Sepax offers three different SEC phase chemistries, SRT®, SRT®-C and Nanofilm®, for different membrane protein/detergent complex separations. Due to the unique features of membrane protein/detergent complexes, one of the phases may provide best resolution/recovery for your sample.
Due to the different hydrodynamic sizes of these complexes, a larger pore size may need to be evaluated to achieve the best HMW aggregate and monomer separation.
With 5 and 10µm particle choices and various analytical and prep column dimensions, Sepax SEC columns can be used for detergent screening, high resolution analytical separation, and purification of membrane protein in crystallography processes.
Antibody drug conjugates are created by linking a potent small molecule to a monoclonal antibody (mAb). It combines the potency of cytotoxic drugs with the high specificity of monoclonal antibody to target many oncology diseases. Due to the small molecule property, the chemical linking chemistry and different amino acid conjugation, antibody drug conjugate (ADC) exhibits a more complex and heterogeneous structure than the parent monoclonal antibody.
In this webinar, we would like to present the ADC characterization in the following four areas.
Size exclusion chromatography (SEC):
ADC aggregate, monomer and fragment analysis using Zenix®-C SEC-300 size exclusion chromatography. This analysis can be part of the ADC lot release and stability assays.
Free small molecule drugs analysis after the conjugation reaction can be achieved with Zenix®-C SEC-80 (the smallest pore size 80 Å in the Sepax SEC product line).
Volatile mass spec friendly mobile phase can be applied to determine the intact mass of ADC
Hydrophobic interaction chromatography (HIC): ADC with cysteine conjugates drug to antibody ratio (DAR) can be monitored by Proteomix® HIC Butyl, fractions can be collected and further analyzed by other analytical methods.
Cation exchange chromatography (CEX): ADC charge variants can be characterized by CEX.
Reversed-phase chromatography (RP): mAb and ADC large fragments such as HC/LC, Fab/Fc and fragments from IDES digestion can be analyzed by Proteomix® RP and detected by LC/MS.
Recombinant monoclonal antibodies (mAbs) have become a very important segment of protein drug therapeutics. Post-translational modifications including glycosylation, oxidation, deamidation and incomplete C-terminal processing can contribute to antibody heterogeneity. Degradation reactions during manufacturing, formulation, and storage can also cause structural and functional changes in mAbs. It is important to characterize and evaluate the heterogeneities and monitor the stability of mAbs for shelf-life studies. Antibodix weak cation exchange (ABX WCX), Proteomix strong cation exchange (SCX) and Proteomix weak cation exchange (WCX) are applicable in separating antibodies with minor forms. These three resin surfaces are grafted different cation exchangers on a hydrophilic layer which offer different selectivity toward different antibodies.
More specifically, this webinar will cover:
Introduction to CEX method development and optimization
AntibodixTM WCX-NP5, ProteomixTM SCX-NP5, and ProteomixTM WCX-NP5 for separations of variants and stability studies with a number of mAbs
Monoclonal antibodies have been increasingly becoming drug candidates for disease therapeutics. Due to the molecular complexity of monoclonal antibodies, the characterization remains a challenge and required step throughout the development and manufacturing process. In order to determine the efficacy of the molecules, aggregation, heterogeneity such as charge variants, C-terminal lysine processing, deamidation, glycosylation must be screened for their structural and biological changes. Antibody solution kit offers a complete separation solution for monoclonal antibody analysis and characterization. Sepax offers a complete set of tools for monoclonal antibody analysis, including Zenix SEC-300 and Antibodix NP5 columns.
Sepax Zenix-300 size exclusion chromatographic (SEC) column is designed for high efficiency and resolution separation of monoclonal antibody monomers, aggregates, fragments such as heavy/light, fab/fc and f(ab')2 fragments. With its uniform, hydrophilic, and neutral nanometer thick films chemically bonded on high purity silica, the non-specific interaction between proteins and column surface is minimized to result in high resolution of monoclonal antibody separation. With volatile mobile phase systems, Zenix-300 is able to separate MAb fragments, which can be analyzed by on-line mass spectrometry. Molecular weight information of MAb fragments is then obtained with SEC-LC/MS work flow.
Sepax Antibodix NP5 weak cation exchange column to separate the charge variants. Multiple mobile phase systems were investigated for optimum charge variant separation. With its non-porous polymer bead, the Antibodix WCX is suitable for resolving slightly different structures of monoclonal antibodies within a wide pH range of 2-12.