Methods for predicting protein expression stability
By measuring recloning success rate and protein expression variability through double single-cell cloning, the method predicts stable protein expression in clonal cell lines, reducing development time and enhancing biopharmaceutical production efficiency.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- CHITOSE LAB
- Filing Date
- 2024-11-21
- Publication Date
- 2026-06-02
AI Technical Summary
Existing methods for selecting clonal cell lines with stable protein expression require multiple subculturing cycles, which prolong the development time and are not suitable for subsequent development processes.
A method involving single-cell cloning twice on genetically modified animal cells to measure the recloning success rate and protein expression variability, allowing prediction of expression stability after multiple generations without subculturing.
Enables the selection of clonal cell lines with stable expression after more than 50 generations in a short period, improving productivity and reducing costs in biopharmaceutical production.
Smart Images
Figure 2026090147000001_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for predicting the expression stability of a target protein after subculture of genetically modified cells.
Background Art
[0002] In recent years, mainly for the purpose of manufacturing biopharmaceuticals, a large amount of desired proteins such as antibody pharmaceuticals have been manufactured. For this purpose, a gene expression vector encoding a recombinant protein is incorporated into animal cells, and the animal cells are made to produce the recombinant protein. These genetically modified animal cells are required to meet various criteria in manufacturing pharmaceutical raw materials. One of the criteria is the stability of the expression of the recombinant protein introduced into the animal cells.
[0003] This is because genetically modified animal cells for pharmaceutical raw materials are grown over several generations for mass production, and it is important that the expression level of the desired protein does not decrease during the growth period and the actual production process.
[0004] Therefore, when creating genetically modified animal cells for pharmaceutical raw materials, among the many clone cell lines obtained in the creation process, even in cells cultured up to the upper limit of the cell age assumed until pharmaceutical production, it is necessary to select a clone cell line in which the expression of the recombinant protein does not decrease.
[0005] In conventional expression stability evaluations, in order to confirm that the expression does not decrease even after culturing for 60 generations or more, the cells were actually subcultured over multiple generations to confirm the expression stability. However, with such a procedure, the period for creating genetically modified animal cells becomes longer by the period of subculture. Therefore, a technique for selecting a clone cell line with stable expression without performing subculture is required.
[0006] Japanese Patent Publication No. 2022-519237 describes a method for predicting the production stability and / or production instability of a clonal cell line, comprising the steps of: a) growing two or more clonal cell lines in separate cell cultures; b) performing karyotype analysis of the cells in each cell culture; and c) deriving a genomic instability value from the karyotype analysis in step (b). Using this method, it is possible to select cell lines whose expression does not decrease through subculturing without performing subculturing.
[0007] [Prior art documents] [Patent Documents]
[0008] [Patent Document 1] Japanese Patent Publication No. 2022-519237 [Overview of the Initiative] [Problems that the invention aims to solve]
[0009] However, in the method described in Japanese Patent Publication No. 2022-519237, if a cloned cell line is used for karyotype analysis of cells in each cell culture, it cannot be used in subsequent development processes, thus requiring two or more cell clones. Furthermore, the cells used for karyotype analysis must be subcultured for at least 10 generations.
[0010] Therefore, there is a continuing need for methods to select clonal cell lines with stable expression without performing such subculturing. The present invention aims to provide a method for predicting expression stability after multiple generations without performing multiple subculturing cycles. [Means for solving the problem]
[0011] To solve the above problems, the inventors performed single-cell cloning twice on genetically modified animal cells and focused on the variability in protein expression levels of the progeny cell lines after the second single-cell cloning. From these measurement results, the inventors found that the number of progeny cell lines produced and the variability in their protein expression levels correlated with the stability of the target protein expression after multiple generations. Based on these findings, they developed a method to predict the stability of the target protein expression after multiple generations.
[0012] In other words, according to the first aspect of the present invention, (a) A first clone cell line acquisition step, which includes the steps of: isolating one or more cells from animal cells genetically modified to express a target protein to obtain a first isolated cell; and growing the first isolated cell to obtain a clone cell line of the first isolated cell; (b) A success rate measurement step comprising the steps of: isolating one or more cells from a clone cell line of first isolated cells to obtain second isolated cells; growing the second isolated cells to obtain a clone cell line of second isolated cells; and measuring the recloning success rate of second isolated cells, wherein the recloning success rate of second isolated cells is a value relating to the ratio of the number of cells of second isolated cells to the number of cells of the clone cell line of second isolated cells. (c) An expression level-related information acquisition step, which is a step of measuring the expression level of the target protein in the second isolated cell and obtaining expression level-related information which is information regarding the expression level of the target protein, (d) An expression stability prediction step, which is a step of predicting the expression stability of the clone cell line of the second isolated cell using the recloning success rate of the second isolated cell and the expression level-related information, A method is provided that includes this.
[0013] In one embodiment, the animal cells may be CHO cells.
[0014] In addition, in one embodiment, the target protein may be any of a mouse antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
[0015] In one embodiment, the target protein is a humanized IgG.
[0016] In one embodiment, the target protein is a humanized IgG encoding trastuzumab.
[0017] In one embodiment, the expression level-related information is the coefficient of variation regarding the variation in the expression level of the target protein.
[0018] In one embodiment, Let X be the cloning success rate of the second isolated cell, When the coefficient of variation regarding the variation in the expression level of the target protein is Y, the cloned cell line of the second isolated cell is (1) When X is 20% or more and Y is 15% or less, (2) When X is 5% or less and Y is 20% or more, and (3) Other three stages, It may further include an analysis step of analyzing that it belongs to a class with higher expression stability in the order of (1), (2), and (3).
[0019] Also provided is a method for producing a target protein, including a step of predicting the expression stability of a cloned cell line of a second isolated cell by any of the above methods.
Effect of the Invention
[0020] According to the present invention, a method for predicting a cloned cell line that can still be stably expressed even after more than 50 generations of subculture is provided. By this method, it has become possible to select a strain whose expression does not decrease by subculture in a short period without performing subculture. As a result, in the production of biopharmaceuticals, effects such as improved productivity, cost reduction, and medical cost suppression can be obtained.
Brief Description of the Drawings
[0021] [Figure 1] Figure 1 is a graph showing the production culture results by the fed-batch culture method of cloned cells. [Figure 2] Figure 2 is a graph showing the measurement results of the antibody concentration of recloned cells by the batch culture method. [Figure 3] Figure 3 is a graph showing the production culture results of recloned cells by the fed-batch culture method. [Figure 4] Figure 4 is a graph showing the 15 subculture histories of recloned cells. [Figure 5] Figure 5 is a graph showing the production culture results after 15 subcultures of recloned cells by the fed-batch culture method.
Mode for Carrying Out the Invention
[0022] Hereinafter, the mode for carrying out the present invention will be described. The present invention is not limited to the modes described below, and includes those appropriately modified by those skilled in the art within the obvious range from the following modes.
[0023] (a) First cloned cell line acquisition step The first cloned cell line acquisition step includes a step of isolating one or more cells from a genetically modified animal cell for expressing a target protein to obtain a first isolated cell, and a step of growing the first isolated cell to obtain a cloned cell line of the first isolated cell.
[0024] In the step of obtaining the first isolated cell, one or more cells are isolated from the genetically modified animal cell.
[0025] Examples of animal cells include mammalian cells. These include mouse myeloma cells (NSO), baby hamster kidney cells (BHK), and Chinese hamster ovary cells (CHO). These animal cells are used for the production of recombinant therapeutic proteins. More than 80% of currently approved recombinant proteins are expressed on the CHO platform. CHO cell lines are known to be able to be cultured at high densities and readily incorporate exogenous DNA. Therefore, CHO cells can also be used in this invention.
[0026] The target protein can be any protein produced by a host cell. Examples of target proteins include mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. An example of a target protein is humanized IgG, specifically humanized IgG encoding trastuzumab.
[0027] Because the method of the present invention uses a polyclonal fluorescent (FITC)-labeled anti-human IgG antibody, it can, in principle, be used with humanized IgG antibodies other than those encoding trastuzumab. For example, it can be applied to cells expressing fully humanized antibodies or chimeric antibodies in which only the antigen-recognition portion is a mouse antibody and the rest is a human antibody.
[0028] Known methods can be used to modify the genes of the animal cells described above. For example, the nucleic acid sequence encoding the target protein is cloned into an expression vector, which is then introduced into the host animal cells. An expression vector refers to a vector containing a recombinant nucleic acid sequence that includes an expression regulatory sequence functionally linked to the nucleic acid sequence to be expressed. Expression vectors include plasmids, adenoviruses, adeno-associated viruses, viral vectors including retroviruses, cosmids, etc.
[0029] To introduce an expression vector into host cells, any suitable means may be used, including, for example, electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances, a microparticle gun, lipofection, and infection. In the present invention, electroporation is preferred, but is not limited thereto.
[0030] After introducing the expression vector, one or more cells are isolated. The following methods can be used to isolate the cells: First, doublet cells are removed from the cell population into which the expression vector has been introduced using flow cytometry or the like. Next, cells are fractionated using the forward scatter signal (FSC) area and BSC area by flow cytometry, and the cells are gated and isolated based on fluorescence intensity. In this way, the first isolated cells are obtained.
[0031] Next, the first isolated cells are used as seed cells and grown to obtain a clone cell line of the first isolated cells. To obtain a clone cell line, seed cells can be grown by single-cell cloning. Single-cell cloning refers to the creation of a genetically identical monoclonal population from an isolated single cell. Known cloning techniques for this purpose include limiting dilution, microfluidic devices, spotting, and flow cytometry, and any of these may be used. For example, using limiting dilution, one to several dozen of the first isolated cells can be seeded on a microplate covered with a special culture medium and grown to obtain a clone cell line.
[0032] The culture conditions for growing seeded cells, such as temperature, CO2 concentration, dissolved oxygen concentration, and pH, should be appropriately selected based on techniques conventionally used for culturing animal cells. For example, a culture medium containing Geneticin may be used, and the culture temperature may be 33-39°C or 36-37°C. The CO2 concentration may be 1-10% or 2-5%. Under these conditions, the plate can be left to stand for 10-20 days for cultivation.
[0033] (b) Success rate measurement process The success rate measurement step includes the steps of: isolating one or more cells from the clone cell line of the first isolated cells to obtain the second isolated cells; growing the second isolated cells to obtain the clone cell line of the second isolated cells; and measuring the recloning success rate of the second isolated cells. The recloning success rate of the second isolated cells is a value relating to the ratio of the number of cells in the second isolated cells to the number of cells in the clone cell line of the second isolated cells.
[0034] To isolate one or more cells from the clonal cell line of the first isolated cells, the same method as the method for obtaining the first isolated cells described above is used. For example, a second isolated cell can be obtained by using flow cytometry or the like.
[0035] The second isolated cells obtained are propagated to obtain a clonal cell line of the second isolated cells. Cell proliferation can be carried out using the same method as for the proliferation of the first isolated cells. For example, one to several dozen second isolated cells can be seeded and propagated in a microwell plate filled with a special medium using the limiting dilution method. For example, the cells can be cultured in a medium containing geneticin, under conditions of 30-40°C and a CO2 concentration of 3-7%, by allowing the plate to stand for 10-20 days.
[0036] Before obtaining the second isolated cells, the cells obtained from the cloned cell line of the first isolated cells may be further cultured. For example, the cells may be further cultured by adding culture medium or transferring the proliferated cells to a plate with more microwells. The cell line obtained by the expanded culture may be evaluated for IgG productivity using, for example, a fed-batch culture method, and cloned cells with higher productivity may be selected as the second isolated cells.
[0037] After culturing, a step is performed to measure the recloning success rate of the second isolated cells. To do this, the number of cells seeded in each microwell during the proliferation of the second isolated cells is recorded. After the cell culture period, the number of cells in the cloned cell line of the second isolated cells is measured in each microwell. Methods for measuring the cell count include accurately diluting with 0.25% trypan blue and measuring under a microscope, measuring under a microscope after fluorescent staining, using a flow cytometer, or using an image recognition device. The recloning success rate is calculated as (number of cells in the cloned cell line of the second isolated cells) / (number of seeded cells).
[0038] (c) Process for obtaining expression level-related information The expression level-related information acquisition step involves measuring the expression level of the target protein in the second isolated cell and obtaining expression level-related information, which is information regarding the expression level of the target protein.
[0039] When measuring the expression level of the target protein, the target protein may be isolated and / or purified beforehand. If the target protein is secreted into the culture medium, cells and other solid matter may be removed by centrifugation or filtration. If the target protein remains in the cells or is bound to the cell surface, the culture medium may be removed and the cells may be lysed by physical disruption with glass beads or exposure to high pH conditions. The target protein can be purified by chromatography, gel filtration, centrifugation, ethanol precipitation, etc.
[0040] There are no particular restrictions on the method for measuring the expression level of the target protein, and conventional methods can be used. For example, antibody reactions such as enzyme-labeled immunosorbent assay (ELISA), Western blotting, immunofluorescence staining, fluorescence-activated cell sorting (FACS), and multiplex assays using fluorescently labeled beads can be used, or high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), and turbidimetric methods can be used.
[0041] As expression level-related information, other measured or calculated values that directly or indirectly reflect the expression level of the target protein measured by the method described above can be used as expression level-related information. Of these, the expression level-related information is preferably the coefficient of variation relating to the variability of the expression level of the target protein.
[0042] More specifically, if the target protein is a specific antibody, the antibody concentration can be measured using a batch culture method, and the mean, standard deviation, and coefficient of variation of the antibody concentration can be calculated. In the batch culture method, the culture medium is not changed, and seeded cells are cultured in a closed system container such as a flask or bioreactor. After starting the culture, a fixed amount of culture medium sample is taken at regular intervals, and the antibody concentration is measured over several days. The antibody concentration can be measured using one of the methods described above for measuring the expression level of the target protein. Using this time-based antibody concentration plot, the mean, standard deviation, and coefficient of variation of the antibody concentration can be calculated.
[0043] Furthermore, before measuring the expression level of the target protein, the second isolated cell line (clonal cell line) may be further cultured. For example, the cells may be further cultured by adding culture medium or transferring the proliferated cells to a plate with a larger number of microwells.
[0044] (d) Expression stability prediction process The expression stability prediction step is a step in which the expression stability of the cloned cell line of the second isolated cell is predicted using the recloning success rate and expression level-related information of the second isolated cell.
[0045] This invention focuses on the fact that the number of cloned cell lines of the second isolated cells and the variability in the expression level of the target protein among those cell lines correlate with the stability of the target protein expression after subculturing of those cell lines. In other words, by using the recloning success rate and expression level information of the second isolated cells, it is possible to predict whether the expression level of the target protein will be stable after subculturing of the cloned cell lines of the second isolated cells. According to this invention, it is possible to select cloned cell lines in which the expression level of the target protein does not decrease even after subculturing, after performing two single-cell clonings. The expression stability prediction step may further include the following analytical steps. An example of this analytical step is to divide the expected expression stability into multiple classes and analyze which class each cell belongs to. An example of the analytical step is to determine the cloned cell line of the second isolated cell, where X is the recloning success rate of the second isolated cell and Y is the coefficient of variation for the variability of the expression level of the target protein. (1) X is 20% or more and Y is 15% or less, (2) X is 5% or less and Y is 20% or more, (3) Divide into the other three stages, (1), (2), and (3) are analyzed to belong to the rank of highest expression stability in that order. Focusing on the recloning success rate of the second isolated cells (X) and the coefficient of variation (Y) related to the variability of the expression level of the target protein, the higher the recloning success rate and the lower the variability of the expression level of the target protein, the higher the expression stability of the target protein. More specifically, if X is 20% or more, 30% or more, or 40% or more, and Y is 15% or less, 10% or less, or 5% or less (1), the recloning cell line belongs to the class with high expression stability. On the other hand, even if the recloning success rate is low, if the variability of the expression level of the target protein is high, the expression stability will be the next highest. If X is 5% or less, 4% or less, or 3% or less, and Y is 20% or more, 25% or more, or 30% or more (2), the recloning cell line belongs to the class with the next highest expression stability. If X and Y are outside these ranges (3), the recloning cell line belongs to the class with the third highest expression stability.
[0046] The expression stability of the second isolated cell clone line can be evaluated as follows: First, the productivity of the target protein in the second isolated cell clone line is evaluated using the fed-batch culture method (evaluation value A). Next, the second isolated cell clone line is subcultured for, for example, 15 generations, and the productivity of the target protein is evaluated using the same fed-batch culture method (evaluation value B). A higher value of evaluation value B / evaluation value A indicates higher expression stability.
[0047] In fed-batch culture, seeded cells are cultured while nutrients (glucose, amino acids, etc.) are added to the culture medium at regular intervals. Samples are taken at regular intervals during culture, and the concentration of the target protein is measured. Because nutrients are not depleted, cells can proliferate and produce antibodies over a long period of time.
[0048] The present invention may involve a computer performing the expression stability prediction process. In other words, the present invention also provides a method for predicting expression stability using a computer, and a computer-based device for predicting expression stability. Furthermore, the present invention also provides a program that causes a computer to execute a method for predicting expression stability, and a non-temporary information recording medium that stores such a program.
[0049] A computer has an input unit, an output unit, a control unit, an arithmetic unit, and a memory unit, and each element is connected by a bus or the like to enable the exchange of information. Computers usually handle digital information. For example, a computer may store programs or various kinds of information in its memory unit. When predetermined information is input from the input unit, the control unit reads the program stored in the memory unit. The control unit then reads the information stored in the memory unit as appropriate and transmits it to the arithmetic unit. The control unit also transmits the input information to the arithmetic unit as appropriate. The arithmetic unit performs calculations using the received information and stores it in the memory unit. The control unit reads the calculation results stored in the memory unit and outputs them from the output unit. In this way, various processes and steps are executed. Each unit and each means is responsible for executing these various processes. A computer may have a processor, and the processor may implement various functions and steps. A computer may be standalone. A computer may have some of its functions distributed between a server and terminals. In that case, it is preferable that the server and terminals can exchange information via a network such as the internet or an intranet. A computer may include a processor and memory connected to the processor. The memory may store instructions, and when executed by the processor, these instructions may cause the computer to perform various processes and function as various components. The computer may build a learning model by providing various training data and perform various calculations through machine learning. In this case, the computer may perform various analyses and interpretations using the learning model created by AI (artificial intelligence) machine learning and deep learning.
[0050] The present invention also provides a method for producing a target protein, which includes a step of predicting the expression stability of a second isolated cell clonal cell line.
[0051] [Examples]
[0052] The present invention will be described in detail below with reference to examples.
[0053] [Example 1] The sequence of a humanized IgG antibody encoding trastuzumab was incorporated into an expression vector containing a drug (geneticin) resistance cassette, and then the vector was introduced into host CHO cells by electroporation.
[0054] The day after vector introduction, the cells were stained with a fluorescently labeled (FITC) anti-human IgG antibody and prepared as samples for single-cell cloning. Single-cell cloning was performed using a Sony cell sorter.
[0055] The sample gating procedure was as follows: First, doublet cells were removed to obtain single cells. Furthermore, the single cells were fractionated based on the FSC area and BSC area signals. Cells from these fractions were gated based on fluorescence intensity to obtain seeded cells for single-cell cloning.
[0056] Seeded cells were seeded in 96-well culture plates lined with an isolation medium containing geneticin, with several tens of cells per well. The seeded cells were cultured for 14 days under static conditions at 37°C and 5% CO2 concentration. Cells were harvested from wells where cell proliferation was confirmed, and after expansion culture, they were evaluated using a 10-day fed-batch culture method to select six cloned cells with high productivity (IgG production) (Figure 1).
[0057] To re-cultivate the six types of cloned cells into single cells, single-cell seeding was performed in 96-well culture plates after confirming that one cell was seeded in each well with a probability of over 90%.
[0058] Seeded cells were seeded one cell per well in a 96-well culture plate lined with isolation medium. The seeded cells were cultured for 14 days under static conditions at 37°C and 5% CO2 concentration. After static culture, cells were harvested from wells where cell proliferation was confirmed and were converted into recloned cells. Monoclonality was ensured for the recloned cells by recording the cell division process from a single cell using a high-resolution cell imaging device (Solentim Cell Metric CLD). The ratio of recloned cells obtained from each cloned cell to the seeded cells is shown in Table 1 as the recloning success rate.
[0059] The licloned cells were subsequently cultured in a larger culture medium and divided into two cell groups. In the first cell group, the antibody concentration in the culture medium was measured using Protein A affinity HPLC after a 5-day batch culture. Figure 2 shows the results of the antibody concentration measurements for each licloned cell group using the batch culture method. Table 1 shows the mean, standard deviation, and coefficient of variation of the antibody concentration for each licloned cell group calculated from the batch culture results.
[0060] [Table 1]
[0061] From the second cell group, recloned cells showing the highest value for each clonal cell group were selected based on the batch culture results. These cells underwent further expansion culture to create frozen stocks, and their productivity was evaluated using the fed-batch culture method, followed by 15 subculturing cycles. Figure 3 shows the productivity evaluation of the selected recloned cells using the fed-batch culture method (A). Figure 4 shows the history of viable cell density after 15 subculturing cycles of the selected recloned cells, and Figure 5 shows the productivity evaluation of these subculted cells using the fed-batch culture method after 15 subculturing cycles (B). In summary, the value obtained by dividing the maximum value of productivity evaluation (B) by the maximum value of productivity evaluation (A) is shown in Table 2 as the subculturing stability.
[0062] [Table 2]
[0063] Here, using the probability of recloning success (X) and coefficient of variation (Y), we predicted the subculturing stability to be 70% or higher when X was 20% or more and Y was 15% or less, less than 40% when X was 5% or less and Y was 20% or more, and less than 70% or more and 40% or more otherwise. The results matched the actual subculturing stability. Therefore, the classification in the analytical process is considered appropriate. Furthermore, this classification is considered to be generally valid, not limited to the specific examples in the examples. [Industrial applicability]
[0064] The method of the present invention is useful for the production of biopharmaceuticals.
Claims
1. (a) A first clone cell line acquisition step, which includes the steps of: isolating one or more cells from animal cells genetically modified to express a target protein to obtain a first isolated cell; and growing the first isolated cell to obtain a clone cell line of the first isolated cell; (b) A success rate measurement step comprising the steps of: isolating one or more cells from a clone cell line of first isolated cells to obtain second isolated cells; growing the second isolated cells to obtain a clone cell line of second isolated cells; and measuring the recloning success rate of second isolated cells, wherein the recloning success rate of second isolated cells is a value relating to the ratio of the number of cells of second isolated cells to the number of cells of the clone cell line of second isolated cells. (c) An expression level-related information acquisition step, which is a step of measuring the expression level of the target protein in the second isolated cell and obtaining expression level-related information which is information regarding the expression level of the target protein, (d) An expression stability prediction step, which is a step of predicting the expression stability of the clone cell line of the second isolated cell using the recloning success rate of the second isolated cell and the expression level-related information, Methods that include...
2. The method according to claim 1, wherein the animal cells are CHO cells.
3. A method according to claim 1, wherein the target protein is one of a mouse antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
4. A method according to claim 1, wherein the target protein is humanized IgG.
5. A method according to claim 1, wherein the target protein is a humanized IgG encoding trastuzumab.
6. A method according to claim 1, wherein the expression level-related information is the coefficient of variation relating to the variability of the expression level of the target protein.
7. The method according to claim 6, Let X be the success rate of recloning the second isolated cell. When Y is the coefficient of variation for the variability in the expression level of the target protein, the clonal cell line of the second isolated cell is, (1) X is 20% or more and Y is 15% or less, (2) X is 5% or less and Y is 20% or more, (3) Divide into the other three stages, A method further comprising an analytical step of analyzing which of the following classes has the highest expression stability in the order of (1), (2), and (3).
8. A method for producing a target protein, comprising the step of predicting the expression stability of a second isolated cell clonal cell line by any one of claims 1 to 7.