The development of highly expressed recombinant proteins in cell culture is consequential of using high producing cell lines. One of the most commonly used cell line for high level heterologous protein expression is Chinese hamster ovary (CHO), which produces proteins with glycoforms that are both compatible and bioactive in humans. The higher yields are accompanied by the protein quality issues and one of the most critical one pertains to the protein degradation during the production of glycoproteins. Protein degradation or clipping commonly attributed to the activity of host cell proteases in certain cell culture cannot be avoided but could only be controlled or inhibited. Due to regulatory and safety prospects, the protease inhibition by the addition of inhibitors or substitute substrates is not appropriate, thus it is critical to monitor the protein clipping during the production in order to ensure the quality of the product.In order to optimize process development or perform formulation effective monitoring of the clipped fragments is required.Here we present, a novel reducing size exclusion chromatography (R-SEC) method to monitor the proteolytic activity of a recombinant glycoprotein antigen. To achieve this, R-SEC method was performed using optimized conditions on an ACQUITY UPLC H-Class System using BEH200 SEC column (4.6 mm X 300 mm) under isocratic conditions. The mobile phase used was 20% acetonitrile containing trifluoroacetic acid and formic acid and sample treatment was performed using urea and dithiothreitol (DTT) at RT.The R-SEC detects the glycoprotein clipped fragments quantitatively with significant higher resolutions as compared to the current gold standard SDS PAGE. In addition to being quantitative in nature the R-SEC is a less labor intensive method and can be online coupled to multiangle light scattering (MALS) detector or mass spectrometry for further structural characterization. The reported method was successfully used during process development and formulation development of a Novartis viral vaccine candidate.

Separation of glycoproteins clipped products attributed to residual cell protease activity using a novel reducing size exclusion chromatography (R-SEC) method

248th National Meeting (2014)

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