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WO2018173463A1 - Procédé d'évaluation et programme d'évaluation d'un dispositif de culture de cellules - Google Patents

Procédé d'évaluation et programme d'évaluation d'un dispositif de culture de cellules Download PDF

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Publication number
WO2018173463A1
WO2018173463A1 PCT/JP2018/002063 JP2018002063W WO2018173463A1 WO 2018173463 A1 WO2018173463 A1 WO 2018173463A1 JP 2018002063 W JP2018002063 W JP 2018002063W WO 2018173463 A1 WO2018173463 A1 WO 2018173463A1
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Prior art keywords
cell culture
function
cells
evaluation
evaluating
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English (en)
Japanese (ja)
Inventor
憲一郎 岡
聖 村上
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Hitachi Ltd
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Hitachi Ltd
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  • the present invention relates to a cell culture device evaluation method and an evaluation program.
  • proteins including antibodies used as pharmaceuticals are produced using genetically modified animal cells. Since proteins depend only on the base sequence of a gene until the primary structure, they can be produced by microorganisms, but many proteins such as antibodies are modified with a sugar chain after polypeptide synthesis. Many of these proteins do not show physiological activity only with their primary structure, and it is difficult to completely synthesize them with microorganisms, and it is necessary to use animal cells with functions of secondary and tertiary structure construction and sugar chain modification. .
  • a large cell culture apparatus requires a sufficient amount of aeration and agitation in order to supply oxygen absorbed by the cells and remove carbon dioxide exhaled by the cells.
  • aeration and agitation in order to supply oxygen absorbed by the cells and remove carbon dioxide exhaled by the cells.
  • fluid shear stress and bubble aeration caused by excessive stirring damage cells and kill the cells. Technologies corresponding to this are described in Patent Document 1 and Non-Patent Document 1.
  • Patent Document 1 describes a method for evaluating cultured cells when performing monolayer culture of adhesion-dependent cells in a culture vessel.
  • Deriving cell-specific parameters in at least one stage selected from the four stages of the logarithmic growth phase until reaching the confluent state and the stationary phase after becoming the confluent state It is described that the cell monolayer culture process is quantitatively evaluated.
  • Non-Patent Document 1 describes a numerical simulation technique of a cell culture tank for the purpose of scale-up and optimization design of an animal cell culture tank.
  • FIG. 7 is explanatory drawing explaining the conventional numerical simulation technique regarding a small cell culture apparatus.
  • FIG. 8 is an explanatory diagram for explaining a conventional numerical simulation technique related to a large cell culture apparatus.
  • Non-Patent Document 1 in any of the small cell culture tank and the large cell culture tank, cell death 10 caused by fluid shear stress is used, using the numerical simulation technique described above. It is described that the design of the cell culture tank is optimized by finding the operable region 100 surrounded by the mixing performance 11 and the gas exchange performance 12.
  • a small cell culture apparatus of 1mL to 100L is prepared in advance, and the results of cell culture in the small cell culture apparatus are obtained. Based on this, the operating conditions are evaluated. If the technique described in Patent Document 1 or the technique described in Non-Patent Document 1 is applied, the cells can be more reliably grown.
  • Patent Document 1 can only consider the cell growth rate. Further, in Non-Patent Document 1, as physical quantities to be taken as evaluation indices, cell killing due to fluid shear stress 10, mixing performance 11, and gas exchange performance 12 are listed. It is a discrete time-dependent change in physical quantity measured at each time interval.
  • the phenomenon of cell proliferation can be explained or evaluated by discrete time-dependent changes in cell growth rate and physical quantity, but it relates to the internal state of cells such as gene expression of cells. It was newly found that the phenomenon cannot be explained. In other words, it has been found that with the conventional technology, there is a case where a target product such as a protein is not produced even if the cell grows.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide an evaluation method and an evaluation program for a cell culture apparatus that can evaluate with high accuracy whether or not a target product is produced by the cell culture apparatus.
  • the present inventor has found that the accumulated value of the physical quantity received by the cell, or the time change of the time average value of the accumulated value can be calculated from the transport equation using the physical quantity as the source term. . Then, based on the result calculated by the transport equation, calculate the cumulative value of the physical quantity that the cell receives, or the time change of the time average value of the cumulative value, and use it as one of the influencing factors on the cells in the cell culture device.
  • the present invention has been made based on these findings.
  • the method for evaluating a cell culture device that has solved the above problems is a method for evaluating a cell culture device that performs cell culture, and independently determines one or more physical quantities that cells in the cell culture device receive during culture.
  • an evaluation step for evaluating the cell culture device in this order is a method for evaluating a cell culture device that performs cell culture, and independently determines one or more physical quantities that cells in the cell culture device receive during culture.
  • An evaluation program for a cell culture device is a program for evaluating a cell culture device that performs cell culture, and the computer independently determines one or more physical quantities that the cells in the cell culture device receive during culture.
  • Definition means for defining a function as a variable
  • first calculation means for calculating a time change of the cumulative integral distribution of the function with respect to a group of the cells using a transport equation, and a time change of the cumulative integral distribution of the function as an integration time
  • a second calculating means for calculating a temporal change in the cumulative average distribution of the function by dividing the time lapse of the cumulative average distribution of the function as one of influencing factors on the cells in the cell culture apparatus. It functions as an evaluation means for evaluating the apparatus.
  • the horizontal axis represents the number of stirring rotations
  • the vertical axis represents the amount of ventilation.
  • the horizontal axis represents the number of stirring rotations
  • the vertical axis represents the amount of ventilation.
  • the horizontal axis represents the number of stirring rotations
  • the vertical axis represents the amount of ventilation.
  • FIG. 1 is a flowchart for explaining an embodiment of a method for evaluating a cell culture device of the present invention (hereinafter referred to as “the present evaluation method”).
  • This evaluation method is a method for evaluating a cell culture apparatus that performs cell culture.
  • “Evaluating the cell culture device” means evaluating the operating conditions such as the shape of the cell culture device (cell culture tank), the shape of the stirring blade, the rotational speed of the stirring blade, the air flow rate, and pH. is there. As shown in FIG. 1, this evaluation method includes a definition step S1, a first calculation step S2, a second calculation step S3, and an evaluation step S4 in this order, and each step is performed in this order. .
  • target products examples include substances that are main raw materials such as pharmaceuticals, for example, proteins such as antibodies and enzymes, and physiologically active substances such as low molecular compounds and high molecular compounds. Can be, but is not limited to.
  • Examples of cells to be cultured include animal cells, plant cells, insect cells, bacteria, yeasts, fungi and algae, but are not limited thereto.
  • the cell culture device according to the present embodiment can target animal cells that produce proteins such as antibodies and enzymes as suitable culture targets.
  • the defining step S1 defines a function having one or a plurality of physical quantities received by the cells in the cell culture apparatus during culture as independent variables.
  • physical quantities include fluid shear stress, flow velocity, kinematic viscosity, turbulent energy, turbulent energy dissipation rate, Kolmogorov scale, oxygen concentration, carbon dioxide concentration, component fraction of medium components, pH, temperature, and the like.
  • the first calculation step S2 calculates a time change of the cumulative integral distribution of the function for a group of cells (cell group) using a transport equation. That is, in the first calculation step S2, the accumulated value of the physical quantity received by the cell is calculated by the transport equation.
  • the transport equation For example, when considering the fluid shear stress as the physical quantity, without calculating the fluid shear stress cumulative value of each cell directly, how much of the cells having a specific fluid shear stress cumulative value is It is possible to know the existence ratio, that is, whether or not the ratio exists.
  • the general transport equation takes the form of the following formula (2), and there is a diffusion coefficient D representing molecular diffusion.
  • D the diffusion coefficient
  • weight function for example, a sigmoid function (the following formulas (6) and (7)) and a heavy side function (the following formulas (8) and (9)) can be preferably applied.
  • a small cell culture apparatus is prepared in advance, and the operating conditions are based on the results of cell culture in the small cell culture apparatus. Is evaluated. That is, an operable region where cells can be cultured is investigated for various evaluation indexes in a small cell culture apparatus.
  • FIG. 7 in the prior art, as an evaluation index as to whether or not the cell culture apparatus has an appropriate capability for cell culture, cell death 10 due to fluid shear stress, mixing performance 11, Three gas exchange performances 12 are listed.
  • the operation variables of the cell culture device are two types of stirring rotation speed and aeration amount.
  • the upper limit value of the operable region 100 is represented for the cell death 10 due to the fluid shear stress
  • the lower limit value of the operable region 100 is represented for the mixing performance 11 and the gas exchange performance 12.
  • an operable region 100 of the culture apparatus is determined and investigated.
  • the operable region 100 of the large cell culture apparatus for actual production shown in FIG. 8 corresponds to the operable region 100 of the small cell culture apparatus of FIG. 7, and similar cell culture results can be obtained with apparatuses having different capacities. It is done.
  • examples of the cell culture result include physical quantities such as the number of living cells.
  • FIG. 2 is a block diagram for explaining an embodiment of the evaluation program for the cell culture device of the present invention (hereinafter referred to as “the evaluation program”).
  • This evaluation program is a program for evaluating a cell culture apparatus that performs cell culture.
  • this evaluation program uses the computer 5 as the definition means 1, the first calculation means 2, the second calculation means 3, and the evaluation means 4 in order to practice the evaluation method described above. Make it work.
  • the definition means 1, the first calculation means 2, the second calculation means 3, and the evaluation means 4 in the evaluation program are respectively defined in the definition step S1 and the first calculation step S2 of the evaluation method described with reference to FIG. Since this corresponds to the second calculation step S3 and the evaluation step S4, a detailed description of the significance, operation, action, etc. of each means is omitted.
  • the evaluation program is recorded on a computer-readable recording medium (not shown) such as a CD-ROM or a flexible disk, and the evaluation program is read from the recording medium by a recording medium driving device (not shown), and a hard disk (not shown). It can be installed and executed in a storage means such as a drive.
  • a computer-readable recording medium such as a CD-ROM or a flexible disk
  • a recording medium driving device not shown
  • a hard disk not shown
  • the evaluation program is stored in another computer (server) connected via the communication network, and the book is transmitted from the computer via the communication network.
  • the evaluation program can be downloaded and executed, or the evaluation program stored in the server can be executed.
  • the computer 5 when the computer 5 is caused to function by this evaluation program, it can be evaluated with high accuracy whether the cell culture apparatus to be evaluated can produce the target product.
  • the result of numerical analysis may be stored in storage means (not shown) provided in the server.
  • cells were cultured using a cell culture device, and the number of living cells and protein in a predetermined culture period were measured.
  • CRL-1606 cells purchased from American Type Culture Collection, which are mouse-mouse hybridomas, were used. These cells are floating cells that secrete anti-fibronectin antibodies.
  • FBS Fetal bovine serum
  • IMDM medium Iscove's Modified Dulbecco's Medium
  • a glass cylindrical culture tank having an inner diameter of 150 mm and a culture volume of 5 L was used.
  • the cell culture apparatus is connected to a rubber heater for heating, a magnet-driven stirring blade, a temperature measurement electrode, a pH electrode, a dissolved oxygen (DO) electrode, and a control device that measures and adjusts these.
  • a sintered metal sparger with an average pore diameter of 100 ⁇ m was incorporated for aeration in the liquid.
  • the pH of the culture solution was automatically adjusted by increasing / decreasing the carbon dioxide concentration in the mixed gas (air, nitrogen, oxygen, carbon dioxide) supplied to the gas phase part of the cell culture apparatus.
  • the temperature of the culture solution was adjusted to 37 ° C.
  • the DO concentration was adjusted by increasing or decreasing the oxygen partial pressure of the gas supplied to the gas phase portion and the liquid.
  • the rotation speed of the stirring blade was adjusted at 100 rpm by adjusting the output of the drive motor directly connected to the drive shaft to which the stirring blade was attached.
  • the culture solution in the cell culture apparatus was aseptically sampled at a frequency of 2 to 3 times a day from the start of the culture, and the number of viable cells was measured.
  • the number of viable cells was counted by image processing using a live cell counter BioProfile 100Plus manufactured by Beckman Coulter, which was distinguished from dead cells stained with trypan blue.
  • the sampling solution was subjected to two-dimensional electrophoresis to separate the total protein in the cell into thousands or more spots.
  • the protein in each spot was decomposed with trypsin and analyzed by mass spectrometry to obtain peptide mass and partial sequence information, and the intracellular protein was identified by peptide mass fingerprinting.
  • the physical quantity is a fluid shear stress
  • the cumulative average fluid shear stress is obtained as a scalar value calculated from the fluid shear stress by the above formula.
  • FIG. 7 and FIG. 5 and FIG. 8 and FIG. 6 are compared, respectively, in the operable region 100 of the culture apparatus based on three evaluation indices consisting of cell killing due to fluid shear stress 10, mixing performance 11 and gas exchange performance In the number of living cells, the same result could be obtained.
  • FIGS. 5 and 6 there is an operable region 200 for obtaining the same result regarding protein expression, which is narrower than the operable region 100 shown in FIGS. 7 and 8. This is because, regarding protein expression, in addition to three evaluation indexes consisting of cell killing due to fluid shear stress 10, mixing performance 11 and gas exchange performance 12, cumulative average fluid shear stress must be considered as an evaluation index. Is shown.
  • the cell culture device evaluation method and evaluation program according to the present invention have been described in detail according to the embodiments and examples.
  • the gist of the present invention is not limited thereto, and various modifications are included.
  • the above-described embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to one having all the configurations described.
  • a part of the configuration of an embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of an embodiment.
  • Each of the above-described configurations, functions, processing units, processing means, control means, and the like may be realized by hardware by designing a part or all of them, for example, with an integrated circuit.
  • Each of the above-described configurations, functions, and the like may be realized by software by interpreting and executing a program that realizes each function by the processor.
  • Information such as programs, tables, and files that realize each function can be stored in a recording device such as a memory, a hard disk, or an SSD (Solid State Drive), or a recording medium such as an IC card, an SD card, or a DVD.
  • the control lines and information lines indicate what is considered necessary for the explanation, and not all the control lines and information lines on the product are necessarily shown. Actually, it may be considered that almost all the components are connected to each other.

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Abstract

La présente invention concerne un procédé d'évaluation et un programme d'évaluation d'un dispositif de culture de cellules aptes à évaluer, avec une précision élevée, si un produit cible a été produit par un dispositif de culture de cellules. Le procédé d'évaluation du dispositif de culture de cellules selon la présente invention est un procédé d'évaluation d'un dispositif de culture de cellules qui effectue la culture de cellules, comprenant, dans cet ordre : une étape de définition (S1) consistant à définir une fonction qui utilise comme variables indépendantes une ou une pluralité de quantités physiques reçues durant la culture par les cellules à l'intérieur du dispositif de culture de cellules; une première étape de calcul (S2) consistant à utiliser une équation de transport afin de calculer le changement temporel dans la distribution de l'intégrale cumulée de la fonction pour les cellules dans un groupe; une seconde étape de calcul (S3) consistant à diviser le changement temporel dans la distribution de l'intégrale cumulée de la fonction par le temps d'intégration afin de calculer le changement de temps dans la distribution de la moyenne cumulée de la fonction; et une étape d'évaluation (S4) consistant à évaluer le dispositif de culture de cellules en utilisant le changement temporel dans la distribution de la moyenne cumulée de la fonction comme facteur affectant les cellules dans le dispositif de culture de cellules.
PCT/JP2018/002063 2017-03-24 2018-01-24 Procédé d'évaluation et programme d'évaluation d'un dispositif de culture de cellules Ceased WO2018173463A1 (fr)

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CN117932382A (zh) * 2024-01-26 2024-04-26 深圳市勃新生物科技有限公司 一种环境可控的新型细胞培养方法

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JP2024146482A (ja) * 2023-03-31 2024-10-15 株式会社日立プラントサービス 培養スケールアップ用培養データ収集装置および培養スケールアップ用培養データ収集方法

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JP2006296423A (ja) * 2005-03-24 2006-11-02 Hitachi Ltd 培養槽の制御装置及び培養装置

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JP4883067B2 (ja) * 2008-09-29 2012-02-22 株式会社日立プラントテクノロジー 培養装置及び培養方法

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JP2006296423A (ja) * 2005-03-24 2006-11-02 Hitachi Ltd 培養槽の制御装置及び培養装置

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117932382A (zh) * 2024-01-26 2024-04-26 深圳市勃新生物科技有限公司 一种环境可控的新型细胞培养方法

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