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 x UMG, where a 1 x concentration of U was 1 mM, a 1 x concentration of M was 0.002 mM, and a 1 x concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0 x UMG up to 21% at 8 x UMG and then more slowly to 23% at 20 x 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 x UMG except for suppression of glucose consumption and lactate production at 16 and 20 x UMG and a slight drop in antibody concentration at 20 x UMG. Higher accumulation of free galactose in the medium was observed at 8 x UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4 x 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 x 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 x UMG and no further increase or impact on culture performance up to 12 x 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. Biotechnol. Bioeng. 2011; 108: 1591-1602. (C) 2011 Wiley Periodicals, Inc.