title: Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production.
abstract: MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
authors.forename: Chia
authors.initials: C
authors.lastname: Chu
authors.forename: Vladimir
authors.initials: V
authors.lastname: Lugovtsev
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
authors.forename: Michael
authors.initials: M
authors.lastname: Betenbaugh
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis; D006801:Humans; D009981:Influenza B virus / Q000502:physiology*; D007700:Kinetics; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014779:Virus Replication
chemical_list: D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012333:RNA, Messenger; D012799:Sialyltransferases; C038525:CMP-N-acetylneuraminate-alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase
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