阅读说明：本技术 用于病毒载体分离的方法和试剂盒 (Methods and kits for viral vector isolation ) 是由 T.史密斯 于 2018-12-20 设计创作，主要内容包括：本发明涉及用于纯化病毒载体的方法,更具体地,它涉及通过使用单一自动化过程,从生产者细胞中纯化病毒载体。该方法包括下述步骤：a)将生产者细胞和细胞裂解缓冲液加入处理容器中；b)在所述处理容器中混合所述生产者细胞和细胞裂解缓冲液,以获得混合物；c)使所述混合物流过用于纯化病毒载体的色谱柱,其中所述病毒载体被吸附到所述色谱柱上；和d)将病毒载体从色谱柱中洗脱到产物容器内。(The present invention relates to a method for purifying viral vectors, more specifically it relates to the purification of viral vectors from producer cells by using a single automated process. The method comprises the following steps: a) adding the producer cells and a cell lysis buffer to a processing vessel; b) mixing the producer cells and cell lysis buffer in the processing vessel to obtain a mixture; c) flowing the mixture through a chromatography column for purifying a viral vector, wherein the viral vector is adsorbed onto the chromatography column; and d) eluting the viral vector from the chromatography column into a product container.)

1. A method for isolating a viral vector comprising a gene of interest after production of said viral vector by culture in a producer cell comprising the following steps in a continuous workflow: a) adding producer cells (1) and a cell lysis buffer (2) to a treatment vessel (5); b) mixing the producer cells and cell lysis buffer in the processing vessel (5) to form a mixture; c) flowing the mixture from the treatment vessel (5) through a chromatography column (8) comprising an affinity resin for purifying a viral vector, wherein the viral vector is adsorbed onto the resin in the chromatography column (8); and d) eluting the viral vector from the resin in the chromatographic column (8) into the product container (7).

2. The method according to claim 1, wherein the viral vector obtained in step d) in the product container (7) is used to transfect fresh producer cells, which are cultured after transfection, and then steps a) -d) are repeated at least once.

3. The method according to claim 1 or 2, wherein the number of viral vectors obtained from said method is determined by the volume and saturation point of the resin in the chromatography column (8).

4. Method according to one or more of the preceding claims, comprising the following steps after step c) or d): the wash buffer (3) is released into the processing container (5) to wash out the cells/debris and sprayed into the waste container (6).

5. The method according to claim 4, wherein the processing vessel (5), the product vessel (7) and the column (8) are mounted in a cartridge, and wherein the cell lysis buffer (2), the wash buffer (3), the elution buffer (4) and optionally the waste vessel (6) are located outside the cartridge.

6. The method according to one or more of the preceding claims, wherein said chromatography column (8) is interchangeable in said cartridge and comprises an affinity resin specific for the selected viral vector.

7. The method according to one or more of the preceding claims, wherein said treatment vessel (5) is provided with an outlet made by a filter to retain any intact cells/cell debris within the treatment vessel (5).

8. The method according to one or more of the preceding claims, wherein the viral vector is a lentivirus or a gammaretrovirus.

9. The method according to one or more of the preceding claims, wherein the gene of interest comprises a disease-regulating gene, such as a gene encoding a Chimeric Antigen (CAR).

10. The method according to one or more of the preceding claims 2-9, wherein the purity of the isolated viral vectors in the product container (7) is tested in a quality assay involving SPR (surface plasmon resonance) detection before repeating steps a) -d).

11. Use of the isolated viral vector according to one or more of claims 1 to 10 for cell therapy.

12. Use according to claim 10, wherein the viral vector is for converting T cells into CAR T cells.

13. A kit comprising a processing container (5), a product container (7) and an affinity column (8) mounted in one and the same cassette, and further comprising a source container (1) for producer cells, a cell lysis buffer (2), a wash buffer (3), an elution buffer (4) and optionally a waste container (6) located outside the cassette.

Technical Field

The present invention relates to purification of viral vectors, more specifically it relates to a method and kit for purification of viral vectors from producer cells by using a single automated process.

Background

Cell therapy is an emerging field for the treatment of serious diseases, such as cancer, and represents a complement to traditional therapies. For decades, cancer treatment has been limited to chemotherapy and radiation therapy. Recently, biopharmaceuticals such as imatinib (Gleevec) and trastuzumab (Herceptin) have become increasingly popular. These drugs target cancer cells by homing to specific molecular changes that are primarily seen in those cells.

However, in the last few years new forms of therapy, cell therapy or immunotherapy, have emerged as options for e.g. cancer treatment.

The rapidly growing immunotherapy is called Adoptive Cell Transfer (ACT): the patient's own immune cells are collected and used to treat their cancer. There are several types of ACT (TIL, TCR, and CAR), but ACT that is closest to producing a treatment approved by the united states Food and Drug Administration (FDA) is called CAR T cell therapy.

Until recently, the use of CAR T cell therapy has been limited to small clinical trials largely in patients with advanced hematologic cancer. However, these therapies have also received attention from researchers and the public due to the significant responses these therapies have produced in some patients (both children and adults for whom all other therapies have ceased to work).

One CAR T cell therapy was approved for the treatment of children with Acute Lymphoblastic Leukemia (ALL) in 8 months 2017. And the second for adults with advanced lymphoma was approved in 2017 at month 10.

CAR T cell therapy relies on T cells, which have a key role in coordinating immune responses and killing cells infected by pathogens. This therapy requires drawing blood from the patient and isolating the T cells. Next, using the viral vector, T cells are genetically engineered to produce receptors on their surface called chimeric antigen receptors or CARs.

Currently, purification of viral vectors for cell transformation is not an automated process.

Purification of viral vectors typically requires a column to reliably separate the particles. For example, lentivirus purification requires antibodies specific for its protein coat. As with cell isolation, each type of strain requires antibody-specific purification methods. However, separation of viral particles from cells and cell debris can also be accomplished by centrifugation steps using density gradients or similar solution-based techniques.

Like many other biological processes, contaminants in this step can delay treatment of the patient at best and negatively impact its health at worst. The creation of a trustworthy "once and for all" approach will ensure the safety and efficiency of the downstream process.

It would be highly desirable to be able to automate the process of generating purified viral vectors for converting T cells into CAR T cells. This is particularly relevant in the current cell therapy market, as lentiviral vectors are the most common strategy for gene insertion into T cells for CAR expression.

To date, there is no such desired purification method available that is widely applicable to a variety of viral strains capable of transforming cells.

Thus, there remains a need for a widely applicable method for viral vector purification.

Summary of The Invention

The present invention avoids the disadvantages in the prior art by providing a broadly applicable method and kit for viral vector purification in an automated process, preferably using an apparatus that has been contemplated to be included in a Cell therapy workflow, such as Xuri Cell Harvester.

Using a combination of solution-based cell lysis, column extraction of viral particles, washing and elution steps, the method can be used to purify any type of viral vector in an all-in-one kit product, which offers several advantages over the prior art. The methods and kits of the invention provide a closed, sterile system for purification of viral vectors in an automated process.

In a first aspect, the present invention relates to a method for isolating a viral vector comprising a gene of interest after production of said viral vector by culture in a producer cell, comprising the following steps in a continuous workflow: a) adding the producer cells and a cell lysis buffer to a processing vessel; b) mixing the producer cells and cell lysis buffer in the processing vessel to form a mixture; c) flowing the mixture through a chromatography column comprising an affinity resin for purifying a viral vector, wherein the viral vector is adsorbed onto the resin in the chromatography column; and d) eluting the viral vector from the resin in the chromatography column into a product container.

The viral vector obtained in step d) in the product container is used to transfect fresh producer cells which are then cultured and then steps a) -d) are repeated at least once, for example 3-6 times or 3-6 passes (passage).

The scale of production, i.e. the number of viral vectors obtained from the process, is determined by the saturation point of the affinity resin and the volume of the chromatography column. The column volume is in a preferred configuration at most 200 mL.

Optionally, the method comprises the following steps after step c) or d): the wash buffer is released into the processing container to flush out the cells/debris and spray it into the waste container.

Preferably, the processing container, the product container and the chromatography column are mounted in one and the same cassette, and wherein the cell lysis buffer, the wash buffer, the elution buffer and optionally the waste container are located outside the cassette. All components 1-8, including the cassettes, are sometimes referred to herein as viral vector kits. Preferably, the container is a plastic bag container. The producer cells may be provided in a source bag or any other suitable container. The container may be a plastic bag or any other suitable container. The cassette is mounted on a device with a pump that facilitates the flow of liquid through the kit, as described below in fig. 2-7. The pinch valve allows for the selection of a particular line for different inputs and allows for centralized processing of the culture in the processing vessel.

The method and kit of the present invention are used with an instrument, preferably Xuri cell Harvester, having a digital interface that allows the user to set specific process parameters to optimize their own workflow. While the cassette allows automation of traditional viral vector separation with affinity columns, the method also involves the use of cell lysis buffers to release viral particles, and the use of specific viral binding to antibodies, ligands or other anchorable molecules in the same process. The method and kit can also be used intermediately to purify existing virus cultures, making their versatility quite novel in the field of virus vector production.

The chromatography column is an interchangeable affinity column specific for the selected viral vector. The affinity column may include, but is not limited to, antibodies, ligands, or any other anchorable molecule specific for binding to the viral vector within the column.

In one embodiment, the processing vessel is provided with an outlet completed by a filter to retain any intact cells/cell debris within the processing vessel.

The viral vector may be any virus, such as a lentivirus or a gammaretrovirus.

Preferably, the gene of interest comprises a disease-regulating gene, such as a gene encoding a Chimeric Antigen (CAR).

The purity of the isolated viral vectors in the product container can be tested in a mass assay involving SPR (surface plasmon resonance) detection before repeating steps a) -d). In such an assay, affinity ligands of the same type as in the affinity column may be immobilized to the sensor surface.

In a second aspect, the invention relates to the use of a viral vector isolated according to the invention for cell therapy, e.g. for converting T cells into CAR T cells.

In a third aspect, the invention relates to a kit comprising the process container, the product container and the chromatography column mounted in one and the same cassette, and further comprising a source container for producer cells, a cell lysis buffer, a wash buffer, an elution buffer and optionally a waste container located outside the cassette. All containers are preferably plastic bag containers.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing the different components of a viral vector isolation kit for use in the method according to the present invention.

FIG. 2 shows the first step (lysis of producer cells) in the isolation kit using viral vectors.

Figure 3 shows a mixing step, which may be applied between any steps using the kit as desired.

FIG. 4 shows the second step in the isolation kit using viral vectors (the supernatant was washed through the column).

FIG. 5 shows the third step (treatment bag washing) in the isolation kit using a viral vector.

FIG. 6 shows the fourth step (column elution) in the isolation kit using viral vectors.

FIG. 7 is a more detailed view of the fluid path of the viral vector isolation kit used in the present invention.

Detailed Description

With the advent of multiple FDA-approved CAR T-cell therapies, the most effective candidate viruses, such as lentiviruses and gamma retroviruses, are known.

The present inventors provide a platform for the purification of any viral vector in small to medium scale comprising a system of bags and chromatography columns, wherein the platform is limited only by the saturation point and volume of the resin in the chromatography column, and wherein the columns are interchangeable to allow the purification of different types of viral vectors.

The present invention will be described more specifically with reference to the accompanying drawings.

The present invention will be described with lentiviruses as the preferred viral vector, but any viral vector can be purified on the platform of the present invention.

The components of the kit according to the invention comprise a source bag containing producer cells previously infected with lentivirus, and any lentivirus in the supernatant that has been produced in culture. In addition, the cell lysis buffer lyses the producer cells, releasing additional virus particles, thus maximizing yield. The cell debris in the kit is washed with a wash buffer prior to final column elution. Finally, a column elution solution is required to release the purified virus particles into the final product bag.

FIG. 1 shows the different components of a viral vector isolation kit. The kit comprises eight parts or containers, preferably seven bag-type containers and one cylindrical container.

First is a source container, preferably source bag 1, comprising producer cells for producing viral vectors. The volume of this bag is scalable, limited only by the saturation point of the column.

Cell lysis buffer 2 is provided in a cell lysis buffer container or bag for cell lysis of producer cells from a source bag, wherein lysis is mediated by pH or enzymatically. The cell lysis buffer will lyse the cells but preserve the viral vector. Lysis buffer 2 contains a small volume of highly concentrated lysis agent for ease of use and flexibility with respect to the variable volume seen in the source bag. Examples of cell lysis buffers are typically detergent based to disrupt lipid bilayer cell membranes. The cell lysis buffer must be sufficiently aggressive to lyse the cells, but not interfere with antibody binding or protein interactions in downstream column separations. It is important that the cell lysis buffer is highly concentrated so that its final concentration is appropriate when mixed with the producer cell suspension.

The wash buffer 3 is preferably provided in the bag and contains a wash buffer to wash the cells, cell debris in the kit. The volume will be a sufficient number to remove all traces of lysis buffer 2.

The elution buffer 4 is preferably provided in a column elution buffer bag containing the elution buffer used to wash the column 8. The volume should be large enough to allow multiple elution steps from the column to maximize virus yield.

The container 5 is a Processing container or bag and should be about 0.5-1L, e.g., 850 mL, which corresponds to the Processing bag volume in the Xuri CellHarvester ® Processing Kit.

The container 6 is a waste container or bag and should be similar in volume to the current Xuri Cell Harvester ® Processing bags of up to 6L.

The container or bag 7 is a product bag for the purified viral vector. The volume should be sufficient to accommodate the entire volume of elution buffer 4.

The column 8 is a chromatographic column packed with a chromatographic medium or resin provided with antibodies or ligands specific for the desired viral vector to be isolated.

The posts 8 may be interchanged using any technique, such as GE Healthcare "ReadyMate" connector systems.

As shown in fig. 2, in the first step of the method, producer cells 1 and cell lysis buffer 2 are added to a processing bag 5. As shown in fig. 3, cells and buffer were circulated through the kit to ensure mixing and subsequent lysis occurred. After cell lysis, the mixture is pushed through the column 8 as shown in fig. 4. The outlet for this step is accomplished by a filter which retains any intact cells/cell debris within the processing bag 5. To ensure that all cells/debris are flushed out of the system, the wash buffer 3 is released into the processing bag 5, circulated through the system, and finally sprayed into the waste bag 6, as shown in fig. 5. After this, the elution buffer is released to column 8, pushing the viral vectors into the final product bag 7, as shown in fig. 6.

FIG. 7 is a more detailed view of a virus isolation kit used in the method of the present invention. The valve, in this case a pinch valve, is indicated by a triangular hourglass symbol, while the pump head is indicated by a circle with six rollers. All components of the kit are mounted to the cartridge, except for the input components (1-4) and the waste bag (6). Preferably, the cassettes are mounted within Xuri Cell Harvester described in WO2017/109071 (incorporated herein by reference), preferably by a track mechanism.

The instrument for use with the viral vector kit according to the invention is the same as described in WO 2017/109071. In operation, the instrument includes reusable mechanical elements, including pumps, pinch valves and weighing mechanisms, along with a removable and disposable low cost viral vector kit that includes all of the fluidic elements (e.g., fluidic pathways, buffer bags and columns) necessary for viral vector harvesting. The combination of these features results in a method for viral vector isolation wherein the selected viral vector kit is easily mounted on an instrument. No mechanical parts of the instrument will encounter the fluid, which means that no mechanical parts cleaning between different virus vector separation runs is required. This is particularly important for subsequent use in cell therapy applications where the easily achievable sterile operating conditions of the apparatus provide a much improved chance of therapeutic success, as well as reduced cost and turnaround time.

The viral vector isolation Kit shares several similarities with the schematic fluid pathway from Xuri Cell Harvester manufacturing Kit described in WO2017/109071, but there are several additions. That is, bags for wash buffer and elution buffer must be added to contain the wash buffer and elution solution for the method. Tubing connecting these bags to the main circulation of the kit was also added, feeding pinch valves and junction blocks, which selected which input bag could flow through the system without adding the pump heads currently installed in Xuri Cell Harvester. The chromatographic column (8) will replace the typical location of a hollow fiber filter cartridge.

Examples

Specific examples of the viral vector isolation procedure of the invention that fits within the CAR T cell therapy workflow consist of: by machines encoding viruses, e.g.gag/pol、revLentiviral plasmids of envelope genes, andtraditional stationary culture of human embryonic kidney 293T (HEK 293T) cells transfected with a transfer plasmid encoding the gene of interest, in this case a Chimeric Antigen Receptor (CAR). After about 72 hours, when the viral load within the cell culture medium has reached the appropriate concentration, the culture of producer cells is transferred from stationary culture to a source container, which may be a source bag that may be welded or luer connected to a cassette. This is different from the prior art, as conventional workflows typically require the user to transfer only the supernatant to harvest the virus particles. According to the invention, both producer cells and supernatant are used for harvesting. Other input bags, cell lysis buffer, wash buffer and column elution buffer were connected in a similar manner. The user then selects a column with a specific affinity for the viral vector to be harvested and aseptically attaches it to the cassette using a single-use sterile connector, such as a ReadyMate-system. When all the components were attached, the cartridges were inserted into Xuri Cell Harvester instruments and the protocol was selected.

First, a source bag containing producer cells and viral vectors is added to the processing bag. Once emptied, cell lysis buffer was added to the treatment bag. The fluids of the processing bags are then mixed by pushing the fluids of the processing bags through the circuits within the cassette. This step is optional, depending on the workflow required. The processing bag is then emptied into a chromatography column, wherein the anchored virus-specific ligand binds the virus and cell debris is passed to the waste bag. When the treatment bag volume is completely through the column, the column elution buffer then enters the cassette, passes through the treatment bag to the column, and elutes the virus particles into the final product bag.

The product is then introduced into an existing T cell culture to convert T cells into CAR T cells. Alternatively, the product can be resuspended with a fresh culture of producer cells to transfect the cells with the purified product, and the producer cells cultured with the intent of producing a more purified product, since the defective virus has been discarded from the column. This is consistent with a cGMP process that requires multiple purification steps. Once the viral vector is deemed sufficiently pure and stable as judged by mass determination, a new viral vector isolation kit can be employed to re-isolate the vector once or several times for transformation of T cells.

Current methodologies require harvesting of viral supernatant, a process that involves only removal of media from HEK-293T cell cultures. The virus particles still inside the cell are left behind at this step. The supernatant was then clarified using depth filtration and ultrafiltered with a hollow fiber cartridge. The viral vector specific column (8) is selected to have sufficiently significant affinity for the viral particles to reduce the process to one step. This in combination with cell lysis provides higher virus yields compared to current methods.