|Novel ways to introduce the traditional salt based chromatography technique of Ion Exchange Chromatography of biopharmaceutical proteins into High Resolution Mass Spectrometry
|Hyphenated Techniques for Comprehensive Analysis
|Mr Florian Fussl
|Dr Jonathan Bones
Dr ken cook
|Thermo Fisher Scientific
Abstract Information :
Thorough characterisation of Bio-therapeutic proteins is essential at all stages of development through to manufacture and final product quality control. Monoclonal Antibodies [Mab] are the fastest growing class of these new drugs due to their high specificity to the targets they are used against. Each Mab will have several different variant forms due to multiple post translational modifications that can occur during production, purification and storage. These modifications can often alter the charge distribution on the surface of the protein and so can be characterised by charge variant analysis using ion exchange chromatography. All modification variants on the Mab require characterisation and control to ensure product quality and reproducibility as they could have an impact on efficacy or safety. The charged variant profile specific to each biopharmaceutical product is also used as an indication of similarity in Biosimilar analysis. Identification of structural variants is a critical challenge and Mass Spectrometry [MS] is used as an essential tool in the characterisation and identification of the protein variants. However, the techniques of ion exchange and size exclusion of proteins both require high salt eluents in the chromatography which is incompatible with MS so the structural variants exposed by these techniques must be collected separately off-line, then desalted before further characterisation by MS. Here we describe novel direct on-line coupling of ion exchange to the MS instrument in the characterisation of Mab variants. The technique has a fast run time and greatly reduces analysis time and sample handling by avoiding fraction collection and separate desalting injections by reverse phase LCMS. The chromatographic resolution of MAb charged variants using pH gradient elution with a novel volatile buffer preparation compares favourably with traditional salt elution. The proteins enter the Orbitrap MS system in the native state with a reduced charge distribution and an elevated mass to charge ratio. Variants found with this direct on-line coupling include glycosylation, deamidation, oxidation and lysine truncation. However the final analysis results will also give a more accurate intact mass and a charged variant profile, elevating this novel methodology to a true multi attribute method with no sample preparation necessary.