Downstream processing of a plant-made recombinant human therapeutic enzyme

Mucopolysaccharidosis I is a lysosomal storage disease that is characterized by a deficiency of the lysosomal hydrolase alpha-L-iduronidase. Enzyme replacement therapy with purified, recombinant IDUA is one treatment option available for individuals that inherit this disease. The goal of my thesis was to engineer a transgenic line of Arabidopsis thaliana that could express, in the seed tissue, human iduronidase with a therapeutically acceptable N-glycan profile. Human iduronidase was expressed at 5.7% of total soluble protein (TSP) in the Arabidopsis cgl mutant line, which is deficient in N-acetylglucosamine transferase I, and at 1.5% of TSP in the Arabidopsis Golgi-Mannosidase I (GM-I) knockout line. The iduronidase purified from both lines was able to hydrolyze the fluorescent iduronide compound, 4-methylumbelliferyliduronide (4-MUI), at a rate comparable to that of iduronidase produced in Chinese Hamster Ovary cells, commercially available as Aldurazyme. Both plant-derived forms of iduronidase possessed primarily high-mannose N-glycans (at 95% for cgl-iduronidase and 100% for GM1-iduronidase); however, the dominant glycoform in the N-glycan profile of cgl-iduronidase was Man5GlcNAc2, whereas most of the N-glycans on GM1-iduronidase contained six to eight mannose sugars. Additionally, cgl-iduronidase contained a small percentage of complex glycans (and thus potentially immunogenic xylose and/or fucose sugars), which were not found on GM1-iduronidase. Surprisingly GM1-iduronidase did not contain an N-glycan at Asn336, which is normally present on the native human iduronidase and on the recombinant iduronidase of cgl Arabidopsis seeds and Chinese hamster ovary cells. Of importance to creating a product that is of therapeutic value, another goal was to determine whether the plant-produced iduronidase was amenable to in vitro phosphorylation to create the mannose-6-phosphate tag necessary for sequestration and lysosomal delivery in human cells. Both plant-derived forms of the enzymes were able to be phosphorylated in vitro by the UDP-GlcNAc:lysosomal enzyme (GlcNAc)-1-phosphotransferase at a rate similar to that of Aldurazyme.