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Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We(More)
The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have(More)
The structural features of the asymmetric activated states of the insulin receptor family are still poorly understood. We investigated hydrogen/deuterium (H/D)-exchange within the extracellular domain of the type-I insulin-like growth factor receptor (IGF-1R) in the absence and presence of IGF-1 (active state) and in the presence of antibody inhibitors(More)
Protein function is dictated by protein conformation. For the protein biopharmaceutical industry, therefore, it is important to have analytical tools that can detect changes in protein conformation rapidly, accurately, and with high sensitivity. In this paper we show that hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can play an important role in(More)
A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic(More)
Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both(More)
Characterization and quantitative analysis of oxidation plays an important role in biopharmaceutical development. This study demonstrates an approach to the assessment of susceptible to oxidation methionine residues in monoclonal antibodies and recombinant proteins. A method for the determination of oxidation levels by peptide mapping with mass(More)
To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is,(More)
A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro(More)
In this work several aspects of imaging capillary IEF (icIEF) application for charge heterogeneity analysis of recombinant proteins and monoclonal antibodies have been discussed. Advantages of the method as compared with traditional approaches for determination of biomolecule charge heterogeneity, such as gel and IEC, have been demonstrated. Correlation of(More)