Transferrin (Tf) is a glycoprotein that transports iron to cells and has two N-glycosylation sites in humans – at asparagine 432 and asparagine 630. Carbohydrate-deficient Tf, which lacks one or both N-glycans, is the most common marker for congenital disorders of glycosylation.1 Altered Tf glycosylation has also been reported in hepatocellular carcinoma2 and chronic alcohol consumption.3,4 High-throughput Tf purification and glycan characterisation methods are under extensive development in order to facilitate screening of glycosylation patterns for population, genetic and clinical studies.
This application note describes the development of an immunoaffinity purification method on a CIMac™ analytical column with immobilised anti-transferrin antibodies (@Tf) and the successful transfer of the method to the monolithic 96-well plate (CIM® @Tf-0.2 monolithic 96-well plate). The affinity purification method has been used for Tf isolation from human blood plasma followed by ultra-performance liquid chromatography (UPLC) analysis of Tf N-glycosylation.
PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.
Differently pre-activated CIM® monoliths enable immobilisation of a range of proteins, peptides, nucleotides and other affinity ligands. The first parameters to be defined for a new immobilisation are the activation chemistry of the matrix and the linkage protocol. Due to the varying physico-chemical characteristics of different ligands, however, a platform process cannot be achieved.