Process integration of cell disruption and fluidised bed adsorption of microbial enzymes: application to the retro-design of the purification of L-asparaginase

The practical feasibility and generic applicability of the direct integration in the same time frame of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption. In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished. Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration. Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the designof adsorbents for specific applications. In any purification of L-asparaginase (pI=8.6), product-debris interactions commonly diminish the recovery of available product. It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of overall product yield

Site-specific N-and O-glycosylation analysis of atacicept

International audienceThe Fc-fusion protein atacicept is currently under clinical investigation for its biotherapeutic application in autoimmune diseases owing to its ability to bind the two cytokines B-Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL). Like typical recombinant IgG-based therapeutics, atacicept is a glycoprotein whose glycosylation-related heterogeneity arises from the glycosylation-site localization, site-specific occupation and structural diversity of the attached glycans. Here, we present a first comprehensive site-specific N- and O-glycosylation characterization of atacicept using mass spectrometry-based workflows. First, N- and O-glycosylation sites and their corresponding glycoforms were identified. Second, a relative quantitation of the N-glycosylation site microheterogeneity was achieved by glycopeptide analysis, which was further supported by analysis of the released N-glycans. We confirmed the presence of one N-glycosylation site, carrying 47 glycoforms covering 34 different compositions, next to two hinge region O-glycosylation sites with core 1-type glycans. The relative O-glycan distribution was analyzed based on the de-N-glycosylated intact protein species. Overall, N- and O-glycosylation were consistent between two individual production batches