Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.