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There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences.

A theoretically correct breakdown of the molecular wave function into Hartree-Fock and electron correlation components leads to the identification of four parameter problems important in approximate molecular orbital theory.The effects of the intramolecular environment on isolated atom optimized atomic orbitals, the deviation of simple basis atomic orbitals from true Hartree-Fock atomic orbitals, differences made in transforming to a basis of orthogonal atomic orbitals, and the estimation of the molecular electron correlation energy are considered. Order-of-magnitude corrections are suggested for each of these problems. Comparative NDDO all electron calculations on the carbon monoxide molecule illustrate the improved results obtained. Additional information on the performance of methods more approximate than the NDDO all electron one indicates that quantitatively they leave much to be desired.

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