pH gradient analysis of mAbs using 3 μm monodisperse particle strong cation exchange chromatography column

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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. 

The challenge with mAbs

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.  

The IEX solution

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. 

The tools

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. 

The optimal 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:   

• The packing material is based on a monodisperse 3 μm, nonporous divinylbenzene polymer particle to provide exceptionally high resolving power.  
• A thin, hydrophilic layer is grafted to the particle to reduce secondary interactions of protein samples with the hydrophobic core. This paired with precisely controlled grafting of SCX functionality minimizes band broadening for maximal resolution.  
• The column chemistry has been developed to be compatible with the CX-1 pH gradient buffers.  
• The reproducible resin chemistry and manufacturing processes eliminate column variability as a concern in method development and data analysis.  

Bottom line

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. 

Learn more

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.