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A new concept in reaction-based chemical analysis is introduced and theoretically described. By utilization of the variability in electrophoretic mobilities among charged species, spatially distinct zones of chemical reagents can be electrophoretically merged under the influence of an applied electric field. Electrophoretically mediated microanalysis (EMMA) exploits this phenomenon as a basis for chemical analysis utilizing capillary electrophoretic systems. EMMA is described in terms of the four stages required for reaction-based analysis: (1) analyte and analytical reagent metering; (2) initiation of reaction; (3) control of reaction conditions and product formation; (4) detection of species whose production or depletion is indicative of the concentration or quantity of the analyte of interest. The method is illustrated by the enzymatic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase with the concurrent reduction of NAD+ to NADH monitored at 340 nm. Experimental results for both substrate and enzyme determinations are shown to agree with the presented theory.

An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing the potential glycosylation sites. Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-specific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.

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Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site-and branchspecific differences in sialylation of human interferon-␥ (IFN-␥) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-␥'s two potential N-linked glycosylation sites (i.e., Asn 25 and Asn 97). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn 25 were more heavily sialylated than those of Asn 97. Furthermore, Man(␣1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(␣1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product.