429398 Improving the Separation Performance of Modified Proteins By Electrostatic Interaction Chromatography

Monday, November 9, 2015: 1:50 PM
155F (Salt Palace Convention Center)
Daisuke Itoh1, Yu Isakari1, Noriko Yoshimoto1, Shuichi Yamamoto1 and Ales Podgornik2, (1)Bio-Process Engineering Laboratory, Yamaguchi University, Ube, Japan, (2)COBIK, Solkan, Slovenia

When native proteins are modified (intendedly or unintendedly), modified forms must be separated from the native protein and other unwanted contaminants. A well-known intended modification is PEGylation, where a PEG molecule is attached to a protein. Unintended modified proteins are dimer and aggregates, which must be removed by chromatography during downstream processes of antibody drugs.

Electrostatic interaction based chromatography (IEC) is known to be most efficient for PEGylated protein separations. PEGylated proteins are much more weakly retained compared with the native form in IEC. PEGylated protein positional isomers can be separated by IEC and the binding sites for mono-PEGylated proteins are similar to those for native proteins. Weak retention of PEGylated proteins is likely to be due to a steric hindrance between the ion-exchange ligand and charges of PEGylated proteins.

Since dimer and aggregates are more hydrophobic and have more charges, their retention volumes are larger in IEC. We investigated the number of binding sites B for a model protein monomer and dimer. As the mobile phase pH increased both the B values and the difference between the B values of monomer and dimer (aggregates) increased for an-ion exchange chromatography. Consequently, the removal of dimer became better with increasing pH. Another efficient method for improving the separation performance is to add polyethylene glycol (PEG) into the mobile phase. As PEG increased the viscosity, the pore diffusion coefficient decreased, which resulted in peak broadening. We determined the pore diffusion coefficient in the presence of PEG in the mobile phase of IEC. Based on the pore diffusion coefficient and the mobile phase viscosity the optimum conditions were detetemined for the monomer separation.

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See more of this Session: AIChE-SCEJ Joint Session: Bioseparations and Bionanotechnology
See more of this Group/Topical: Separations Division