Detection of protein heterogeneity using different
modes of analytical chromatography
Dr. Atis Chakrabarti, Technical Service Manager, Tosoh Bioscience
ABSTRACT
Antibodies and recombinant proteins are now widely used for the
therapeutic treatment. So the evaluation of the heterogeneity of the
therapeutic antibody is essential for the development, stability study
and quality control of the final product. Protein heterogeneity is
generated by posit-translational modifications, decomposition or
chemical modification. There is increased risk of anaphylaxis or
immunoreaction if these impurities are not separated.
In this presentation we have shown the Size exclusion chromatography (SEC) is suitably used to separate
the monomer (150 kDa) and the dimer (300kDa) of a human monoclonal antibody to a very good
baseline resolution. Papain and pepsin digested antibodies could be resolved to separate Fc, Fab and
intact protein to the baseline resolution by size exclusion chromatography. Analysis of Aggregates,
fragments and PEGylated proteins and the analysis of hapten conjugated protein using SEC are also
reported in this presentation. A set of 3 novel columns designed for the analysis of mAbs will be
introduced in this presentation. The first one for the separation of higher order aggregates, the second
one for high throughput separation and the third for high resolution separation. A new reversed-phase
chromatography (RPC) column with a large pore size of 300A is shown to be applicable for the protein
analysis and detection of heterogeneity.
Overall this presentation is focused on the overview of the columns available for the detection and
separation of protein heterogeneity by HPLC using four different modes of chromatography such as SEC,
IEX, RPC and HIC.
BIOGRAPHY
Dr. Chakrabarti completed his Ph.D. thesis work on the design and synthesis of anti-proliferative probes.
His post-doctoral research at the Indian Institute of Science, Bangalore, focused on the molecular
biology and biophysical characterization of bacterial thioredoxin. In 2000, he joined Thomas Jefferson
Medical College researching diagnostic and therapeutic probes for PET imaging. Throughout his
academic and industry career, he utilized different modes of chromatography for the purification of the
synthetic conjugate probes, small molecules, API's for bulk and dosage use and proteins. He is an
adjunct faculty member in the department of chemistry at Camden County College, NJ. Atis joined Tosoh
Bioscience in July 2008 as Technical Service Specialist. Since October 2011 he is the manager of the
Technical Service group for laboratory products with special emphasis on the Tosoh Bioscience line of
chromatography columns. Atis' research group is focused on expanding the range of applications of
TSKgel columns. In his leisure time Atis enjoys spending time with his family, traveling, and camping.