Monoclonal antibodies (mAbs) are monospecific proteins that are extensively used as a research tool in molecular biology and to develop them as therapeutics in medicine.Frequently, mAbs are present in various heterogenic impurities which may have formed during various stages of processing, purification or even during storage.  The presence of such heterogenic impurities can lead to deleterious effects on the efficacy and stability of the final product, and must be removed from the bio therapeutic protein formulations in order to comply with strict regulatory guidelines and maintain product quality.  Analytical Size Exclusion Chromatography (SEC) if often used to separate heterogenic impurities of proteins differing on the basis of molar mass particularly in the polishing step in the large scale chromatography and in Quality Control. Optimization of the SEC methods through the investigation of various chromatographic conditions can lead to highly resolved peaks with a target of maximizing the molecular sieving mechanism. Mobile phase ionic strength and composition, flow rate etc., are among the various factors having a profound effect on the separation of the mAb variants. We have recently introduced a 7.8 mm ID � 30 cm analytical column packed with 25 nm pore size, 4 �m particles, which is designed for high throughput analysis of mAbs. The column dimension is compatible with conventional LC systems with relatively large extra-column dead volume and is suitable for high resolution analysis of mAb monomers and dimers. Here, we report results on the study of the effect of flow rates, salt and organic solvent etc. on the resolution of the monoclonal antibody monomer from its molecular mass protein variants using this new column.

Optimization of chromatographic conditions during the analysis of monoclonal antibody using a 25 nm pore size, 4 �m particle size, Size Exclusion Chromatography (SEC) column in the separation on heterogenic impurities

247th National Meeting (2014)

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