title: Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose.
abstract: Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 × UMG, where a 1× concentration of U was 1 mM, a 1× concentration of M was 0.002 mM, and a 1× concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0× UMG up to 21% at 8× UMG and then more slowly to 23% at 20× UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20× UMG except for suppression of glucose consumption and lactate production at 16 and 20× UMG and a slight drop in antibody concentration at 20× UMG. Higher accumulation of free galactose in the medium was observed at 8× UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4× UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12× concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8× UMG and no further increase or impact on culture performance up to 12× UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium.
authors.affiliation: Genmab MN, Inc., 9450 Winnetka Ave N, Brooklyn Park, Minnesota 55445, USA. m.gramer@genmab.com
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gramer
authors.forename: Jackie J
authors.initials: JJ
authors.lastname: Eckblad
authors.forename: Ruth
authors.initials: R
authors.lastname: Donahue
authors.forename: Joseph
authors.initials: J
authors.lastname: Brown
authors.forename: Carrie
authors.initials: C
authors.lastname: Shultz
authors.forename: Kent
authors.initials: K
authors.lastname: Vickerman
authors.forename: Patrick
authors.initials: P
authors.lastname: Priem
authors.forename: Ewald T J
authors.initials: ET
authors.lastname: van den Bremer
authors.forename: Jolanda
authors.initials: J
authors.lastname: Gerritsen
authors.forename: Patrick H C
authors.initials: PH
authors.lastname: van Berkel
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D019149:Bioreactors; D016466:CHO Cells; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D005690:Galactose / Q000378:metabolism*; D006031:Glycosylation; D017895:Manganese Compounds / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
chemical_list: D000906:Antibodies; D002712:Chlorides; D003470:Culture Media; D017895:Manganese Compounds; D011994:Recombinant Proteins; C025340:manganese chloride; D014529:Uridine; D005690:Galactose