A high-resolution HIC application for the separation of fusion protein therapeutics. This application, based on protein surface hydrophobicity, allows separation of a fusion protein from a truncated version and structural isomer.
Cysteine-conjugated ADC mimics are successfully separated based on their drug-to-antibody ratio (DAR) using a Thermo Scientific™ MAbPac™ HIC-10 column. Separation of ADC mimics was optimized by adjusting temperature, solvent content, and gradient.
This application note demonstrates the rapid separation of a wide mass range of peptides and proteins using a reversed-phase monolithic column with both UV and mass spectrometry (MS) detection. The separation is conducted on Thermo Scientific™ ProSwift™ RP-4H columns using conventional water/acetonitrile-based eluents. These simple, straightforward methods can be used to optimize chromatographic separations for analysis time, resolution, and MS detection especially for top-down proteomics.
By combining HPAE-PAD analysis with acid hydrolysis and enzymatic digestion of protein glycans, information about the glycan identity as well as terminal carbohydrate linkage isomers can be determined from small amounts of protein.
In this Application Note, an Orbitrap-based mass spectrometer is used for high-confidence analysis of the intact and reduced forms of rituximab (monoclonal antibody), in biopharmaceutical product development and characterization.
The Thermo Scientific™ MAbPac™ Protein A column is a high-performance affinity chromatography column specifically designed for the determination of monoclonal antibody concentration in harvest cell culture. The nonporous, hydrophilic stationary phase enables fast and accurate MAb titer analysis. The ability to collect the IgG fraction during the HPLC analysis allows the analyst to characterize the IgG charge variants using a novel pH gradient approach.