Gel Electrophoresis

Polyacrylamide Gel Electrophoresis (PAGE) provides a relatively inexpensive yet efficient method for visualising protein mixtures. The technique involves applying a protein sample to a gel matrix (usually acrylamide) and applying a voltage to the gel, causing protein in the mixture to separate according to physical properties, such as net charge and molecular weight.

Gel electrophoresis may be used for various applications in biopharmaceutical product characterisation:

  • SDS-PAGE - to assess the molecular weight and purity of a protein, including the identification of the presence of truncated forms of a protein product.

  • Isoelectric focusing (IEF) - to determine the isoelectric point of a protein and to establish the presence of charge heterogeneity, due to post-translational modifications such as glycosylation or deamidation. Capillary isoelectric focusing (cIEF), offering superior data to conventional CE, is also available for this purpose.

  • Native-PAGE - to assess product-related impurities, such as aggregation.

  • 2D-PAGE - the combination of two electrophoretic techniques, such as IEF and SDS-PAGE in two dimensions, can provide a resolving power for protein mixtures greater than either technique by itself. This approach is commonly used in proteomic applications where there is a need to study cellular protein expression and, typically, tens to hundreds of proteins can be resolved using this methodology.

SDS-PAGE and isoelectric focusing are key parts of the ICH Q6B guidelines for characterisation and confirmation of biopharmaceuticals in support of new marketing applications.