on 05-23-2023 10:07 AM
Monoclonal antibodies (mAbs) are a preferred class of protein therapeutics used for the treatment of various diseases because of their ability to target specific tissues for drug delivery or the modulation of cellular activities.
Cellular production and downstream manufacturing processes commonly introduce heterogeneity to the mAb structure by way of post-translational or chemical modifications that can have potential effects on product efficacy, safety and stability. Therefore, thorough characterization of mAbs is required to fulfill regulatory requirements to bring new therapeutics to market.
Common modifications to the mAb structure (including lysine truncation, asparagine deamidation and glycosylation) alter the charge of the biomolecule by addition or elimination of cationic or anionic sites, resulting in increased charge heterogeneity.
Ion exchange (IEX) chromatography separates components based on their charge and is a standard technique for analyzing mAbs and associated variants. When using a pH gradient, the cationic protein is adsorbed to the stationary phase at low pH conditions followed by a gradient of increasing mobile phase pH. As the pH of the buffer increases, the charge of the protein shifts from cationic to neutral and then anionic at higher buffer pH values. The change in protein charge results in desorption from the anionic surface and elution from the column.
To easily facilitate these types of separations, you can use Thermo Scientific CX-1 pH gradient buffers to generate a linear pH gradient that simplifies method optimization for high-resolution separations of mAbs and their charge variants. However, to achieve the best separations with these buffers, you need to use an appropriate cation exchange column.
One smart choice is the Thermo Scientific ProPac 3R SCX column, which can analyze complex proteins with high resolution and excellent reproducibility. Here’s how:
When used together, the combination of the Thermo Scientific ProPac 3R SCX 3μm 2 x 50 mm column and Thermo Scientific CX-1 pH gradient buffers provide a high-resolution, fast, easy to optimize, and reproducible platform method for charge variant characterization of mAbs.
To read two practical examples of method design for NISTmAb and associated variants using ProPac 3R SCX2 x 50 mm column in combination with CX-1 pH gradient buffers, read this application note.