Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucos amine-6-phosphate isomerase (GPI) has been proposed as the key enzyme respo nsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc ) by accepting ammonium from the medium of cultured mammalian cells. As pre viously reported, the increased UDPGNAc pool then affects the N-glycan comp lexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK -21 cells and characterized. Thus, the complete pathway by which ammonium e nters the cellular metabolism was elucidated. To reduce the negative effect s of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expres sion. In both cases, the cellular UDPGNAc pool was suppressed in the presen ce of high ammonium concentrations in the medium. However, constant suppres sion of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed t hat the main reason for instability was the inducibility of GPI by its subs trate ammonium. GPI was induced to a factor of two under ammonium-containin g medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the prese nce of ammonium. (C) 1999 John Wiley & Sons, Inc.