Early Production of rhIDU

A series of expression vectors for human a-L-iduronidase were created using the human cDNA for the enzyme and a variety of common expression elements. Based on a series of transient and stable cell lines, a recombinant cell line was developed using the DUX B11 strain of CHO cells [9]. The recombinant cell line was studied in vitro and was found to produce a-L-iduronidase at the level of 0.6 to 2.4% of the total cellular protein, and to secrete the enzyme at levels 3000- to 7000-fold higher than the background secretion in normal cells. Although the production was impressive compared with natural sources, the amount of enzyme produced was still relatively limited; although many clones were screened, achieving a higher level of production was not possible. The key finding however, was that the enzyme produced was properly glycosylated and had high uptake in Hurler fibroblasts in culture with a Kuptake of about 1 nM. This finding was a prerequisite before proceeding further in the development of a recombinant enzyme for ERT. The early production process utilized a gelatin macroporous microcarrier (Cultisphere G) initially and later a dextran solid microcarrier with modified surface charge (Cytodex 2; Pharmacia). The cells were grown in cell culture flasks using 10% fetal bovine serum (FBS)-supplemented DME/F12 medium (Dulbecco's modified Eagle Medium mixed in 1:1 ratio with F12 medium). Once the cells reached near-confluence, the cells were harvested from the flasks and inoculated into a 5 L wand-stirred microcarrier flask. After the cells reached near-confluence on the microcarrier beads at 4 to 5 d, we began removing and replacing 3 to 4 L with DME/F12 containing 2% FBS (production medium) every 12 h. The culture was controlled for temperature and stir rate, but not for oxygen, carbon dioxide, or pH; it lasted less than 20 d. The harvested medium was purified over a sequence of columns including concanavalin A-Sepharose, heparin-CL4B Sepharose and finally, two passes over Sephacryl S-200. The yield from the purification process was about 25%.

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