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A comparative method for the quantitative analysis of the ratio of oxonium fragment (reporter) ions derived from sialic acid and N-acetylhexosamine residues on a large intact glycoprotein, the B domain of recombinant human factor VIII (rhFVIII), was developed. The method utilized liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) on a single-quadrupole instrument. During development, systematic approaches such as full-matrix and simplex strategies were used for the optimization of the signal-to-noise ratio by controlling source temperature and cone voltage. The method was found to be precise (RSD = 0.84%), sensitive (capable of differentiating 1 sialic acid residue change among at least 29 sialic acids on a 103-kDa glycoprotein that is 38% carbohydrate), applicable to a wide range of loading (11.6-372 micrograms of FVIII), and accurate according to a comparison to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Combining the method with enzymatic removal of N-glycans, selective O-glycan analysis was also performed leading to differential fragment ion analysis ascribed to N- and O-glycans. Quantitative ESI in-source dissociation MS combined with LC can generally be used for glycoproteins, as one of the indicators, to compare the distribution of carbohydrate residues over N- and O-glycans, to investigate their isoforms, and compare batch-to-batch characteristics of biopharmaceuticals.

This article examines retention in preparative chromatography in terms of the stoichiometric displacement model. This model is based on an equilibrium in which proteins and small displacing agents compete for ligands on the surface of chromatographic sorbents. Both the equilibrium constant for this process and the three‐dimensional structure of the macromolecule being separated are shown to be major determinants of retention in the analytical (linear) mode. A theoretical application of this model to preparative (nonlinear) separations shows a high probability that there will be perturbations of the chromatographic distribution coefficient. The activity coefficients of proteins, molecular orientation of proteins toward the sorbent surface, and adsorption kinetics are both load and sample dependent. In addition, displacement effects by other macromolecular species are predicted at high proteins concentrations. This theoretical analysis concludes that mathematical modeling of the retention process under nonlinear conditions will be more sample specific and require significantly more data on column operating conditions, interfering sample components, sample load, and the chemical nature of the stationary phase.