Effect of Glycosylation On the Partition Behavior of a Human Antibody On Aqueous Two-Phase Systems

Tuesday, October 18, 2011: 1:50 PM
202 A (Minneapolis Convention Center)
Daniel Forciniti, Cbe, Missouri University of Science and Technology, Rolla, MO and J.-W. Lee, Cbe, Umr, Roll, MO

Human proteins are expressed in some hosts wrongly glycosylated or non-glycosylated.  Although it is accepted that glycosylation contributes to the stability of the protein in solution, the effect of glycosylation on the stability of human antibodies is not fully understood.  In this manuscript, we investigate the effect of glycosylation on the partition behavior of two transgenic (the host was corn) antibody preparations.  One of them consisted of 95% non-glycosylated antibody.  The other was a mixture of 25% non-glycosylated antibody, 50% of single GlcNac glycosylated antibody, and 25% of fully glycosylated monoclonal antibody; the purity of this preparation was also 95%.   

The studies were done by monitoring the partitioning behavior of both proteins in a series of aqueous two-phase systems at and away the isoelectric point of the proteins and at different temperatures.  Our studies show that in the absence of direct electrostatic forces, the partitioning behavior of the antibodies depends on the presence or absence of the polysaccharide chains.  Overall, the non-glycosylated protein is less soluble than the glycosylated one.  The non-glycosylated antibody precipitates heavily at the interface between the two phases whereas the glycosylated precipitates to a lesser extent.  The differences in the partition behavior of both preparations are analyzed by accounting for the energetic and entropic contribution to the Gibbs free of each phase.  The potential of aqueous two-phase systems for the separation of the glycosylated and non-glycosylated proteins was also explored. A simple series of extractions seems to be enough to separate the glycosylated variety from the non-glycosylated one at high purity but relatively low yields.


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See more of this Session: Extractions In Bioprocessing
See more of this Group/Topical: Separations Division