Abstract
Separation and analysis of charge heterogeneity in recombinant protein or monoclonal antibody (mAb) production is a prime quality control step in the biopharmaceutical industry. This step is often carried out by a combination of separation techniques followed by mass spectrometric detection. Two dimensional gel electrophoresis (2D-GE) is unrivalled in terms of resolution but is a tedious procedure. Here we present a combination of two easy methods that separate proteins in analogy to 2D-GE according to pI and molecular weight (kDa) with high reproducibility for the analysis of mAb's and recombinant proteins followed by mass spectrometry (MS) analysis. For the 1st dimension, OFFGEL electrophoresis was used. This method takes advantage of the impressive resolving power of immobilized pH gradient (IPG) gels but in contrast to conventional IEF delivers samples in liquid-phase. Fractions with charged isoforms in solution can directly be analyzed by MS. For the 2nd dimension, a highly sensitive microfluidic on-chip protein sizing method was employed. This method allows protein separation from 5 to 250 kDa and offers a sensitivity equivalent or better than silver staining and a linear dynamic range across four orders of magnitude. The charge heterogeneity in a mAb sample was evaluated under native conditions and with addition of a mild detergent (Tween-20). The difference in focusing patterns between the two conditions is clearly visualized with the Bioanalyzer protein assay. The combination of OFFGEL and lab-onchip protein analysis allows separation and identification of isoforms based on pI and molecular weight. Protein isoforms differing only by a few kDa in apparent molecular weight were successfully separated and enriched by OFFGEL electrophoresis for further downstream analysis by LC/MS-MS. The combination of OFFGEL, Bioanalyzer, and mass spectrometry is thus an efficient combination for detailed characterization of recombinant proteins such as mAb's.