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Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

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Abstract

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.

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Abbreviations

8-AG:

8-azaguanine

BSA:

Bovine Serum Albumin

EBV:

Epstein-Barr Virus

EIA:

Enzyme Immunoassay

FCS:

Fetal Calf Serum

FGF:

Fibroblast Growth Factor

HBsAg:

Hepatitis B virus surface Antigen

HBV:

Hepatitis B Virus

HDL:

High Density Lipoprotein

h-h:

human-human

IL-2:

Interleukin-2

IMDM:

Iscove Modified Dulbecco's Medium

LDL:

Low Density Lipoprotein

m-h:

mouse-human

m·h-h:

mouse·human-human

MoAb:

Monoclonal Antibody

PBL:

Peripheral Blood Lymphocyte

PEG:

Polyethylene Glycol

PVA:

Polyvinyl Alcohol

6-TG:

6-Thioguanine

T.T.:

Tetanus Toxoid

References

  1. AltshulerGL, DziewulskiDM, SowekJA and BelfortG (1986) Continous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators. Biotech. Bioeng. 28: 646–658.

    Google Scholar 

  2. BarnesDW, SirbaskuDA and SatoGH (1984) Cell culture methods for molecular and cell biology Vol. 1–4. Alan R. Liss, Inc. New York.

    Google Scholar 

  3. BödekerBGD, HuberGE, HewlettG and SchlumbergerHD (1987) Production of human monoclonal antibodies from immobilized cells in the opticell culture system. Develop. Biol. Standard. 66: 473–479.

    Google Scholar 

  4. BronD, FeinbergMB, TengNNH and KaplanHS (1984) Production of human monoclonal IgG antibodies against Rhesus (D) antigen. Proc. Natl. Acad. Sci. U.S.A. 81: 3214–3217.

    Google Scholar 

  5. BurnettKG, LeungJP and MartinisJ (1985) Human monoclonal antibodies to defined antigens: Towards clinical applications. In: EuglemanEG, FoungSKH, LarrickJ and RaubitschekA (eds) Human hybridomas and monoclonal antibodies pp. 113–133 Prenum Press, New York.

    Google Scholar 

  6. ChiorazziN, WassermannRL and KunkelHG (1982) Use of Epstein-Barr transformed B cell lines for the generation of immunoglobulin producing human B cell hybridomas. J. Exp. Med. 156: 930–935.

    Google Scholar 

  7. CoteRJ, MorrisseyDM, HoughtonAN, BeattleEJ, OettgenHF and OldLJ (1983) Generation of human monoclonal antibodies reactive with cellular antigens. Proc. Natl. Acad. Sci. U.S.A. 80: 2026–2030.

    Google Scholar 

  8. CroceCM, LinnenbachA, HallW, SteplewskiZ and KoprowskiH (1980) Production of human hybridomas secreting antibodies to measles virus. Nature 288: 488–489.

    Google Scholar 

  9. DarferFJ, MurakamiH, InselPA (1980) Growth of T-lymphoma cells in serum-free medium: Lack of involvement of the cyclic AMP pathway in long-term cultures of murine T-lymphoma cells. Proc. Natl. Acad. Sci. U.S.A. 77: 5993–5997.

    Google Scholar 

  10. EdwardsPAW, SmithCM, NevilleAM and O'HareMJ (1982) A human-human hybridoma system based on a fast growing mutant of the ARH-77 plasma cell leukemia derived line. Eur. J. Immunol. 12: 641–648.

    Google Scholar 

  11. EmanuelD, GoldJ, ColancinoJ. LopezC and HammerlingU (1984) A human monoclonal antibody to cytomegalovirus (CMV). J. Immunol. 133: 2202–2205.

    Google Scholar 

  12. ForsgrenA and SjoquistJ (1966) Protein A fromS. aureus, pseudoimmune reaction with human γ-globulin. J. Immunol. 97: 822–827.

    Google Scholar 

  13. FujiiDK and GospodarowiczD (1983) Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells. In Vitro 19: 811–817.

    Google Scholar 

  14. GigliottiF and InselRA (1982) Protective human hybridoma antibody to tetanus toxin. J. Clin. Invest. 70: 1306–1309.

    Google Scholar 

  15. GinsbergHS, GoldE and JordanWS (1955) Tryptose phosphate broth as supplementary factor for maintenance of HeLa cell tissue culture. Proc. Sci. Exp. Biol. Med. 89: 66–71.

    Google Scholar 

  16. HaradaK, IchimoriY, SasanoK, SasaiS, KitanoK, IwasaS, TsukamotoK and SuginoY (1989) Human-human hybridomas secreting hepatitis B virus-neutralizing antibodies. Bio/Technology 7: 374–377.

    Google Scholar 

  17. HimmelfarbP, ThayerPS and MartinHE (1969) Spin filter culture: The propagation of mammalian cells in suspension. Science 164: 555–557.

    Google Scholar 

  18. HosoiS, MiohH, AnzaiC, SatoS and FujyoshiN (1988) Establishment of Namalva cell lines which grow continuously in glutamine-free medium. Cytotechnology 1: 151–158.

    Google Scholar 

  19. IchimoriY, SasanoK, ItohH, HitotsumachiS, KimuraY, KanekoK, KidaM and TsukamotoK (1985) Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen. Biochem. Biophys. Res. Commun. 129: 26–33.

    Google Scholar 

  20. IchimoriY, HaradaK, HitotsumachiS and TsukamotoK (1987) Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen. Biochem. Biophys. Res. Commun. 142: 805–812.

    Google Scholar 

  21. Iwamoto K, Shintani Y, Sasada R, Honjo T and Kitano K (1990) Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene. Cytotechnology, in press.

  22. KatsukiT, HinumaY, YamamotoN, AboT and KumagaiK (1977) Identification of the target cells in human B lymphocytes for transformation by Epstein-Barr virus. Virology 83: 287–294.

    Google Scholar 

  23. KatsutaH, TakaokaT, FurukawaT and KawanaM (1960) Cultivation of strain HeLa cells (cervical carcinoma of human uterus) in a protein-free medium. Jpn. J. Exp. Med. 30: 147–157.

    Google Scholar 

  24. KatsutaH and TakaokaT (1973) Cultivation of cells in protein- and lipid-free synthetic media. Methods in Cell Biol. 6: 1–42.

    Google Scholar 

  25. KitanoK, ShintaniY, IchimoriY, TsukamotoK, SasaiS and KidaM (1986) Production of human monoclonal antibodies by heterohybridomas. Appl. Microbiol. Biotechnol. 24: 282–286.

    Google Scholar 

  26. KitanoK, IwamotoK, ShintaniY and AkiyamaS (1988) Effective production of a human monoclonal antibody against tetanus toxoid by selection of high productivity clones of a heterohybridoma. J. Immunol. Methods 109: 9–16.

    Google Scholar 

  27. KitanoK, IwamotoK, ShintaniY and SasadaR (1990) Improvement in cell lines for effective production of human monoclonal antibodies by human-human hybridomas. In: MurakakiH (eds.) Trends in animal cell technology, Kodansha, Tokyo and VCH, Weinheim.

    Google Scholar 

  28. KnazekRA, GullinePM, KohlerPO and DedrickRL (1972) Cell culture on artificial capillaries: An approach to tissue growth in vitro. Science 178: 65–66.

    Google Scholar 

  29. KöhlerG and MilsteinC (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.

    Google Scholar 

  30. KozborD and RoderJC (1981) Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique. J. Immunol. 127: 1275–1280.

    Google Scholar 

  31. KozborD, LagardeAE and RoderJC (1982a) Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines. Proc. Natl. Acad. Sci. U.S.A. 79: 6651–6655.

    Google Scholar 

  32. KozborD, RoderJC, ChangTH, SteplewskiZ and KoprowskiH (1982b) Human anti-tetanus toxoid monoclonal antibody secreted by EBV-transformed human B cells fused with murine myeloma. Hybridoma 1: 323–328.

    Google Scholar 

  33. KudoT, MorishitaR, SuzukiR and TachibanaT (1987) A great improvement of fusion efficiency in mouse B cell hybridoma production by use of the new culture medium, GIT. Tohoku J. Exp. Med. 153: 55–66.

    Google Scholar 

  34. KudoT, AsanoA and TachibanaT (1988) Highly efficient procedure for production of human monoclonal antibodies: Establishment of hybrids between Epstein-Barr virus-transfected B lymphocytes and heterohybridoma cells by use of GIT culture medium. Tohoku J. Exp. Med. 154: 345–355.

    Google Scholar 

  35. LarrickJW, TruittKE, RaubitschekAA, SenykG and WangJCN (1983) Characterization of human hybridomas secreting antibody to tetanus toxin. Proc. Natl. Acad. Sci. U.S.A. 80: 6376–6380.

    Google Scholar 

  36. LeeSM, GustafsonME, PickleDJ, FlickingerMC, MuschikGM and MorganACJr. (1986) Large-scale purification of a murine antimelanoma monoclonal antibody. J. Biotechnol. 4: 189–204.

    Google Scholar 

  37. LevyJA, VirollainenM and DefendiV (1968) Human lymphoblastoid lines from lymph node and spleen. Cancer 22: 517–524.

    Google Scholar 

  38. LydersenB, PughG, ParisM, SharmaB and NollL (1985) Ceraminc matrix for large scale animal cell culture. Biol/Technology 3: 63–67.

    Google Scholar 

  39. MaedaT, EdaY, NishiyamaK, IshikawaY, TashiroA and WatanabeT (1986) Production of stable mouse x human hybridomas secreting HBs antigen-specific human monoclonal antibody by using in vitro sensitization. Hybridoma 5: 33–41.

    Google Scholar 

  40. MizrahiA (1977) Primatone RL in mammalian cell culture media. Biotechnol. Bioeng. 19: 1557–1561.

    Google Scholar 

  41. MurakamiH (1989) Serum-free media used for cultivation of hybridomas. In: Monoclonal antibodies: production and application, pp. 107–141 Alan R. Lis, Inc. New York.

    Google Scholar 

  42. NeumannRE and TytellAA (1960) Serum less medium for cultivation of cells of normal and malignant origin. Proc. Soc. Exp. Biol. Med. 104: 252–256.

    Google Scholar 

  43. NowinskiR, BerglundC, LaneJ, LostromM, BernsteinI, YoungW, HakomoriS, HillL and CooneyM (1980) Human monoclonal antibody against Forssman antigen. Science 210: 537–539.

    Google Scholar 

  44. OhashiH, HashizumeS, MurakamiH, AiharaK, ShinoharaK and OmuraH (1986) HO-323, a human B-lymphoblastoid cell line useful for making human-human hybridomas. Cell Biology International Reports 10: 77–83.

    Google Scholar 

  45. OlssonK and KaplanHS (1980) Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc. Natl. Acad. Sci. U.S.A. 77: 5429–5431.

    Google Scholar 

  46. OlssonL, MazauricT, Vincent-FalquetJC and ArmandA (1984) A human monoclonal antibody specific for tetanus toxoid. Develop. Biol. Standard 57: 87–91.

    Google Scholar 

  47. OnoderaK, EtohY, TakanoS, MoriokaH and ShibaiH (1987) Structure and function of Ela genes of adenovirus type 12. In: UmedaM et al. (eds.) Biotechnology of mammalian cells, Jpn. Sci. Soc. Press, Tokyo and Springer-Verlag, Berlin.

    Google Scholar 

  48. PutnamJE (1987) Monoclonal antibody production in a ceramic matrix. In: Commercial Production of monoclonal antibodies — a guide for scale-up pp. 119–138. Marcell Dekker Inc., New York and Basel.

    Google Scholar 

  49. RukerF, ReiterS, JungbauerA, LiegelW, HimmlerG, SteinkellnerH, WenischE, SteindlF, WagnerK and KatingerH (1987) Self-hybridization of hybridomas leads to stabilization of clones and increased yield of monoclonal antibodies. Develop. Biol. Standard 66: 71–74.

    Google Scholar 

  50. Sasai S, Fujimoto T and Tsukamoto K (1987) Composition for cell cultivation, production and use thereof, US patent 4,654,304.

  51. SatoGH (1975) The role of serum in cell culture. In: LitwickG (eds.) Biochemical action of hormones. Vol. 3, pp. 391–396. Academic Press, New York.

    Google Scholar 

  52. SatoS, KawamotoK and FujiyoshiN (1983) Animal cell cultivation for production of biological substances with a novel perfusion culture apparatus. J. Tissue Culture Methods 8: 167–171.

    Google Scholar 

  53. SeaverSS (1987) Commercial production of monoclonal antibodies, a guide to scale-up. Marcel Dekker, Inc., New York and Basel.

    Google Scholar 

  54. SchlomJ, WunderlichD and TeramotoYA (1980) Generation of human monoclonal antibodies reactive with human mammary carcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 77: 6841–6845.

    Google Scholar 

  55. SeppalaI, SarvasH, PeterfyF and MakelaO (1981) The four subclasses of IgG can be isolated from mouse serum by using protein A-Sepharose. Scand. J. Immunol. 14: 335–342.

    Google Scholar 

  56. ShintaniY, IwamotoK and KitanoK (1989) Polyethylene glycols for promoting the growth of mammalian cells. Appl. Microbiol. Biotechnol. 27: 533–537.

    Google Scholar 

  57. ShintaniY, IwamotoK and KitanoK (1989) SSGF-I, a potent growth-promoting substance for mammalian cells from swine serum. Cytotechnology 2: 9–17.

    Google Scholar 

  58. SteinitzM, KleinG, KoskimiesS and MakelO (1977) EB virus induced B lymphocyte cell lines producing specific antibody. Nature 269: 420–422.

    Google Scholar 

  59. StrickerEAM, TieboutRF, LeliePN and ZeijlemakerWP (1985) A human monoclonal IgG anti-hepatitis B surface antibody: Production, properties and applications. Scand. J. Immunol. 22: 337–340.

    Google Scholar 

  60. TakazawaY, TokashikiM, HamamotoK and MurakamiH (1988) High cell density perfusion culture of hybridoma cells recycling high molecular weight components. Cytotechnology 1: 171–178.

    Google Scholar 

  61. TieboutRF, StrickerEAM, HagenaarsR and ZeijlemakerWP (1984) Human lymphoblastoid cell line producing protective monoclonal IgG1, x anti-tetanus toxin. Eur. J. Immunol. 14: 399–404.

    Google Scholar 

  62. TengNNH, LamKS, RieraFC and KaplanHS (1983) Construction and testing of mouse-human heteromyelomas for human monoclonal antibody production. Proc. Natl. Acad. Sci. U.S.A. 80: 7308–7312.

    Google Scholar 

  63. TengNNH, KaplanHS, HebertJM, MooseC, DouglasH, WunderlichA and BraudalA (1985) Protection against Gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies. Proc. Natl. Acad. Sci. U.S.A. 82: 1790–1794.

    Google Scholar 

  64. vonWedelRJ (1987) Mass culture of mouse and human hybridoma cells in hollow-fiber culture. In: SeaverSS (eds) Commercial production of monoclonal antibodies. Marcel Dekker Inc., New York and Basel.

    Google Scholar 

  65. WagnerR and LehmannJ (1988) The growth and productivity of recombinant animal cells in a bubble-free aeration system. Trends in Biotechnol. 6: 101–104.

    Google Scholar 

  66. WaymouthC (1956) A serum-free nutrient solution sustaining rapid and continuous proliferation of strain L (Earle) mouse cells. J. Nat. Cancer Institute 17: 315–325.

    Google Scholar 

  67. WeissMC and GreenH (1967) Human-mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl. Acad. Sci. U.S.A. 58: 1104–1111.

    Google Scholar 

  68. YeltonDE, DiamondBA, KwanSP and ScharffMD (1978) Fusion of mouse myeloma and spleen cells. Cur. Top. Microbiol. Immunol. 81: 1–7.

    Google Scholar 

  69. Ziegler-HeitbrockHWL, ReiterC, TrenkmannJ, FuttererA and RiethmullerG (1986) Protection of mice against tetanus toxin by combination of two human monoclonal antibodies recognizing distinct epitopes on the toxin molecule. Hybridoma 5: 21–31.

    Google Scholar 

  70. ZurawskiJrVR, HaberE and BlackPH (1978) Production of antibody to tetanus toxoid by continuous human lymphoblastoid cell lines. Science 199: 1439–1441.

    Google Scholar 

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Authors and Affiliations

  1. Research and Development Division, Takeda Chemical Industries, Ltd., Jusohonmachi 2-chome, Yodogawa-ku, 532, Osaka, Japan

    Kazuaki Kitano, Yuzo Ichimori, Hidekazu Sawada, Susumu Iwasa & Kyozo Tsukamoto

  2. Hikari Plant, Takeda Chemical Industries, Ltd., 743, Hikari-shi, Yamaguchi, Japan

    Seijiro Sasai

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  1. Kazuaki Kitano
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  2. Yuzo Ichimori
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Kitano, K., Ichimori, Y., Sawada, H. et al. Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas. Cytotechnology 5 (Suppl 2), 53–74 (1991). https://doi.org/10.1007/BF00573880

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