JP5810478B2 - CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD - Google Patents

CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD Download PDF

Info

Publication number
JP5810478B2
JP5810478B2 JP2010015844A JP2010015844A JP5810478B2 JP 5810478 B2 JP5810478 B2 JP 5810478B2 JP 2010015844 A JP2010015844 A JP 2010015844A JP 2010015844 A JP2010015844 A JP 2010015844A JP 5810478 B2 JP5810478 B2 JP 5810478B2
Authority
JP
Japan
Prior art keywords
culture
virus
concentration
tank
carbon dioxide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
JP2010015844A
Other languages
Japanese (ja)
Other versions
JP2011152078A (en
Inventor
和夫 菅谷
和夫 菅谷
誉人 水沼
誉人 水沼
岡本　智之
智之 岡本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IHI Corp
Original Assignee
IHI Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP2010015844A priority Critical patent/JP5810478B2/en
Application filed by IHI Corp filed Critical IHI Corp
Priority to CN2011800070761A priority patent/CN102712891A/en
Priority to PCT/JP2011/050828 priority patent/WO2011093180A1/en
Priority to SG2012055422A priority patent/SG182744A1/en
Priority to KR1020147013968A priority patent/KR101511029B1/en
Priority to KR1020127019064A priority patent/KR101436134B1/en
Priority to TW100102580A priority patent/TWI429743B/en
Publication of JP2011152078A publication Critical patent/JP2011152078A/en
Application granted granted Critical
Publication of JP5810478B2 publication Critical patent/JP5810478B2/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00051—Methods of production or purification of viral material

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

本発明は、培養情報生成装置及び培養装置並びに培養方法に関する。   The present invention relates to a culture information generation device, a culture device, and a culture method.

ウイルスを用いて目的とする蛋白質等を生産する場合、特定の蛋白質を生成するための遺伝子を組み込んだウイルスを動物細胞等に播種して培養し、培養後のウイルスから上記蛋白質を分離することが行われている。また、ウイルスを用いてワクチンを生産する場合には、ウイルス自体が生産対象であることもあり、適度に培養した動物細胞等にウイルスを播種して増殖させることが行われている。そして、このようなウイルスの培養では、培養状態を評価するための手法として、ウイルス含有培養液をサンプリングしてウイルス数を計数する方法（タイター測定）や顕微鏡観察によってサンプル中の細胞の状態を確認することが一般的に行われている。下記特許文献１及び非特許文献１〜３には、このようなウイルスの培養技術の一例が開示されている。   When producing a target protein using a virus, it is possible to disseminate and culture a virus in which a gene for producing a specific protein is incorporated into an animal cell, etc., and to isolate the protein from the cultured virus. Has been done. In addition, when a vaccine is produced using a virus, the virus itself may be a production target, and the virus is seeded and propagated in appropriately cultured animal cells or the like. In such virus culture, as a method for evaluating the culture state, the state of cells in the sample is confirmed by sampling the virus-containing culture solution and counting the number of viruses (titer measurement) or by microscopic observation. It is generally done. The following Patent Document 1 and Non-Patent Documents 1 to 3 disclose examples of such virus culturing techniques.

特開２００２−１４８２５８号公報JP 2002-148258 A

「バキュロウイルス-昆虫細胞発現系を用いた蛋白質の大量生産」蛋白質科学会愛カーブ＃022"Mass production of protein using baculovirus-insect cell expression system" Protein Science Society Ai Curve # 022 「細胞培養におけるカイコ核多角体病ウイルスの増殖」養蚕昆虫研報 ７号；9-29（1993）“Propagation of silkworm nucleopolyhedrovirus in cell culture”, Sericultural insect research bulletin 7; 9-29 (1993) 「Expresion and purification of an infuluenza hemagglutinin - one step closer to a recombinant protein-based influenza vaccine」 Vaccine 24 P2176(2006)`` Expresion and purification of an infuluenza hemagglutinin-one step closer to a recombinant protein-based influenza vaccine '' Vaccine 24 P2176 (2006)

ところで、上記従来の培養状態の評価技術では、培養対象がウイルスであるために無菌及び閉鎖系での操作によるサンプリング操作が必須であり、よって当該サンプリング操作が煩雑であると共に、培養槽等が雑菌によって汚染される虞もある。また、タイター測定には数日を要すること、また測定操作が難しいために測定者によっては誤差が大きくなるので、タイムリーな測定かつ測定精度の確保が困難である。さらに、顕微鏡を用いる評価では観察者の個人差が生じ易く、よって客観性を担保した評価ができないという問題もある。   By the way, in the conventional culture state evaluation technique, since the culture target is a virus, a sampling operation by an operation in a sterile and closed system is essential. Therefore, the sampling operation is complicated, and the culture tank or the like is a germ. There is also a risk of contamination. In addition, it takes several days for the titer measurement, and the measurement operation is difficult, so that an error increases depending on the measurer. Therefore, it is difficult to ensure timely measurement and measurement accuracy. Furthermore, in the evaluation using a microscope, there is a problem that individual differences among observers are likely to occur, so that evaluation with guaranteed objectivity cannot be performed.

本発明は、上述した事情に鑑みてなされたものであり、以下の点を目的とするものである。
（１）ウイルスの培養状態をタイムリーに評価する。
（２）従来よりも客観性を担保し得るウイルスの培養状態の評価手法を提供する。
（３）培養槽が雑菌で汚染されることを防止する。 The present invention has been made in view of the above-described circumstances, and has the following objects.
(1) Evaluate the culture state of the virus in a timely manner.
(2) To provide a method for evaluating a virus culture state that can ensure objectivity as compared with the prior art.
(3) Prevent the culture tank from being contaminated with various bacteria.

上記目的を達成するために、本発明では、培養情報生成装置に係る第１の解決手段として、ウイルス含有培養液を保有する培養槽から排気されるガス中の対象成分の濃度変化を示す測定信号を入力とし、当該測定信号に所定の情報処理を施すことによりウイルスの培養状態を示す培養情報を生成する、という手段を採用する。   In order to achieve the above object, in the present invention, as a first solving means related to the culture information generating apparatus, a measurement signal indicating a change in concentration of a target component in a gas exhausted from a culture tank holding a virus-containing culture solution Is used as input, and culture information indicating the culture state of the virus is generated by performing predetermined information processing on the measurement signal.

培養情報生成装置に係る第２の解決手段として、上記第１の解決手段において、培養槽内でウイルスの栄養となる所定の細胞にウイルスを播種して培養する場合には、ガスの発生濃度の予め決定された回数の極大値の発生を示す培養情報を生成する、という手段を採用する。   As a second solving means relating to the culture information generating apparatus, in the first solving means, when the seed cells are seeded and cultured in the culture tank and become a nutrient of the virus, A means of generating culture information indicating the occurrence of a maximum number of times determined in advance is adopted.

培養情報生成装置に係る第３の解決手段として、上記第１または第２の解決手段において、ガスは二酸化炭素（ＣＯ２）である、という手段を採用する。 As a third solving means relating to the culture information generating apparatus, a means is adopted in which the gas is carbon dioxide (CO 2 ) in the first or second solving means.

また、本発明では、培養装置に係る第１の解決手段として、ウイルスを培養する培養槽と、該培養槽から排出される排気ガスを分析・測定し、測定結果を示す測定信号を出力する排気ガス分析装置と、上記第１〜第３のいずれかの培養情報生成装置とを具備する、という手段を採用する。   Further, in the present invention, as a first solving means related to the culture apparatus, a culture tank for culturing viruses, and an exhaust gas for analyzing and measuring exhaust gas discharged from the culture tank and outputting a measurement signal indicating the measurement result A means is provided that includes a gas analyzer and any one of the first to third culture information generation apparatuses.

培養装置に係る第２の解決手段として、上記第１の解決手段において、培養槽は培養情報に基づいてウイルスの培養を終了する、という手段を採用する。   As the second solving means relating to the culture apparatus, in the first solving means, a means is adopted in which the culture tank ends the virus culture based on the culture information.

また、本発明では、培養方法に係る第１の解決手段として、ウイルスの培養で発生したガス中の対象成分の濃度変化に基づいてウイルスの培養を終了する、という手段を採用する。   Moreover, in this invention, the 1st solution means which concerns on the culture | cultivation method employ | adopts the means of ending the culture | cultivation of a virus based on the density | concentration change of the target component in the gas which generate | occur | produced by the culture | cultivation of a virus.

培養方法に係る第２の解決手段として、上記第１の解決手段において、ウイルスの栄養となる所定の細胞にウイルスを播種して培養する場合には、ガスの発生濃度の予め決定された回数の極大値の発生をもってウイルスの培養を終了する、という手段を採用する。   As a second solution relating to the culture method, in the first solution, in the case where the virus is seeded and cultured in a predetermined cell serving as a virus nutrient, a predetermined number of gas generation concentrations are determined. A method is adopted in which the virus culture is terminated when the maximum value is generated.

培養方法に係る第３の解決手段として、上記第１または第２の解決手段において、ガスは二酸化炭素（ＣＯ２）である、という手段を採用する。 As a third solving means relating to the culture method, a means is adopted in which the gas is carbon dioxide (CO 2 ) in the first or second solving means.

本発明によれば、培養槽から排出される排気ガスに基づいて培養槽の培養状態をオンライン状態で自動的に検知することができるので、ウイルスの培養状態をタイムリーに評価することが可能であると共に、従来よりも客観性を担保し得るウイルスの培養状態の評価手法を提供することができ、また培養槽が雑菌で汚染されることを防止することができる。   According to the present invention, since the culture state of the culture tank can be automatically detected on-line based on the exhaust gas discharged from the culture tank, the virus culture state can be evaluated in a timely manner. In addition, it is possible to provide a method for evaluating the culture state of a virus that can ensure objectivity more than before, and to prevent the culture tank from being contaminated with various bacteria.

本発明の一実施形態に係るウイルス培養装置Ａの構成を示すブロック図である。It is a block diagram which shows the structure of the virus culture apparatus A which concerns on one Embodiment of this invention. 本発明の一実施形態に係るウイルス培養装置Ａの要部動作を示すフローチャートである。It is a flowchart which shows the principal part operation | movement of the virus culture apparatus A which concerns on one Embodiment of this invention. 本発明の一実施形態において、培養槽１から排出される二酸化炭素（ＣＯ２）及び酸素（Ｏ２）の濃度変化を示す測定結果である。In one embodiment of the present invention, a measured result indicating a change in concentration of carbon dioxide discharged from the culture tank 1 (CO 2) and oxygen (O 2). 本発明の一実施形態において、培養時間による生産物濃度比の変化を示す特性図である。In one Embodiment of this invention, it is a characteristic view which shows the change of the product concentration ratio by culture | cultivation time.

以下、図面を参照して、本発明の一実施形態について説明する。
図１に示すように、本実施形態に係るウイルス培養装置Ａは、培養槽１、ウイルス供給装置２、排気ガス分析装置３及び培養情報生成装置（培養状態情報生成装置）４から構成されている。このようなウイルス培養装置Ａは、所定の目的蛋白質を生成するための遺伝子を組み込んだウイルスを昆虫細胞に播種して培養することにより、上記目的蛋白質を生産する、またはウイルスそのものを増殖させるためのものである。 Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the virus culture apparatus A according to the present embodiment includes a culture tank 1, a virus supply apparatus 2, an exhaust gas analysis apparatus 3, and a culture information generation apparatus (culture state information generation apparatus) 4. . Such a virus culture apparatus A is used for producing the above-mentioned target protein or for propagating the virus itself by seeding and culturing a virus incorporating a gene for producing a predetermined target protein in insect cells. Is.

培養槽１は、培養対象である上記昆虫細胞及びウイルスを培養するための設備であり、槽本体内の環境（培養環境）を培養に最適な環境を維持しつつ培養対象を培養する装置である。すなわち、この培養槽１は、槽本体、槽本体内の培養液を攪拌する攪拌装置、槽本体に培養に必要な酸素を供給する酸素供給装置、槽本体を培養に最適な温度に維持するための温度管理装置、等々から構成されている。培養槽の形態としては、攪拌機がなく、通気で攪拌を行うもの等、一般にいくつかの形態のものが知られているが、本実施形態における培養槽１は何れの形態のものでも良い。   The culture tank 1 is a facility for culturing the insect cells and viruses to be cultured, and is an apparatus for culturing the culture object while maintaining the environment (culture environment) in the tank body optimal for the culture. . That is, the culture tank 1 has a tank body, a stirring device for stirring the culture solution in the tank body, an oxygen supply device for supplying oxygen necessary for the culture to the tank body, and the tank body for maintaining the temperature at an optimum temperature for the culture. Temperature control device, and so on. As the form of the culture tank, there are generally known several forms such as those without a stirrer and stirring by aeration, but the culture tank 1 in this embodiment may be of any form.

ウイルス供給装置２は、培養開始後の所定のタイミングで上記培養槽１にウイルスを供給するものである。このウイルスは、特定の蛋白質を生成するための遺伝子が組み込まれたものである。上記タンパク質は、本ウイルス培養装置Ａの最終的な生産物である。培養槽１は初期的には昆虫細胞を培養するが、培養開始後の所定のタイミングでウイルス供給装置２からウイルスが供給されると、これ以降は昆虫細胞に播種したウイルスが槽本体内で培養される。   The virus supply device 2 supplies virus to the culture tank 1 at a predetermined timing after the start of culture. This virus is a gene in which a gene for producing a specific protein is incorporated. The protein is the final product of the virus culture apparatus A. The culture tank 1 initially cultivates insect cells. When virus is supplied from the virus supply device 2 at a predetermined timing after the start of culture, the virus seeded on the insect cells is then cultured in the tank body. Is done.

排気ガス分析装置３は、このような培養槽１における培養過程において時々刻々と培養槽１から排出される排気ガスの成分及び濃度をオンラインで分析・測定する装置である。この排気ガス分析装置３は、排気ガスに含まれるいくつかの成分のうち、例えば二酸化炭素（ＣＯ２）の濃度（ＣＯ２濃度）を分析・測定し、測定信号として培養情報生成装置４に出力する。このような排気ガス分析装置３としては、例えばガス成分の違いによる光吸収波長の違い等を利用した光学的なオンラインガス測定器が用いられる。 The exhaust gas analyzer 3 is an apparatus that analyzes and measures on-line the components and concentration of exhaust gas discharged from the culture tank 1 every moment in the culture process in the culture tank 1. This exhaust gas analyzer 3 analyzes and measures, for example, the concentration (CO 2 concentration) of carbon dioxide (CO 2 ) among several components contained in the exhaust gas, and outputs it to the culture information generator 4 as a measurement signal. To do. As such an exhaust gas analyzer 3, for example, an optical online gas measuring device using a difference in light absorption wavelength due to a difference in gas components is used.

培養情報生成装置４は、本ウイルス培養装置Ａにおける最も特徴的な構成要素である。この培養情報生成装置４は、図示するように情報処理部４ａと記憶部４ｂとを主な構成要素とする一種のコンピュータである。情報処理部４ａは、所定の培養情報生成プログラムに基づいて上記測定信号を信号処理することにより、上記培養槽１における培養対象の培養状態を示す培養情報を生成して外部、自らに備えられた表示装置（図示略）あるいは／及び培養槽１に出力する。記憶部４ｂは、上記培養情報生成プログラムを記憶する不揮発性記憶領域及び演算結果を一時的に記憶する揮発性記憶領域とからなる半導体メモリである。   The culture information generation device 4 is the most characteristic component in the virus culture device A. The culture information generating device 4 is a kind of computer having an information processing unit 4a and a storage unit 4b as main components as shown in the figure. The information processing unit 4a generates culture information indicating the culture state of the culture target in the culture tank 1 by signal processing the measurement signal based on a predetermined culture information generation program, and is provided outside and itself. The data is output to a display device (not shown) or / and the culture tank 1. The storage unit 4b is a semiconductor memory including a nonvolatile storage area for storing the culture information generation program and a volatile storage area for temporarily storing calculation results.

次に、このように構成された本ウイルス培養装置Ａの動作について、図２〜図４をも参照して詳しく説明する。   Next, operation | movement of this virus culture apparatus A comprised in this way is demonstrated in detail with reference also to FIGS.

本ウイルス培養装置Ａでは、培養槽１に昆虫細胞のみを収容して培養を開始する。そして、培養開始から所定時間が経過した時点でウイルス供給装置２が作動して培養槽１にウイルスが供給され、培養槽１では、攪拌装置によって昆虫細胞とウイルスとが攪拌混合されつつ培養が進行し、培養開始から一定の時間が経過した時点で培養を終了する。   In this virus culturing apparatus A, only insect cells are accommodated in the culture tank 1 and culture is started. Then, when a predetermined time has elapsed from the start of the culture, the virus supply device 2 operates to supply the virus to the culture tank 1, and the culture proceeds in the culture tank 1 while the insect cells and the virus are stirred and mixed by the stirring device. The culture is terminated when a certain time has elapsed from the start of the culture.

図３は、このような培養開始から培養終了までの間に培養槽１から排出される二酸化炭素（ＣＯ２）及び酸素（Ｏ２）の濃度変化、つまり培養開始から培養終了までの間における排気ガス分析装置３の測定結果は、図３に示す通りである。二酸化炭素（ＣＯ２）の濃度変化に着目すると、培養開始から４５時間が経過した時点でウイルス供給装置２が作動することによりウイルスが培養槽１に供給され、ウイルスの昆虫細胞への播種が発生する。培養開始から昆虫細胞が順調に培養されることにより二酸化炭素（ＣＯ２）の濃度は徐々に上昇するが、上記播種のタイミングで二酸化炭素（ＣＯ２）の濃度の上昇カーブに若干の乱れが発生している。 FIG. 3 shows changes in the concentrations of carbon dioxide (CO 2 ) and oxygen (O 2 ) discharged from the culture tank 1 between the start of culture and the end of culture, that is, exhaust between the start of culture and the end of culture. The measurement result of the gas analyzer 3 is as shown in FIG. Focusing on the change in the concentration of carbon dioxide (CO 2 ), the virus is supplied to the culture tank 1 by operating the virus supply device 2 when 45 hours have elapsed from the start of the culture, and the seeding of the virus on the insect cells occurs. To do. The concentration of carbon dioxide (CO 2 ) gradually rises as insect cells are cultivated smoothly from the beginning of the culture, but a slight disturbance occurs in the carbon dioxide (CO 2 ) concentration rise curve at the timing of seeding. doing.

二酸化炭素（ＣＯ２）の濃度は、播種後も昆虫細胞が継続して増殖するので上昇カーブを描くが、その後、二酸化炭素（ＣＯ２）の濃度の上昇率は徐々に小さくなり、培養開始から７５時間が経過した時点で下降カーブを描くようになる。すなわち、二酸化炭素（ＣＯ２）の濃度は、丸印で示すようにが播種後に１回目の極大値を示す。このような二酸化炭素（ＣＯ２）の濃度の変化は、播種後にウイルスが徐々に昆虫細胞に感染（一次感染）して昆虫細胞の増殖が妨げられ、また昆虫細胞内でのウイルスの増殖に伴って死滅する昆虫細胞が徐々に増加するためである。 The concentration of carbon dioxide (CO 2 ) draws a rising curve because insect cells continue to proliferate after seeding, but thereafter, the rate of increase in the concentration of carbon dioxide (CO 2 ) gradually decreases, from the start of culture. When 75 hours have passed, a downward curve is drawn. That is, the concentration of carbon dioxide (CO 2 ) shows the maximum value for the first time after sowing as indicated by a circle. Such a change in the concentration of carbon dioxide (CO 2 ) causes the virus to gradually infect insect cells (primary infection) after seeding, thereby preventing the growth of the insect cells, and accompanying the growth of the virus in the insect cells. This is because the number of insect cells that die is gradually increased.

そして、昆虫細胞内で所定数まで増殖したウイルスは、培養開始から１００時間程度が経過した時点で、昆虫細胞から離脱して他の昆虫細胞に感染（二次感染）する。この二次感染の際には、昆虫細胞内で増殖していたウイルスが昆虫細胞の外に一旦出てくるので、丸印で示すように二酸化炭素（ＣＯ２）の濃度が急激に増加して減少する。すなわち、二酸化炭素（ＣＯ２）の濃度は、ウイルスの二次感染によって２回目の極大値を示す。 The virus that has grown to a predetermined number in the insect cell leaves the insect cell and infects other insect cells (secondary infection) after about 100 hours have passed since the start of the culture. During this secondary infection, the virus that had grown in the insect cells once comes out of the insect cells, and as shown by the circles, the concentration of carbon dioxide (CO 2 ) increases rapidly. Decrease. That is, the concentration of carbon dioxide (CO 2 ) shows a second maximum value due to secondary infection of the virus.

ウイルス内で生成される蛋白質（生産物）は、ある蛋白質の場合には、図４に示すように、培養開始から４５時間が経過後の播種によってウイルスが培養槽１内で増殖すると共に徐々に増大する。その後、蛋白質（生産物）は、時間の経過とともに序所に増加するが、一次感染時及び二次感染時に飛躍的に増大する。そして、蛋白質（生産物）は、二次感染が終了すると極端に減少する。このような二次感染後における蛋白質（生産物）の減少は、死滅した昆虫細胞から分泌されたプロテアーゼ（蛋白質やペプチドを加水分解する酵素）によって、ウイルス内の蛋白質（生産物）が急激に分解されるためと考えられる。したがって、蛋白質（生産物）を効率（収率）良く生産するためには二次感染の発生を的確かつ正確に検知することが極めて重要である。   In the case of a protein (product) produced in a virus, as shown in FIG. 4, as the protein grows in the culture tank 1 as a result of seeding 45 hours after the start of culture, as shown in FIG. Increase. Thereafter, the protein (product) gradually increases with the passage of time, but dramatically increases during the primary infection and the secondary infection. And protein (product) decreases extremely when the secondary infection ends. The decrease in protein (product) after such secondary infection is caused by rapid degradation of the protein (product) in the virus by proteases (enzymes that hydrolyze proteins and peptides) secreted from dead insect cells. It is thought to be done. Therefore, in order to produce a protein (product) efficiently (yield), it is extremely important to accurately and accurately detect the occurrence of secondary infection.

さて、以上説明した二酸化炭素（ＣＯ２）の濃度変化は、排気ガス分析装置３によって分析・測定されたものであり、排気ガス分析装置３から培養情報生成装置４に測定信号として供給される。培養情報生成装置４の情報処理部４ａは、培養情報生成プログラムに基づいて作動することにより、各時刻で排気ガス分析装置３から入力される上記測定信号が示す二酸化炭素（ＣＯ２）の濃度を時系列データとして記憶部４ｂに順次記憶させつつ、当該時系列データ、つまり現在及び過去の時刻における二酸化炭素（ＣＯ２）の濃度に基づいて上記二次感染の発生を図２のフローチャートに示す手順で検知する。 The concentration change of carbon dioxide (CO 2 ) described above is analyzed and measured by the exhaust gas analyzer 3 and is supplied from the exhaust gas analyzer 3 to the culture information generator 4 as a measurement signal. The information processing unit 4a of the culture information generation device 4 operates based on the culture information generation program, thereby determining the concentration of carbon dioxide (CO 2 ) indicated by the measurement signal input from the exhaust gas analyzer 3 at each time. The procedure shown in the flowchart of FIG. 2 shows the occurrence of the secondary infection based on the time-series data, that is, the concentration of carbon dioxide (CO 2 ) at the current and past times, while being sequentially stored in the storage unit 4b as time-series data. Detect with.

情報処理部４ａは、培養開始後に播種が発生したか否かを判断し（ステップＳ1）、この判断が「Ｙｅｓ」となると、一次感染が発生したか否かを判断する（ステップＳ2）。ここで、情報処理部４ａは、例えば内部タイマー等をセットすることにより培養開始後の経過時間（タイマーの計時時間）が予め設定されたウイルス供給時間に一致したか否かを判断することによって、上記播種の発生を判断する。そして、情報処理部４ａは、二酸化炭素（ＣＯ２）の濃度変化が播種後において上昇カーブから下降カーブに変化したことを上記時系列データに基づいて確認することによって、上記一次感染の発生を判断する。 The information processing unit 4a determines whether or not seeding has occurred after the start of culture (step S1). If this determination is “Yes”, it determines whether or not primary infection has occurred (step S2). Here, the information processing unit 4a, for example, by setting an internal timer or the like, by determining whether or not the elapsed time after the start of culture (timed time of the timer) matches the preset virus supply time, Determine the occurrence of the sowing. Then, the information processing unit 4a determines the occurrence of the primary infection by confirming, based on the time series data, that the change in the concentration of carbon dioxide (CO 2 ) has changed from an ascending curve to a descending curve after sowing. To do.

そして、情報処理部４ａは、ステップＳ2の判断が「Ｙｅｓ」となると、二次感染が発生したか否かを判断する（ステップＳ3）。すなわち、情報処理部４ａは、一次感染後において二酸化炭素（ＣＯ２）の濃度変化が再び上昇して下降したことを時系列データに基づいて確認することによって、上記二次感染の発生を判断する。情報処理部４ａは、このようにして二次感染の発生を検知すると、当該二次感染の発生を示す培養情報を生成して外部及び培養槽１に出力する（ステップＳ4）。そして、培養槽１は、培養情報生成装置４から培養情報が入力されると、を速やかに培養を終了して蛋白質（生産物）の分解を防止する。 Then, when the determination in step S2 is “Yes”, the information processing section 4a determines whether or not a secondary infection has occurred (step S3). That is, the information processing unit 4a determines the occurrence of the secondary infection by confirming, based on the time series data, that the change in the concentration of carbon dioxide (CO 2 ) has increased and decreased again after the primary infection. . When detecting the occurrence of the secondary infection in this way, the information processing unit 4a generates culture information indicating the occurrence of the secondary infection and outputs it to the outside and the culture tank 1 (step S4). Then, when the culture information is input from the culture information generation device 4, the culture tank 1 immediately terminates the culture and prevents the protein (product) from being decomposed.

このように、培養情報生成装置４の情報処理部４ａは、培養情報生成プログラムに基づいて作動することにより、培養槽１から排出される排気ガスの一成分である二酸化炭素（ＣＯ２）の濃度に関する時系列データに基づいて二次感染の発生をオンライン状態で自動的に検知する。したがって、本ウイルス培養装置Ａによれば、従来技術の問題を完全に解決することが可能であり、ウイルスの培養状態をタイムリーに評価することが可能であると共に、従来よりも客観性を担保し得るウイルスの培養状態の評価手法を提供することができる。 As described above, the information processing unit 4a of the culture information generation device 4 operates based on the culture information generation program, whereby the concentration of carbon dioxide (CO 2 ), which is a component of exhaust gas discharged from the culture tank 1, The occurrence of secondary infection is automatically detected on-line based on the time-series data regarding. Therefore, according to this virus culture apparatus A, it is possible to completely solve the problems of the prior art, to evaluate the culture state of the virus in a timely manner, and to ensure the objectivity than before. It is possible to provide a method for evaluating the culture state of a possible virus.

なお、本発明は、上記実施形態に限定されるものではなく、例えば以下のような変形例が考えられる。
（１）上記実施形態では、培養情報生成装置４は、二次感染の発生を示す情報を培養情報として生成したが、本発明における培養情報はこれに限定されない。例えば、培養情報生成装置４で図３に示すような二酸化炭素（ＣＯ２）の濃度変化を示すトレンドグラフを生成し、当該トレンドグラフを培養情報として外部、自らに備えられた表示装置あるいは／及び培養槽１に出力するようにしても良い。このようなトレンドグラフを培養情報として生成する場合、作業者が、このトレンドグラフに基づいて二次感染の発生を判断して所定の措置をとることによって培養槽１における培養が終了する。 In addition, this invention is not limited to the said embodiment, For example, the following modifications can be considered.
(1) In the above embodiment, the culture information generation device 4 generates information indicating the occurrence of secondary infection as culture information, but the culture information in the present invention is not limited to this. For example, the culture information generation device 4 generates a trend graph showing the change in the concentration of carbon dioxide (CO 2 ) as shown in FIG. You may make it output to the culture tank 1. FIG. When such a trend graph is generated as culture information, the operator determines the occurrence of secondary infection based on the trend graph and takes a predetermined measure, whereby the culture in the culture tank 1 is completed.

（２）図２に示したように播種の発生を判断した後に一次感染（１回目の極大値の発生）及び二次感染（２回目の極大値の発生）の発生を判断したが、播種の発生に関する判断を省略しても良い。すなわち、培養の終了を決定するガスの発生濃度の極大値の発生回数は、予め決定された回数であればよく、上記実施形態の２回に限定されない。 (2) After judging the occurrence of sowing as shown in FIG. 2, the occurrence of primary infection (occurrence of the first local maximum) and secondary infection (occurrence of the second local maximum) was determined. The determination regarding occurrence may be omitted. That is, the number of occurrences of the maximum value of the gas generation concentration that determines the end of the culture may be any number determined in advance, and is not limited to twice in the above embodiment.

（３）図２に示した二酸化炭素（ＣＯ２）の濃度変化は、特定の昆虫細胞とウイルスについて測定されたものであるが、一次感染及び二次感染は、各種のウイルス及び各種の動物細胞あるいは昆虫細胞以外の動物細胞等についても同様に発生するので、１回目の極大値及び２回目の極大値は、昆虫細胞等のウイルスの栄養となる細胞とウイルスとの関係に依らず発生するものと考えることができる。したがって、本願発明は、ウイルスの栄養となる細胞とウイルスとの関係を特に限定しないものである。 (3) The change in the concentration of carbon dioxide (CO 2 ) shown in FIG. 2 was measured for specific insect cells and viruses. Primary infections and secondary infections are caused by various viruses and various animal cells. Or it occurs in the same way for animal cells other than insect cells, so the first maximum and the second maximum occur regardless of the relationship between the virus and the cells that serve as nutrients for the virus, such as insect cells. Can be considered. Accordingly, the present invention does not particularly limit the relationship between cells that serve as virus nutrition and viruses.

（４）上記実施形態では、培養槽１は二次感染の発生を示す培養情報が培養情報生成装置４から入力されると培養を終了させたが、培養槽１における培養情報に基づくウイルスの培養状態の制御は、このような培養の終了制御に限定されるものではない。培養情報生成装置４が検知した各種の培養状態を培養槽１に逐次供給することによって、培養の終了制御以外の制御を行うようにしても良い。例えば、培養情報生成装置４は、一次感染（１回目の極大値の発生）を示す培養情報を生成して培養槽１に提供し、培養槽１は、これを受けて培養条件に変更を加えるようにしても良い。 (4) In the above-described embodiment, the culture tank 1 terminates the culture when the culture information indicating the occurrence of the secondary infection is input from the culture information generation device 4, but the virus culture based on the culture information in the culture tank 1 The state control is not limited to such a culture end control. Control other than the end control of the culture may be performed by sequentially supplying various culture states detected by the culture information generation device 4 to the culture tank 1. For example, the culture information generation device 4 generates culture information indicating primary infection (occurrence of the first local maximum) and provides it to the culture tank 1, and the culture tank 1 receives this and changes the culture conditions. You may do it.

（５）上記実施形態では、培養槽１から排出される排気ガス中の二酸化炭素（ＣＯ２）の濃度に基づいて培養情報を生成したが、本発明はこれに限定されない。一次感染あるいあｈ／及び二次感染時に特異な濃度変化を示すガス成分であれば、二酸化炭素（ＣＯ２）以外の成分の濃度を測定しても良い。 (5) In the above embodiment, the culture information is generated based on the concentration of carbon dioxide (CO 2 ) in the exhaust gas discharged from the culture tank 1, but the present invention is not limited to this. The concentration of components other than carbon dioxide (CO 2 ) may be measured as long as it is a gas component that exhibits a specific concentration change at the time of primary infection or h /.

Ａ…ウイルス培養装置、１…培養槽、２…ウイルス供給装置、３…排気ガス分析装置、４…培養情報生成装置、４ａ…情報処理部、４ｂ…記憶部   A ... virus culture device, 1 ... culture tank, 2 ... virus supply device, 3 ... exhaust gas analyzer, 4 ... culture information generating device, 4a ... information processing unit, 4b ... storage unit

Claims (6)

ウイルス含有培養液を保有する培養槽から排気されるガス中の対象成分の濃度変化を示す測定信号を入力とし、前記培養槽内でウイルスの栄養となる所定の細胞にウイルスを播種して培養する場合には、前記測定信号に基づいて前記対象成分の濃度変化の極大値の発生を判断することにより、前記対象成分の発生濃度の予め決定された回数の極大値の発生を示す培養情報を生成することを特徴とする培養情報生成装置。 The measurement signal indicating the concentration change of the target component in the gas exhausted from the culture tank holding the virus-containing culture solution is used as an input, and the cells are seeded and cultured in predetermined cells serving as virus nutrition in the culture tank. In this case, by determining the occurrence of the maximum value of the concentration change of the target component based on the measurement signal, the culture information indicating the generation of the maximum value for the predetermined number of times of the generated concentration of the target component is generated. A culture information generating apparatus characterized by: 前記対象成分は、二酸化炭素（ＣＯ ２ ）であることを特徴とする請求項１記載の培養情報生成装置。 The culture information generating apparatus according to claim 1 , wherein the target component is carbon dioxide (CO 2 ) . ウイルスを培養する培養槽と、A culture vessel for culturing viruses;
該培養槽から排出される排気ガスを分析・測定し、測定結果を示す測定信号を出力する排気ガス分析装置と、An exhaust gas analyzer that analyzes and measures the exhaust gas discharged from the culture tank and outputs a measurement signal indicating a measurement result;
請求項１または２に記載の培養情報生成装置とThe culture information generating apparatus according to claim 1 or 2,
を具備することを特徴とする培養装置。A culture apparatus comprising: 前記培養槽は、培養情報に基づいてウイルスの培養を終了することを特徴とする請求項３記載の培養装置。 The culture apparatus according to claim 3, wherein the culture tank finishes virus culture based on culture information . ウイルスの栄養となる所定の細胞にウイルスを播種して培養する場合には、ウイルスの培養で発生したガス中の対象成分の発生濃度の予め決定された回数の極大値の発生をもってウイルスの培養を終了することを特徴とする培養方法。When inoculating and culturing the virus on the predetermined cells that serve as the nutrition of the virus, the virus is cultured with the occurrence of a maximum value of the predetermined concentration of the target concentration of the target component in the gas generated by the virus culture. A culture method characterized by being terminated. 前記対象成分は、二酸化炭素（ＣＯ ２ ）であることを特徴とする請求項５記載の培養方法。
The culture method according to claim 5 , wherein the target component is carbon dioxide (CO 2 ) .
JP2010015844A 2010-01-27 2010-01-27 CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD Expired - Fee Related JP5810478B2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2010015844A JP5810478B2 (en) 2010-01-27 2010-01-27 CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD
PCT/JP2011/050828 WO2011093180A1 (en) 2010-01-27 2011-01-19 Culture information-generating device, culture device and culture method
SG2012055422A SG182744A1 (en) 2010-01-27 2011-01-19 Culture information-generating device, culture device and culture method
KR1020147013968A KR101511029B1 (en) 2010-01-27 2011-01-19 Culture information-generating device, culture device and culture method
CN2011800070761A CN102712891A (en) 2010-01-27 2011-01-19 Culture information-generating device, culture device and culture method
KR1020127019064A KR101436134B1 (en) 2010-01-27 2011-01-19 Culture information-generating device, culture device and culture method
TW100102580A TWI429743B (en) 2010-01-27 2011-01-25 Incubation information generating apparatus, incubation apparatus and incubation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2010015844A JP5810478B2 (en) 2010-01-27 2010-01-27 CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD

Publications (2)

Publication Number Publication Date
JP2011152078A JP2011152078A (en) 2011-08-11
JP5810478B2 true JP5810478B2 (en) 2015-11-11

Family

ID=44319171

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2010015844A Expired - Fee Related JP5810478B2 (en) 2010-01-27 2010-01-27 CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD

Country Status (6)

Country Link
JP (1) JP5810478B2 (en)
KR (2) KR101436134B1 (en)
CN (1) CN102712891A (en)
SG (1) SG182744A1 (en)
TW (1) TWI429743B (en)
WO (1) WO2011093180A1 (en)

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3641123B2 (en) * 1997-12-26 2005-04-20 帝人株式会社 Virus or cell culture method
US8026096B1 (en) * 1998-10-08 2011-09-27 Protein Sciences Corporation In vivo active erythropoietin produced in insect cells
JP2002148258A (en) * 2000-08-22 2002-05-22 Toto Ltd Culture monitor and incubator
EP1637586A3 (en) * 2000-09-29 2008-10-29 Becton, Dickinson and Company System and method for detecting the growth of microorganisms in a sample.
EP1548099A1 (en) * 2002-07-31 2005-06-29 Japan Science and Technology Agency Automatic culture apparatus for cell or tisse with biological origin
JP2007143492A (en) * 2005-11-29 2007-06-14 Tosoh Corp Method for producing carotenoids
JP2008220235A (en) * 2007-03-12 2008-09-25 Sanyo Electric Co Ltd Culture apparatus
DK2217711T3 (en) * 2007-09-20 2015-09-14 Amyris Inc Production of isoprenoids
JP2010246473A (en) * 2009-04-16 2010-11-04 Sharp Corp Method and apparatus for culturing microalgae

Also Published As

Publication number Publication date
CN102712891A (en) 2012-10-03
KR20120106843A (en) 2012-09-26
KR20140091717A (en) 2014-07-22
TW201144431A (en) 2011-12-16
JP2011152078A (en) 2011-08-11
KR101436134B1 (en) 2014-09-01
TWI429743B (en) 2014-03-11
WO2011093180A1 (en) 2011-08-04
KR101511029B1 (en) 2015-04-13
SG182744A1 (en) 2012-08-30

Similar Documents

Publication Publication Date Title
US20170267964A1 (en) Method for use in monitoring biological material
Riley et al. Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy
Sandor et al. Comparative study of non-invasive monitoring via infrared spectroscopy for mammalian cell cultivations
JP6442398B2 (en) Method and system for detecting microbial growth in a sample container
EP3433686A1 (en) Controlling and monitoring a process to produce a chemical, pharmaceutical or biotechnological product
Kondragunta et al. Advances in clone selection using high‐throughput bioreactors
KR102690117B1 (en) Methods for determining process variables in cell culture processes
Wang et al. Process analytical technologies in cell therapy manufacturing: State‐of‐the‐art and future directions
Dill et al. Cell density effects in different cell culture media and their impact on the propagation of foot-and-mouth disease virus
Tetsuo et al. Development of a high-throughput serum neutralization test using recombinant pestiviruses possessing a small reporter tag
US20220146987A1 (en) Multivariate process chart to control a process to produce a chemical, pharmaceutical, biopharmaceutical and/or biological product
US20130030715A1 (en) Apparatus and method for monitoring autotroph cultivation
JP5810478B2 (en) CULTURE INFORMATION GENERATION DEVICE, CULTURE DEVICE, AND CULTURE METHOD
Martínez‐Monge et al. Soft‐sensors application for automated feeding control in high‐throughput mammalian cell cultures
Fratz‐Berilla et al. Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures
Klinger et al. Novel carbon dioxide-based method for accurate determination of pH and pCO2 in mammalian cell culture processes
EP3702849A1 (en) Optimized process control
Kroll et al. Impact of cell lysis on the description of cell growth and death in cell culture
McKinley et al. A Bayesian approach to analyse genetic variation within RNA viral populations
Bonk et al. In-situ microscopy and 2D fluorescence spectroscopy as online methods for monitoring CHO cells during cultivation
Opalek et al. How to determine microbial lag phase duration?
JP2020006291A (en) Creation method of prediction information for predicting biogas generation amount and utilization of the prediction information
CN104774967A (en) Primers and detection method for pasteurella multocida real-time fluorescence quantification PCR method
JP7153131B2 (en) Method for verifying performance of culture device
EP4425498A1 (en) Apparatus for determining cell culture conditions by using artificial intelligence and method for operating same

Legal Events

Date Code Title Description
RD04 Notification of resignation of power of attorney

Free format text: JAPANESE INTERMEDIATE CODE: A7424

Effective date: 20120117

A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20121126

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20140507

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20140707

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20150106

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20150309

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20150818

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20150831

R151 Written notification of patent or utility model registration

Ref document number: 5810478

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R151

S111 Request for change of ownership or part of ownership

Free format text: JAPANESE INTERMEDIATE CODE: R313113

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

S111 Request for change of ownership or part of ownership

Free format text: JAPANESE INTERMEDIATE CODE: R313111

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

LAPS Cancellation because of no payment of annual fees