclosed production of lentiviral vectors, according to the invention;
FIG. 3 is a schematic diagram of a lentiviral vector packaging device in a system for the closed production of lentiviral vectors of the present invention;
FIG. 4 is a schematic structural diagram of a lentivirus vector purification device in a system for totally enclosed production of lentivirus vectors according to the present invention.
Wherein the reference numbers are as follows:
a first
Detailed Description
In order to make the technical means, the characteristics, the purposes and the functions of the invention easy to understand, the invention is further described with reference to the specific drawings.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.
In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
The invention provides a method and a system for totally-enclosed production of a lentivirus carrier, aiming at using disposable sterile consumables in the whole process, effectively avoiding cross contamination, realizing collinear production of various products, avoiding the used equipment from contacting the used materials, totally-enclosing pipelines through which sample liquid and balance liquid flow, adopting a sterile pipe sealing machine and a sterile pipe connecting machine for disconnection and connection, effectively avoiding contamination and cross contamination in the production process, reducing the influence of environment, equipment, personnel and other external factors on cell products, and improving the quality and stability of the products.
As shown in FIG. 1, a method for the whole-closed production of lentiviral vectors comprises the following steps:
step 1: culturing and propagating the lentivirus vector host cells;
step 2: packaging a lentivirus vector host cell, namely transfecting a plasmid containing a lentivirus vector genome sequence into the lentivirus vector host cell through a transfection reagent to prepare and collect a lentivirus vector culture cell supernatant;
and step 3: and (3) purifying the supernatant of the lentiviral vector, namely sequentially clarifying the supernatant of the lentiviral vector, digesting by enzyme digestion, ultrafiltering and purifying by a chromatographic column to obtain the lentiviral vector. In the method for totally-enclosed production of lentiviral vectors provided in this embodiment, the lentiviral vector host cells in step 1 need to be cultured in a multilayer cell culture flask in a closed manner in advance, and the closed culture in advance comprises recovering, culturing, digesting and passaging the host cells under a closed condition.
In the method for totally-enclosed production of lentiviral vectors provided in this example, the host cells of the lentiviral vectors are 293T cells.
In the method for totally-enclosed production of lentiviral vectors provided in this example, calcium chloride is used as the transfection reagent.
In the totally enclosed production method of the lentiviral vector provided in this embodiment, the step 3 is performed by clarifying with a
The method for totally-enclosed production of lentiviral vectors provided in this example comprises the following specific steps:
firstly, culturing and proliferating a host cell 293T cell of a lentivirus vector, and recovering, culturing, digesting and passaging the cell in a fully-closed multilayer cell culture bottle to obtain a culture supernatant;
packaging the obtained culture supernatant, and transfecting a plasmid containing a lentiviral vector genome sequence into a host cell by a transfection reagent to prepare a lentiviral vector packaging cell supernatant;
purifying the lentivirus vector supernatant, collecting the lentivirus supernatant, clarifying the lentivirus supernatant in a liquid storage bag through a clarification pipeline 312 and a
The invention relates to a system for totally-enclosed production of a lentiviral vector, which comprises a host cell proliferation culture device, a lentiviral vector packaging device and a lentiviral vector purification device which are sequentially connected.
As shown in fig. 2, the system for totally-enclosed production of lentiviral vectors provided by the embodiment adopts a host cell proliferation culture device comprising a first
the digestive juice culture conveying assembly comprises a digestive
the culture medium liquid conveying assembly comprises a culture
the host cell culture assembly comprises a host
in specific use, a host cell proliferation culture device is required to be used for culturing the host cells, and the specific operation process is as follows:
introducing digestive juice in a digestive juice feed
As shown in fig. 3, the system for totally-enclosed production of lentiviral vectors provided in this embodiment adopts a lentiviral vector packaging device comprising a second
in specific use, a lentivirus vector packaging device is used for extracting the generated cell supernatant and adding a transfection reagent, and the specific operation steps are as follows:
optionally selecting one of the first multilayer
As shown in fig. 4, the system for totally-enclosed production of lentiviral vectors provided in this embodiment adopts a lentiviral vector purification device comprising a lentiviral
in specific use, a lentiviral vector purification device is required to be used for purifying the lentiviral vector, and the specific operation process is as follows:
extracting and storing transfected virus supernatant into a virus supernatant
In conclusion, the method and the system for totally-enclosed production of the lentiviral vector use disposable sterile consumables in the whole process, effectively avoid cross contamination, realize collinear production of various products, avoid the contact of used equipment with used materials, totally-enclosed pipelines through which the sample liquid and the balance liquid flow, and adopt the sterile tube sealing machine and the sterile tube connecting machine for disconnection and connection, effectively avoid contamination and cross contamination in the production process, reduce the influence of environment, equipment, personnel and other external factors on cell products, and improve the quality and stability of the products.
Specific embodiments of the invention have been described above. It is to be understood that the invention is not limited to the particular embodiments described above, in that devices and structures not described in detail are understood to be implemented in a manner common in the art; various changes or modifications may be made by one skilled in the art within the scope of the claims without departing from the spirit of the invention, and without affecting the spirit of the invention.
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