Characterization of Humanized Antibody Produced by Apoptosis-Resistant CHO Cells under Sodium Butyrate-Induced Condition

  • Kim, No-Soo (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Chang, Kern-Hee (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Chung, Bo-Sup (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Kim, Sung-Hyun (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Kim, Jung-Hoe (Department of Biological Sciences, Korea Advanced Institute of Science and Technology) ;
  • Lee, Gyun-min (Department of Biological Sciences, Korea Advanced Institute of Science and Technology)
  • Published : 2003.12.01

Abstract

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

Keywords

References

  1. Anal. Biochem. v.230 Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthronilic acid Bigge,J.C.;T.P.Patel;J.A.Bruce;P.N.Goulding;S.M.Charles;P.R.Parekh https://doi.org/10.1006/abio.1995.1468
  2. Protein Methods Bollag,D.M.;S.J.Edelstein
  3. J. Microbiol. Biotechnol. v.11 Effect of sodium butyrate on glycosylation of recombinant erythropoientin Chung,B.S.;Y.T.Jeong;K.H.Chang;J.S.Kim;J.H.Kim
  4. Biochem. Biophys. Res. Commun. v.195 Butyric acid and its monosaccharide ester induce apoptosis in the HL-60 cell line Calabresse,C.;L.Venturini;G.Ronco;P.Villa;C.Chomienne;D.Belpomme https://doi.org/10.1006/bbrc.1993.2005
  5. Free Radic. Res. v.30 N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells Chang,K.H.;K..S.Kim;J.H.Kim https://doi.org/10.1080/10715769900300091
  6. Cytotechnology v.32 Stimulation of monoclonal antibody production of hybridoma cells by butyrate: Evaluation of a feeding strategy and characterization of cell behaviour Cherlet,M.;A.Marc https://doi.org/10.1023/A:1008069523163
  7. Cytotechnology v.15 Role of environmental conditions on the xpression levels, glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells Chotigeat,W.;Y.Watanapokasin;S.Mahler;P.P.Gray https://doi.org/10.1007/BF00762396
  8. Biotechnol. Bioeng. v.69 Metabolic shifts do not influence the glycostylation patterns of a recombinant fusion protein expressed in BHK cells Cruz,H.J.;C.M.Peixoto;M.Nimtz;P.M.Alves;E.M.Dias;J.L.Moreira;M.J.Carrondo https://doi.org/10.1002/(SICI)1097-0290(20000720)69:2<129::AID-BIT2>3.0.CO;2-5
  9. Biotechnol. Bioeng. v.60 Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid Ferrari,J.;J.Gunson;J.Lofgern;L.Krummen;T.G.Warner https://doi.org/10.1002/(SICI)1097-0290(19981205)60:5<589::AID-BIT9>3.0.CO;2-K
  10. J. Immunol. Methods v.77 Measurements of the true affinity constant in solution of antigen-antigen-antibody complexes by enzyme-linked immunosorbent assay Friguet,B.;A.F.Chaffotte;L.Djavadi-Ohaniance;M.G.Goldberg https://doi.org/10.1016/0022-1759(85)90044-4
  11. Glycobiology: A Practical Approach Fukuda,M.;A.Kobata
  12. Biotechnol. Bioeng. v.46 Effect of different cell culture conditions on the polypeptide integrity and N-glycosylation of a recombinant model glycoprotein Gawlitzek,M.;H.S.conradt;R.Wagner https://doi.org/10.1002/bit.260460606
  13. Biotechnol. Bioeng. v.68 Ammonium alters N-glycan structures of recombinant TNFG-IgG: Degradative versus biosynthetic mechanisms Gawlitzek,W.;T.Ryll;J.Lofgren;M.B.Sliwkowski https://doi.org/10.1002/(SICI)1097-0290(20000620)68:6<637::AID-BIT6>3.0.CO;2-C
  14. Biotechnol. Bioeng. v.62 Apoptosis in batch cultures of Chinese hamster ovary cells Goswami,J.;A.J.Inskey;H.Steller;G.N.Stephanopoulos;D.I.Wang https://doi.org/10.1002/(SICI)1097-0290(19990320)62:6<632::AID-BIT2>3.0.CO;2-I
  15. Biotechnol. Bioeng. v.55 Site- and branch-specific sialylation of recombinant human interferon-γ in Chinese hamster ovary cell culture Gu,X.;B.J.Harmon;D.I.Wang https://doi.org/10.1002/(SICI)1097-0290(19970720)55:2<390::AID-BIT16>3.0.CO;2-L
  16. Biotechnol. Bioeng. v.58 Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine Gu,X.;D.I.Wang https://doi.org/10.1002/(SICI)1097-0290(19980620)58:6<642::AID-BIT10>3.0.CO;2-9
  17. Anal. biochem. v.240 A repid high-resolution high-performance liquid chromatographic method for separating glycan imxtures and analyzing oligosaccharide profiles Guile,G.R.;P.M.Rudd;D.R.Wing;S.B.Prime;R.A.Dwek https://doi.org/10.1006/abio.1996.0351
  18. Biotechnology v.13 N-glycosylation of recombinant human interferon-γ produced in different animal expression system James,D.C.;R.B.Freedman;M.Hoare;O.W.Ogonah;B.C.Rooney;O.A.Larionov;V.N.Dobrovolsky;O.V.Lagutin;N.Jenkins https://doi.org/10.1038/nbt0695-592
  19. Biotechnol. Bioeng. v.51 Decreased chimeric antibody productivity of KR12H-1 transfectoma during long-term culture results from decreased antibody gene copy number Kim,J.H.;S.W.Bae;H.J.Hong;G.M.LEe https://doi.org/10.1002/(SICI)1097-0290(19960820)51:4<479::AID-BIT11>3.0.CO;2-D
  20. Biotechnol. Bioeng. v.71 Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells, resulting in enhanced humanized antibody production Kim,N.S.;G.M.Lee https://doi.org/10.1002/1097-0290(2000)71:3<184::AID-BIT1008>3.0.CO;2-W
  21. Cytotechnology v.29 Na-butyrate increases the production and α2,6-sialyltransferase engineered CHO cells Lamotte,D.;L.Buckberry;L.Monaco;M.Soria;N.Jenkins;J.M.Engasser;A.Marc https://doi.org/10.1023/A:1008080432681
  22. Biochem. Biophys. Res. Commun. v.218 High-level expression of human inducible nitric oxide synthase in Chinese hamster overy cells and characterization of the purified enzyme Laubach,V.E.;E.P.Garvey;P.A.Sherman https://doi.org/10.1006/bbrc.1996.0143
  23. J. Microbiol. Biotechnol. v.11 In vitro detection of apoptosis in human promyelocytic leukemia HL 60 cells by ¹H-NMR Lee,C.H;M.A.Lee;Y.H.Cho;H.Y.Lee;J.H.Jung;K.H.Kim;Y.H.Lim
  24. Cell v.72 A positive role for histone acetylation in transcription factor access to nucleosomal DNA Lee,D.Y.;J.J.Hayes;D.Pruss;A.P.Wolffe https://doi.org/10.1016/0092-8674(93)90051-Q
  25. J. Biol. Chem. v.265 The polypeptide of immunoglobulin G influences its galactosylation in vivo Lee,S.O.;J.M.Connolly;D.Ramirez-Soto;R.D.Poretz
  26. J. Microbiol. Biotechnol. v.12 Production of O-GlcNac modified reocmmbinant proteins in Escherichia coli Lim,K.H.;H.H.Chang;H.I.Chang
  27. Biotechnol. Bioeng. v.54 Effect of bench-scale culture conditions on murine IgG heterogeneity Marino,M.;A.Corti;A.Ippolito;G.Cassani;G.Fassina https://doi.org/10.1002/(SICI)1097-0290(19970405)54:1<17::AID-BIT2>3.0.CO;2-0
  28. Cancer Res. v.57 Induction of caspase-3 activity and apoptosis by butyrate and trichostatin A (inhibitor of hstone deacetylase): Dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway Medina,V.;B.Edmonds;G.P.Young;R.James;S.Appleton;P.D.Zalewski
  29. J. Immunol. Methods v.247 Butyrate increases production of human chimeric IgG in CHO-K1 cells whilst maintaining function and glycoform profile Mimura,Y.;J.Lund;S.Church;S.Dong;J.Li;M.Goodall;R.Jeffers https://doi.org/10.1016/S0022-1759(00)00308-2
  30. J. Biotechnol. v.19 Production of analytical quantitites of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression Palermno,D.P.;M.E.DeGraaf;K.R.Marotti;E.Rehberg;L.E.Post https://doi.org/10.1016/0168-1656(91)90073-5
  31. Bitechnol. Bioeng. v.68 Engineering of coordinated up-and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells Prati,E.G.;M.Matasci;T.B.Suter;A.Dinter;A.R.Sburlati;J.E.Bailey https://doi.org/10.1002/(SICI)1097-0290(20000505)68:3<239::AID-BIT1>3.0.CO;2-7
  32. J. Microbiol. Biotechnol. v.11 Flow cytometric analysis of the effect of silkworm heamlymph on the baculvirus induced insect cell apoptosis Rhee,W.J.;T.H.Park
  33. Glycoconj. J. v.14 The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells Routier,F.H.;M.J.Davies;K.Bergemann;E.F.Hounsell https://doi.org/10.1023/A:1018589704981
  34. Biochem. Biophys. Res. Commun. v.258 Aberrant metabolic sialylation of recombinant proteins expressed in chinese hamster ovary cells in high productivity cultures Santell,L.;T.Ryll;T.Etcheverry;M.Santoris;G.Dutina;A.Wang;J.Gunson;T.G.Warner https://doi.org/10.1006/bbrc.1999.0550
  35. Carcinogenesis v.7 n-Butyrate alters chromatin accessiblility to DNA repair enzymes Smith,P.J. https://doi.org/10.1093/carcin/7.3.423
  36. J. Microbiol. Biotechnol. v.12 Adenovirus-mediated antisense expression of telomerase template RNA induces apoptosis in lung cancer cells Song,J.S.;S.B.Kim;Y.H.Lee;K.Y.Lee;H.H.Jung;M.H.Kim;K.T.Kim;R.Brown;Y.T.Kim
  37. Biotechnol. Bioeng. v.68 Influence of bcl-2 on cell death during the cultrivation of a CHinese hamster ovary cell line expressing a chimeric antibody Tey,B.T.;R.P.Singh;L.Piredda;M.Piacentini;M.Al-Rubeai https://doi.org/10.1002/(SICI)1097-0290(20000405)68:1<31::AID-BIT4>3.0.CO;2-L
  38. Hum. Reprod. v.3 Immunological and biological potencies of the different molecular species of gonadotrophins Ulloa-Aguirre,A.;R.Espinoza;P.Damian-Matsumura;S.C.Chappel https://doi.org/10.1093/oxfordjournals.humrep.a136734
  39. Trens Biotechnol. v.15 Effect of glycosylation on antibody function: Implications for genetic engineering Wrigth,A.;S.L.Morrison https://doi.org/10.1016/S0167-7799(96)10062-7
  40. Biotechnol. Bioeng. v.68 Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation Yang,M.;M.Butler https://doi.org/10.1002/(SICI)1097-0290(20000520)68:4<370::AID-BIT2>3.0.CO;2-K