Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose. Glucose depletion occurred in cultures with an initial glucose ≤5.5 mM and seeded at low density (2.5 × 10(5) cells/mL) or at high cell inoculum (≥2.5 × 10(6) cells/mL) at higher glucose concentration (up to 25 mM). Glucose-depleted cultures produced non-glycosylated Mabs (up to 51%), lower galactosylation index (GI <0.43) and decreased sialylation (by 85%) as measured by mass spectrometry and HPLC. At low glucose a reduced intracellular pool of nucleotides (0.03-0.23 fmoles/cell) was measured as well as a low adenylate energy charge (<0.57). Low glucose also reduced GDP-sugars (by 77%) and UDP-hexosamines (by 90%). The data indicate that under glucose deprivation, low levels of intracellular nucleotides and nucleotide sugars reduced the availability of the immediate precursors of glycosylation. These results are important when applied to the design of fed-batch cultures.

For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.

The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol continues with gel electrophoresis. Bands containing the heavy chain are cut and isolated from the gel followed by tryptic digestion to obtain peptides and glycopeptides. The enrichment of glycopeptides by C18 chromatography is described followed by characterization using positive and negative modes MALDI-MS and MS/MS. An exoglycosidase, beta-galactosidase, is used to verify anomericity of linkages in the glycan portion of glycopeptides. In the last step, glycans are detached from glycopeptides using PNGase F labelled with phehylhydrazine and characterized by MALDI-MS/MS. This workflow is reported for the first time for this particular antibody and presents a valuable approach for the analysis of N-glycans on most antibodies and glycoproteins.Résumé : La caractérisation de la portion N-glycane des anticorps a fait l'objet de nombreuses études faisant appel à la spectrométrie de masse. Dans le présent article, nous présentons un protocole méthodologique qui débute par l'expression d'un anticorps monoclonal (EG2-hFc) dans des cellules ovariennes de hamster et se poursuit avec la purification de la protéine A de l'anticorps suivie de l'électrophorèse en gel. Les bandes contenant les chaînes de masse élevée sont découpées et isolées du gel, et une digestion tryptique est effectuée en vue d'obtenir des peptides et des glycopeptides. Nous décrivons l'enrichissement des glycopeptides par chromatographie sur colonne C18, puis la caractérisation au moyen de la MALDI-MS et SM/SM en modes positif et négatif. Nous avons utilisé la bêta-galactosidase, une exoglycosidase, afin de vérifier l'anoméricité des liaisons dans la portion glycane des glycopeptides. En dernière étape, nous avons détaché les glycanes des glycopeptides à l'aide de la PNGase F, les avons marqués à l'aide de la phénylhydrazine et les avons caractérisés par spectroscopie de masse MALDI-SM/SM. Ce protocole pour cet anticorps précis est publié ici pour la première fois et présente une approche intéressante permettant d'effectuer l'analyse des N-glycanes pour la plupart des anticorps et des glycoprotéines. [Traduit par la Rédaction]

A quantitative understanding of the process of retrovirus-mediated gene transfer into mammalian cells should assist the design and optimization of transduction protocols. We present a mathematical model of the process that incorporates the essential rate-limiting transduction steps including diffusion, convection and decay of viral vectors, their binding at the cell surface and entry into the cell cytoplasm, reverse transcription of uncoated RNA to form DNA intermediates, transport of the latter through the cytosol to the cell nucleus and, finally, nuclear import and integration of the delivered DNA into the target cell genome. Cell and virus population balances are used to account for the kinetics of multiple vector infections which influence the transduction efficiency and govern the integrated copy number. The mathematical model is validated using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. Viral intermediate complexes derived from the internalized retroviral vectors are found to remain stable inside the K562 cells and the cytoplasmic trafficking time is consistent with the time scale for retrovirus uncoating, reverse transcription and transport to the cell nucleus. The model predictions of transduction efficiency and integrated copy number agree well with experimental data for both static (i.e., standard gravity) and centrifugation-based gene transfer protocols. The formulation of the model can also be applied to transduction protocols involving lenti- or foamy-viruses and so should prove to be useful for the optimization of several types of gene transfer processes.