Determination of protein purity and heterogeneity with capillary gel electrophoresis and capillary isoelectric focusing
Antibodies, protein-based therapeutics, and other recombinant proteins are final products in biotechnological and pharmaceutical industry. Determination of their purity, stability, and hetero geneity is of utmost importance since post-translational modifications as well as degradation processes can change drastically the biological activity of these proteins.
To determine the purity and heterogeneity of protein samples, two capillary-based methods are proposed – capillary gel electrophoresis (CGE) and capillary isoelectric focusing (cIEF). The former is applied when proteins differing in molecular mass must be resolved. The latter is applied when proteins or proteinisoforms exhibit almost identical molecular masses, but differ in net charges. In CGE, capillary is filled with a buffered polymer solution. Prior to analysis, protein sample is denatured in the presence of SDS by heating either under reducing (with reducing agent added) or non-reducing conditions. After rapid cooling the sample is injected, high voltage is applied, and proteins start migrating through polymer solution.
Similar to SDS-gel electrophoresis in a slab, proteins are separated in a capillary according to their molecular masses due to a sieving effect of the capillary polymer solution. Proteins differing by as little as 4% in molecular mass can be resolved. In cIEF, the entire capillary is filled with a polymer-based ampholyte-sample mixture. At the first stage, named focusing, high voltage is applied, which results in a pH gradient formation and focusing of proteins in narrow zones according to their pI values (where their net charges are equal to zero). At the end of this stage there is almost no any movement in the capillary and current is about zero.
During the second stage, named mobilization, the outlet vial is replaced with mobilization solution, high voltage is applied again, and focused zones start moving toward detector point. Depending on selected ampholytes, proteins and protein isoforms differing by as little as 0.04 pI unit can be resolved and quantified. For both methods a linear regression exists, reflecting the dependence of log Mw of the proteins (for CGE) or their pI values (for cIEF) upon migration time. This is used to determine both parameters of the unknown proteins by adding the corresponding markers to the sample solution.