Proteins In pH Gradients Research Articles

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

A measuring grid for the analysis of isoelectrically focused proteins in polyacrylamide gel slabs

AbstractAn overlay grid was constructed to facilitate the measurement of densiometric scans of proteins in pH gradients formed by electrofocusing. The overlay procedure provides a rapid way for data collection. The relationship between pH and gel distance closely fit a straight line, (R >0.99). The reliability of the system was shown by the close agreement of the linear regressions from six different sets of data. Also, the feasibility of using a similar grid for a nonlinear system was established.