The use of micro Pillar Array Columns (µPAC™) for characterizing glycosylation of therapeutic enzymes is presented. Recombinant human acid α-glucosidase (hGAA) was digested and resulting peptides were separated on a 50 cm µPAC™ capLC C18 column operated at low µL/min flow rate. Glycopeptide peaks were then selectively detected and identified by Orbitrap mass spectrometry (MS) operated in full MS/all-ion fragmentation (AIF) mode.
Human acid α-glucosidase (hGAA) catalyzes the hydrolysis of glycogen to glucose in the lysosomes of the cell. There are around 50,000 people worldwide which have a deficiency of this enzyme leading to glycogen accumulation in the lysosomes, a rare and fatal disorder known as Pompe disease [1-4]. Pompe patients typically receive an enzyme replacement therapy with recombinant human acid α-glucosidase (rhGAA) commercially known as Myozyme. Recombinant hGAA is a heavily N-glycosylated protein with a MW of 110 kDa expressed in Chinese Hamster Ovary (CHO) cells. The therapeutic enzyme contains 7 N-glycosylation sites which are occupied with complex and high mannose glycans [2-4]. The former complex glycans are predominantly sialylated and to a lesser extent acetylated, the latter glycans contain an unusual mannose-6-phosphate structure considered a critical quality attribute as it is responsible for targeting the enzyme to the lysosomal compartment of the cell where it needs to be catalytically active to break down glycogen.
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