Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

CHO3E7 cells were grown in suspension in F17 medium supplemented with 4 mM glutamine and 0.1 % Kolliphor to a cell density of 1.5×10^6 cells/mL (>99 % viability) in ventilated flasks (SF) shaken at 120 rpm in a humidified incubator at 37 °C with 5 % CO2. PEIpro and plasmid DNA solutions were prepared in F17 for a final concentration of 2.5 µg/mL of culture and 1 µg/mL of culture, respectively. The solution of PEIpro was added to the DNA solution to form polyplexes, which were added to the cells after 5 min of incubation at room temperature. One day post-transfection, the cells were fed with peptone TN1 (1 % final) to enhance productivity. Six days post-transfection, the supernatants were collected.

Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human alpha-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble beta-galactosamide alpha-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 muM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.