To demonstrate the CX-1 pH buffer system is capable of charge variant determination for the majority of therapeutic monoclonal antibody species. To show the ease of optimization and improved reproducibility of this buffer system when compared to salt-based gradient systems used for charge variant analysis.
A UHPLC workflow for mAb aggregate analysis that operates continuously for weeks.
Ion exchange separation is applied for charge variant analysis of Bevacizumab using a pH gradient on a Thermo Scientific MAbPac SCX-10 column. The Thermo Scientific Vanquish Flex UHPLC system with UV detection at 280 nm was used with Thermo Scientific CX-1 pH gradient buffers enabled high resolution separation of monoclonal antibody charge variants over a 30 minute gradient.
The Thermo Scientific Dionex UltiMate 3000 LC Bio-LC system is applied for the Hydrophobic Interaction (HIC) analysis of Bevacizumab. The separation was performed on a Thermo Scientific MabPac HIC-20 column at UV detection of 214 nm. With a total run time of 40 minutes the method allows separation of all oxidized and deamidated impurities of Bevacizumab.
The importance of correct UHPLC instrument setup for protein aggregate analysis by size-exclusion chromatography is shown by using the Thermo Scientific Vanquish Flex Quaternary UHPLC system. For this analysis, pre-column dispersion was reduced to show the effects of dispersion in front of the column at different flow rates. After the flow rates are reduced to 300 μL/min with the MAbPac-SEC-1 column formats of 4 mm i.d., the use of low volume tubing and fittings becomes essential in order to maintain resolution of aggregates from the main peak. Refer to attached pdf file for full experiment.
