ಶู་⒪ྥࡅࡓḟୡ௦ࣂ࢜་⸆ရฟᇶ┙ᢏ⾡㛤Ⓨ
ࣉࣟࢪ࢙ࢡࢺ
㨣ሯṇ⩏
ᨻྐ
Next Generation Research and Development Project for Biologics Production
By
Masayoshi ONITSUKA, and Takeshi OMASA
The object of this project is to achieve the next generation research and development for biologics
production. We performed the reparation for research project for development of biopharmaceutical
production. In this project, our Tokushima university group will perform the cell line engineering and
cell culture engineering for production of biopharmaceuticals. In this paper, we reported that the
achievements for these topics and introduce our results.
Keywords; Biologics Production, Biochemical Engineering, Biopharmaceutical, Cell Engineering, Therapeutic Antibody
ࡣࡌࡵ ᨻᗓࡢ᪂ᡂ㛗ᡓ␎㸦ᖹᡂ22 ᖺ 6 ᭶㸧࠾࠸࡚ࠊ㠉᪂ⓗ ᪂⸆࣭་⒪ᶵჾ➼ࡢᐇ⏝࡛2020 ᖺ㎾ᖺ㛫⣙ 7000 ൨ ࡢ⤒῭ຠᯝࡀᮇᚅࡉࢀ࡚࠸ࡿࠋࡇࢀࢆ࠺ࡅ࡚ࠊ2013 ᖺ 1 ᭶⤒῭⏘ᴗ┬ࡽᴫ⟬せồࡋ࡚ࠕಶู་⒪ྥࡅࡓ ḟୡ௦་⸆ရฟᇶ┙ᢏ⾡㛤Ⓨࠖ㸦H25-H29 ᖺᗘࠊయ ᴗ㈝405 ൨ࠊH25 ᖺᗘ 81 ൨()㸧ࡀ࡞ࡉࢀࡓࠋᮏᴫ⟬ せồ࡛ࡣࠊᇶ┙ᢏ⾡ࡋ࡚ձIT ⸆ࠊղḟୡ௦ᢠయ་⸆⏕ ⏘ࠊճయෆືែᢕᥱࡢ3 ࡘࡢ㡯┠ࢆ⾜࠺ࡀࠊᮏࣉࣟࢪ࢙ࢡ ࢺ࡛ࡣࠊ≉ࠕḟୡ௦ᢠయ་⸆➼ࡢᏳᐃ⏕⏘ᢏ⾡ࠖ㛵㐃 ࡍࡿᚨᓥᏛ࠾ࡅࡿᣐⅬᙧᡂࢆ⾜࠺ࡓࡵࡢ‽ഛࢆ࠾ࡇ ࡞ࡗࡓࠋ ḟୡ௦ᢠయ་⸆Ᏻᐃ⏕⏘㛵㐃ࡍࡿ㛤Ⓨ㡯┠ࡋ࡚ࡣ 1ᚨᓥᏛᏛ㝔ࢯࢩ࢜ࢸࢡࣀࢧ࢚ࣥࢫ◊✲㒊
ࣛࣇࢩࢫࢸ࣒㒊㛛 Dep. of Biological Science and Technology, Institute of Technology and Science,
Tokushima University 㐃⤡ඛ㸸770-8506 ᚨᓥᕷ༡ᖖ୕ᓥ⏫ 2-1 ᚨᓥᏛᏛ㝔ࢯࢩ࢜ࢸࢡࣀࢧ࢚ࣥࢫ◊✲㒊 (1)᭱㐺ᐟ㛤Ⓨࠊ(2)Ᏻᐃ⏕⏘ࢩࢫࢸ࣒㛤Ⓨࠊ(3㸧ศ㞳⢭〇 ᢏ⾡㛤Ⓨࠊ(4)⏕⏘≀ရ㉁ホ౯㛤Ⓨࡢ 4 ࡘࡀィ⏬ࡉࢀ࡚࠸ࡿࠋ ࡞࠾ࠊᙜヱᴗᐇ࠶ࡓࡗ࡚ࡣᚨᓥᏛࡶྵࡴ 29 ᶵ㛵 ࡽ࡞ࡿḟୡ௦ࣂ࢜་⸆ရ〇㐀ᢏ⾡◊✲⤌ྜࢆ❧ࡕୖ ࡆࠊḟୡ௦ᢠయ་⸆⏕⏘㛵ࢃࡿᮏᴗࡢࣉࣟࢪ࢙ࢡࢺࣜ ࣮ࢲ࣮ࡋ࡚యࢆ⤫ᣓࡋࠊࡉࡽࡋ࡚᭱㐺ᐟ㛤Ⓨ ࡞ࡽࡧࠊ㛤Ⓨᢏ⾡ࡢ(2)-(4)ࡢ㡯┠ࡢ㐃ᦠࡘ࠸࡚ᐇ ࡍࡿணᐃ࡛࠶ࡿࠋ⌧ᅾࠊᮏࣉࣟࢪ࢙ࢡࢺ࡛ࡣࠊ29 ᶵ㛵㸦㸰 㸲♫ࠊ㸰Ꮫࠊ㸱ἲே㸧࡞ࡽࡧ㸯㸮௨ୖࡢᏛࡽ࡞ࡿ ጤクඛࡽᵓᡂࡉࢀࡿணᐃ࡛࠶ࡿࠋ ᚨᓥᏛ STS ◊✲㒊࠾࠸࡚ࡣࠊᐟ㛤Ⓨࠊࡇࢀ㐃 ືࡋࡓ ᐃ⣔㛤Ⓨࠊࡉࡽࡣ⢾㙐ࠊจ㞟యࢆྵࡴရ㉁ホ౯ ᨵኚࠊᮏศ㔝ࡢᑓ㛛ᐙࡀᥞࡗ࡚࠾ࡾࠊࡇࢀࡽࡢຊࢆ⤖㞟 ࡋ࡚ࠊᮏࣉࣟࢪ࢙ࢡࢺ࠾ࡅࡿᚨᓥᏛᣐⅬᙧᡂࢆ⾜࠸ࠊ ࣉࣟࢪ࢙ࢡࢺᥦࢆ⾜࠸ࠊ᥇ᢥࡉࢀࡓࠋᮏሗ࿌᭩࡛ࡣࠊࡇ ࢀࡽࡢάືࡢᡂᯝࢆ⤂ࡍࡿࠋ ࣉࣟࢪ࢙ࢡࢺάືࡢලయⓗ࡞ᡂᯝ ḟୡ௦ࡢࣂ࢜་⸆⏕⏘ࡢࡓࡵࡢᇶ┙ᢏ⾡㛤Ⓨ
ᮏศ㔝㛵㐃ࡋ࡚ࠊ2013 ᖺ࠾࠸࡚⏕≀ᕤᏛ⛉ A3 ◊✲ ᐊ࠾࠸࡚㛵㐃ㄽᩥ7 ሗࠊゎㄝ⥲ㄝ 1 ⦅ࠊⴭ᭩ 1 ࠊᅜ㝿 ᏛⓎ⾲㸲௳ࠊᅜෆᏛⓎ⾲11 ௳ࠊᅜ㝿࣭ᅜෆᣍᚅㅮ₇ 㸲௳ࢆ⾜ࡗࡓࠋලయⓗ࡞ෆᐜࡣ௨ୗࡢ㏻ࡾ࡛࠶ࡿࠋ
㛵㐃ㄽᩥ
1) Kyoung Ho Lee, MMasayoshi Onitsuka, Kohsuke Honda, Hisao Ohtake, and TTakeshi Omasa* "Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering" Applied Microbiology and Biotechnology,Volume 997, No. 13, pp. 5731-5741(2013).
2) Kyoung Ho Lee, Tomomi Tsutsui, Kohsuke Honda, Ryutaro Asano, Izumi Kumagai, Hisao Ohtake, and Takeshi Omasa* "Generation of high-producing cell lines by overexpression of cell division cycle 25 homolog A in Chinese hamster ovary cells" Journal of Bioscience and Bioengineering Volume 116, Number 6, pp. 754-760 (2013).
3) Ahmad M Haredy, Akitoshi Nishizawa, Kohsuke Honda, Tomoshi Ohya, Hisao Ohtake and TTakeshi Omasa* "Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression" Cytotechnology Volume 65, Number 6, pp. 993-1002 (2013).
4) Kyoung Ho Lee, Tomomi Tsutsui, Kohsuke Honda, Hisao Ohtake, and TTakeshi Omasa* " Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells" Cytotechnology Volume 65, Number 6, pp. 1017-1026 (2013).
5) Kyoung Ho Lee, Kohsuke Honda, Hisao Ohtake and Takeshi Omasa* "Construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering" BMC Proceedings 2013, 7(Suppl 6):O7 doi:10.1186/1753-6561-7-S6-O7 (ᅜ
㝿㆟ㄽᩥ)
6) MMasayoshi Onitsuka, Miki Tatsuzawa, Masahiro Noda and TTakeshi Omasa* "Chemical chaperone suppresses the antibody aggregation in CHO cell culture" BMC Proceedings 2013, 7(Suppl 6):P68 doi:10.1186/1753-6561-7-S6-P68 (ᅜ㝿㆟ㄽᩥ)
7) Masahiro Noda, MMasayoshi Onitsuka, Miki Tatsuzawa, Ichiro Koguma and TTakeshi Omasa* "Dynamical analysis of antibody aggregation in the CHO cell culture with Thermo Responsive Protein A (TRPA) column" BMC Proceedings 2013, 7(Suppl 6):P69 doi:10.1186/1753-6561-7-S6-P69 (ᅜ㝿 ㆟ㄽᩥ) ゎㄝ⥲ㄝ 1) ᨻྐ* 㻌 “ࣂ࢜་⸆ရ⏕⏘࠾ࡅࡿࣉࣟࢲࢡࢩ ࣙࣥࢧ࢚ࣥࢫ”㻌 ᪥ᮏ⏕≀ᕤᏛㄅ㻌 Vol.91 (No.9) pp. 507-510 (2013). ⴭ᭩ 1) ᨻྐࠊⲶ㔝༓⛅ࠊ୰ᓥ⏣㇏ࠊோᐑ୍❶ࠊཱྀ᪼ࠕᇶ ♏ࡽᏛࡪ⏕≀ᏛᕤᏛ₇⩦ࠖ᪥ᮏ⏕≀ᕤᏛ⦅㸦⦅ 㞟ጤဨ㛗 ᨻྐ㸧ࠊࢥࣟࢼ♫(2013) 㸦ᇳ➹㡯┠ 1.1 ᏛᕤᏛࡢᇶ♏ 1-7 㡫ࠊ2.1 㓝⣲ࡢ≉ᛶࡑࡢ ⏝ᢏ⾡ 17-26 㡫ࠊ2.2 ᚤ⏕≀ࡢ≉ᛶࡑࡢᨵⰋᢏ⾡ 26-39 㡫ࠊ5.4 ࣂ࢜ࣉࣟࢲࢡࢺࡢศ㞳࣭⢭〇 135-143 㡫ࠊ㘓144-151 㡫㸧 ᅜ㝿ᅜෆᣍᚅㅮ₇
1) TTakeshi Omasa “Quality control in Chinese hamster ovary cell culture –from cell to antibody-” In; Asian Congress on Biotechnology 2013 (ACB2013), No.519, abstract p.46, New Delhi, India, December 15-19, 2013 ((Invited lecture, Key Note lecture)
2) 㨣ሯṇ⩏ࠊᨻྐ㸦ᣍᚅㅮ₇㸧 “⣽⬊ᵓ⠏ࡽጞ ࡲࡿࣂ࢜་⸆ရ⏕⏘࠾ࡅࡿࣉࣟࢭࢫࡢ㔜せᛶ -AlphaScreen ࡢᛂ⏝” PerkinElmer Japan35 ࿘ᖺ
グᛕ࣋ࣥࢺ RGHSuBiotherapeutics ᢠయ་⸆◊✲ ࡢ ᮍ ᮶ ࢆ ᣅ ࡃ ࢸ ࢡ ࣀ ࣟ ࢪ ࣮ 㸦Revolutionaries for Global Health Summit 2013㸧㸪2013 ᖺ 11/20 ᮾிᅜ 㝿ࣇ࢛࣮࣒ࣛࠊᮾி 3) ᨻ ྐ㸦ᣍᚅㅮ₇㸧 “ࢭࣝ㸤ࢸࢵࢩ࢚ࣗࣥࢪ ࢽࣜࣥࢢ㺎⣽⬊ࡽ⤌⧊ࡢࢿࢵࢺ࣮࣡ࢡ-” ➨ 10 ᅇHBS බ㛤ࢩ࣏ࣥࢪ࣒࢘ࠕ⏕་Ꮫ◊✲ࡢ⌧≧⮫ ᗋᛂ⏝ࡢㄢ㢟ࠖ㸪 せ᪨㞟 pp.10-12, 2013 ᖺ 11/12ࠊ ᚨᓥᏛࠊᚨᓥ 4) ᨻ ྐ㸦ᇶㄪㅮ₇㸧 “ࣂ࢜་⸆ရ⏕⏘࠾ࡅ ࡿࢭ࢚ࣝࣥࢪࢽࣜࣥࢢ” ᏛᕤᏛ➨ 45 ᅇ⛅Ꮨ 㸪E103, CD-ROMࠊ2 ࣮࣌ࢪ, 2013 ᖺ 9/16-18ࠊᒸ ᒣᏛࠊᒸᒣ ᅜ㝿ᏛⓎ⾲
1) Tomomi Tsutsui, Kyoung Ho Lee, Rina Matsuyama, MMasayoshi Onitsuka and TTakeshi Omasa "Cell cycle checkpoint engineering: novel construction method of gene-amplified CHO cell line for therapeutic antibody production" In; The 12th Tunisian-Japanese Symposium on Society, Science and Technology (TJASSST2013) O-13(Life Sciences, Food & Agriculture), abstract p.14,November 15-18, Yasmine Hammamet, Tsunisia (2013).
2) Kyoungho Lee, Kohsuke Honda, Hisao Ohtake, Takeshi Omasa "Rapid onstruction of transgene-amplified COcell lines by cell cycle checkpoint engineering " In; ESACT Meeting 2013 in Lille, No.O-19, p.31, Lille, France, June 23-26, 2013.
3) Masahiro Noda, Miki Tatsuzawa, MMasayoshi Onitsuka, Akihiro Shirai, Hideaki Maseda, TTakeshi Omasa, "Chemical shaperon suppresses the antibody aggregation in CHO cell culture " In; ESACT Meeting 2013 in Lille, No.A121, p.97, Lille, France, June 23-26, 2013.
4) MMasayoshi Onitsuka, Miki Tatsuzawa, Masahiro Noda, Ichiro Koguma, Akihiro Shirai, TTakeshi OMASA "Dynamical analysis of antibody aggregation in the CHO cell culture with thermo responsive protein A (TRPA) column " In; ESACT Meeting 2013 in Lille, No.A122, p.97, Lille, France, June 23-26, 2013.
ᅜෆᏛⓎ⾲
1) 㧗ᶫ ⯙㸪᳃ୗ ᫂ᙪ㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫 ୡ⏣ ⱥ᫂㸪ᨻ ྐ “Chinese hamster ovary ⣽⬊ᰴ࠾ࡅࡿᰁⰍయᏳᐃᛶゎᯒᢠయ⏕⏘ࡢ ᛂ⏝“, ➨ 65 ᅇ᪥ᮏ⏕≀ᕤᏛ, 1P-221, せ᪨㞟 73 㡫, 2013 ᖺࠊ9/18-20ࠊᗈᓥᅜ㝿㆟ሙࠊᗈᓥ. 2) ⟄ ᬛ⨾㸪kyoung Ho Lee㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫ୡ⏣ ⱥ᫂㸪ᨻ ྐ “⣽⬊࿘ᮇไᚚࡼ ࡿᢠయ་⸆ရ㧗⏕⏘CHO ⣽⬊ᰴᵓ⠏⣔ࡢ☜⋡” ➨ 65 ᅇ᪥ᮏ⏕≀ᕤᏛ, 1P-222, せ᪨㞟 73 㡫, 2013 ᖺࠊ 9/18-20ࠊᗈᓥᅜ㝿㆟ሙࠊᗈᓥ. 3) 㔝⏣ ┿ᗈ㸪㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫ୡ⏣ ⱥ᫂㸪 ᨻ ྐ “㔜≉␗ᛶᢠయࡢࢻ࣓ࣥ㓄⨨ࡀจ㞟 ᛶཬࡰࡍᙳ㡪” ➨ 65 ᅇ᪥ᮏ⏕≀ᕤᏛ, 3P-229, せ᪨㞟 245 㡫, 2013 ᖺࠊ9/18-20ࠊᗈᓥᅜ㝿㆟ሙࠊ ᗈᓥ. 4) ᳃ୗ ᫂ᙪ㸪㧗ᶫ ⯙㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫 ୡ⏣ ⱥ᫂㸪ᨻ ྐ “␗ᩘᛶࢆ᭷ࡍࡿ Chinese hamster ovary ⣽⬊࠾ࡅࡿᰁⰍయᏳᐃᛶゎᯒ” ➨ 65 ᅇ᪥ᮏ⏕≀ᕤᏛ, 3P-230, せ᪨㞟 245 㡫, 2013 ᖺࠊ9/18-20ࠊᗈᓥᅜ㝿㆟ሙࠊᗈᓥ. 5) 㨣ሯ ṇ⩏㸪ᨻ ྐ “ࣞࢡࢳࣥࢆ⏝࠸ࡓᢠయ⢾ 㙐ࡢ㎿㏿᳨ฟࡑࡢྍ⬟ᛶ” ᏛᕤᏛ➨ 45 ᅇ⛅ Ꮨ 㸪ZB2P56, CD-ROMࠊ୍࣮࣌ࢪ, 2013 ᖺ 9/16-18ࠊᒸᒣᏛࠊᒸᒣ 6) ⟄ ᬛ⨾㸪Kyoung Ho Lee㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫ୡ⏣ ⱥ᫂㸪ᨻ ྐ “⣽⬊࿘ᮇ࢚ࣥࢪࢽ ࣜࣥࢢࡼࡿChinese hamster ovary (CHO) ⣽⬊
࠾ࡅࡿຠ⋡ⓗ࡞㑇ఏᏊቑᖜࢩࢫࢸ࣒ࡢᵓ⠏” ᪥ᮏ ື≀⣽⬊ᕤᏛ2013 ᖺᗘ㸦JAACT2013㸧せ᪨ 㞟p.50, 2013 ᖺ 7/18-19ࠊ࣍ࢸࣝࣇࢪࢱ⚟ࠊ⚟ 7) ᮌୗ ᖾᜨ㸪⟄ ᬛ⨾㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪 㛫ୡ⏣ ⱥ᫂㸪ᨻ ྐ “ᢠయ⏕⏘ CHO ⣽⬊ᰴ ࠾ࡅࡿ NFKBIZ Ⓨ⌧ࡢᙳ㡪” ᪥ᮏື≀⣽⬊ᕤᏛ 2013 ᖺᗘ㸦JAACT2013㸧せ᪨㞟 p.68, 2013 ᖺ7/18-19ࠊ࣍ࢸࣝࣇࢪࢱ⚟ࠊ⚟ 8) ᯇᮧ ࡋࡎ㸪ᕝ ⩧ኴ㸪㨣ሯ ṇ⩏㸪ⓑ ༤㸪 㛫ୡ⏣ ⱥ᫂㸪ᨻ ྐ “CHO ⣽⬊࠾ࡅࡿᑠ⬊ ฟⱆ㛵㐃ᅉᏊ Arf ࡢࢡ࣮ࣟࢽࣥࢢⓎ⌧ไᚚࡼࡿ ࢱ ࣥ ࣃ ࢡ ㉁ ⏕ ⏘ ࡢ ᙳ 㡪” ᪥ᮏື≀⣽⬊ᕤᏛ 2013 ᖺᗘ㸦JAACT2013㸧せ᪨㞟 p.83, 2013 ᖺ 7/18-19ࠊ࣍ࢸࣝࣇࢪࢱ⚟ࠊ⚟ 9) ᮌୗ ᖾᜨࠊ⟄ ᬛ⨾ࠊ㨣㨣ሯ ṇ⩏ࠊⓑ ༤ࠊ㛫ୡ ⏣ ⱥ᫂ࠊᨻ ྐ "NFKBIZ Ⓨ⌧ࡼࡿ㧗⏕⏘ CHO ⣽⬊ᰴࡢᵓ⠏ゎᯒ",ᏛᕤᏛ➨ 77 ᖺ㸪 K301㸪CD-ROMࠊ୍࣮࣌ࢪࠊ2013 ᖺ 3/17-19ࠊ 㜰Ꮫࠊ㇏୰ 10) 㔝⏣ ┿ᗈࠊ㱟⃝ ᐇᏘࠊ㨣㨣ሯ ṇ⩏ࠊⓑ ༤ࠊ㛫ୡ ⏣ ⱥ᫂ࠊᨻ ྐ "ࢺࣞࣁ࣮ࣟࢫῧຍࡼࡿ CHO ⣽⬊ᇵ㣴㐣⛬ࡢᢠయจ㞟ᢚไ",ᏛᕤᏛ➨ 77 ᖺ㸪 K302㸪CD-ROMࠊ୍࣮࣌ࢪࠊ2013 ᖺ 3/17-19ࠊ 㜰Ꮫࠊ㇏୰ 11) ᳃ୗ ᫂ᙪࠊ㧗ᶫ ⯙ࠊ㨣㨣ሯ ṇ⩏ࠊⓑ ༤ࠊ㛫ୡ⏣ ⱥ᫂ࠊᨻ ྐ "⤌࠼ࣂ࢜་⸆ရ⏕⏘ CHO ⣽ ⬊ᰴᵓ⠏㐣⛬࠾ࡅࡿᰁⰍయᏳᐃᛶゎᯒ",Ꮫᕤ Ꮫ➨77 ᖺ㸪K303㸪CD-ROMࠊ୍࣮࣌ࢪࠊ2013 ᖺ 3/17-19ࠊ㜰Ꮫࠊ㇏୰ άືᡂᯝࡼࡿཷ㈹ ࡇࢀࡽࡢ⤖ᯝࡋ࡚ࠊᮏࣉࣟࢪ࢙ࢡࢺ㛵㐃࡛ୗグࡢᏛ⏕ ࡀཷ㈹ࡉࢀࡓࠋ࡞࠾ࠊࡇࢀ௨እᨻࡣࠊᚨᓥᏛᕤᏛ㒊 㛗 ⾲ ᙲ ࡞ ࡽ ࡧ Young Asian Biochemical Engineers
Community (YABEC) Plaque of Appreciation㸦Asian Federation of Biotechnology (AFOB)㸧ࢆཷ㈹ࡋ࡚࠸ࡿࠋ
1. 㔝⏣ ┿ᗈ බ┈♫ᅋἲே᪥ᮏ⏕≀ᕤᏛす᪥ᮏᨭ㒊 Ꮫ⏕㈹ 2013 ᖺ 12 ᭶
2. 㧗ᶫ ⯙㸪᳃ୗ ᫂ᙪ㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫 ୡ⏣ ⱥ᫂㸪ᨻ ྐ͆Chinese hamster ovary ⣽ ⬊ᰴ࠾ࡅࡿᰁⰍయᏳᐃᛶゎᯒᢠయ⏕⏘ࡢᛂ ⏝͇ ➨㸴㸳ᅇ᪥ᮏ⏕≀ᕤᏛ㸪1P-221㸪せ᪨㞟 p.73ࠊ 2013 ᖺࠊ9/18-20ࠊᗈᓥᅜ㝿㆟ሙࠊᗈᓥ㸦᪥ᮏ⏕≀ ᕤᏛࢭࣝࣉࣟࢭࢵࢩࣥࢢィ ホ౯◊✲㒊ඃ⚽Ꮫ ⏕Ⓨ⾲㈹㸧 3. ⟄ᬛ⨾㸪Kyoung Ho Lee㸪㨣㨣ሯ ṇ⩏㸪ⓑ ༤㸪㛫ୡ⏣ ⱥ᫂㸪ᨻ ྐ “⣽⬊࿘ᮇ࢚ࣥࢪࢽ ࣜࣥࢢࡼࡿChinese hamster ovary (CHO) ⣽⬊ ࠾ࡅࡿຠ⋡ⓗ࡞㑇ఏᏊቑᖜࢩࢫࢸ࣒ࡢᵓ⠏” ᪥ᮏ ື≀⣽⬊ᕤᏛ2013 ᖺᗘ㸦JAACT2013㸧せ᪨ 㞟p.50, 2013 ᖺ 7/18-19ࠊ࣍ࢸࣝࣇࢪࢱ⚟ࠊ⚟ (᭱ඃ⚽Ⓨ⾲㈹ࠊ66 ௳୰➨୍ᖍ) άືᡂᯝࡢㄽᩥ ᮏᥦࣉࣟࢪ࢙ࢡࢺࡢ┤᥋ࡢᡂᯝࡋ࡚ࠊୗグࡢㄽᩥࢆ సᡂࡋࠊᢞ✏ཷ⌮ࡉࢀࡓࠋ
1. MMasayoshi Onitsuka*, Akira Kawaguchi, Ryutaro Asano ,Izumi Kumagai, Kohsuke Honda, Hisao Ohtake, and TTakeshi Omasa "Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture" Journal of Bioscience and Bioengineering, Volume 117, Number 5, pp. 639-644 (2014).
N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there
was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70-80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates. (ࣔࣀࢡ࣮ࣟࢼࣝᢠయࡢ ᢠయࡢจ㞟య࠾ࡅࡿ N-ᆺ⢾㙐ᵓ㐀ࡢゎᯒࢆ⾜ࡗࡓ㸬 N-ᆺ⢾㙐ࡣᏳᐃᛶࡸจ㞟ᛶᙳ㡪ࢆ࠼ࡿࡀ▱ࡽ ࢀ࡚࠸ࡓࡀ㸪ᐇ㝿ࡢ⏕⏘ࣉࣟࢭࢫ࡛⏕ࡌࡿᢠయจ㞟 ⢾㙐ᵓ㐀ࡢ㛵㐃ࡣ᫂ࡢࡲࡲ࡛࠶ࡗࡓ㸬༢㔞యᢠయ ẚ㍑ࡋࡓᡤ㸪จ㞟ᢠయ࡛ࡣᚤ⣽࡞⢾㙐ᵓ㐀ᕪࡣ࡞࠸ ୍᪉࡛㸪N-ᆺ⢾㙐⮬యࡢຍ㔞ࡀῶᑡࡋ࡚࠸ࡿࢆ ぢฟࡋࡓ㸬ࡇࡢ⤖ᯝࡽ N-ᆺ⢾㙐ࡢḞᦆࡼࡿᏳᐃ ᛶࡢῶᑡࡀจ㞟ࡢཎᅉ࡛࠶ࡿࢆሗ࿌ࡋࡓ㸬)
2. MMasayoshi Onitsuka*, Miki Tatsuzawa, Ryutaro Asano, Izumi Kumagai, Akihiro Shirai, Hideaki Maseda and TTakeshi Omasa "Trehalose suppresses antibody aggregation during the culture of Chinese hamster ovary cells" Journal of Bioscience and Bioengineering, Volume 117, Number 5, pp. 632-638 (2014).
The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the
hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies. (ᢠయ⏕⏘ CHO ⣽⬊ࡢ⣽⬊ᇵ㣴㐣⛬࡛⏕ ࡌࡿᢠయจ㞟ࡢᙧᡂࢆ㜵ࡄ᪉ἲࡋ࡚㸪ᢠจ㞟స⏝ࢆ ᣢࡘࢺࣞࣁ࣮ࣟࢫࢆῧຍࡍࡿᇵ㣴ἲࢆᥦࡋࡓ㸬㔜 ≉␗ᛶᢠయEx3-scDb-Fc ⏘⏕⣽⬊ࢆࢺࣞࣁ࣮ࣟࢫࢆ ྵࡴᇵ㣴ᇵᆅ࡛ᇵ㣴ࡋࡓᡤ㸪ᢠయࡢ⏕⏘ᛶࡀୖ᪼ࡍࡿ ඹ㸪㧗ศᏊ㔞จ㞟యࡢᙧᡂࡀ1/3 ᢚไࡉࢀࡿࡇ ࢆぢฟࡋࡓ㸬จ㞟ᙧᡂ㐣⛬ࡢ㏿ᗘㄽⓗゎᯒࡽจ㞟 ᢚไ࣓࢝ࢽࢬ࣒ࢆᥦࡋࡓ㸬) ࡉ࠸ࡈ ᮏᥦࣉࣟࢪ࢙ࢡࢺ࠾࠸࡚ࡣࠊ༢࡞ࡿ◊✲ࡔࡅ࡛ࡣ࡞ ࡃࠊᅜࣉࣟࢪ࢙ࢡࢺࡢ❧ࡕୖࡆࡢࡓࡵࡢᚨᓥᏛࢆཧ⏬ᶵ 㛵ࡍࡿ⏘Ꮫ㐃ᦠࢆࡵࡊࡋࡓᆺࣉࣟࢪ࢙ࢡࢺ❧ࢆど 㔝ධࢀࡓάືࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊᮏࣉࣟࢪ࢙ࢡࢺࡢ⏘ Ꮫ㐃ᦠάືࢆ⥅⥆ࡋ࡚࠸ࡃࡇࡼࡾࠊᮏᏛࡶ୰᰾ཧ⏬ᶵ 㛵࡞ࡿᆺࣉࣟࢪ࢙ࢡࢺࡢ᥇ᢥࡘ࡞ࡀࡗࡓࠋᏛഃ࠾ ࠸࡚ࡣ⏘ᴗഃࡢࢽ࣮ࢬࢆ༑ศࡃࡳྲྀࡗࡓ◊✲㛤Ⓨࡀᚲ せࡉࢀࠊ⏘ᴗഃࡣࠊᏛᑐࡍࡿ┠ⓗᇶ♏◊✲ࢆ᥎㐍ࡍ ࡃࠊᙉ࠸࣓ࢵࢭ࣮ࢪࡀᚲせ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋ
