Figure 3.

The efficiency of cellular uptake for alglucosidase alfa or ATB200 was evaluated using L6 rat skeletal muscle myoblasts. Varying concentrations of each rhGAA preparation were added to cell culture media and incubated for 16 hours. External rhGAA was inactivated with high pH buffer, cells were then washed and lysed with detergent buffer. The amount of internalized rhGAA within cells was measured by enzyme activity assays using the fluorogenic 4-MU-α-Glc substrate and normalized relative to total cellular protein. The amount of internalized rhGAA (y-axis) was graphed relative to the amount of rhGAA added (x-axis) to correlate the uptake efficiency (i.e., competency) for each rhGAA preparation. Results indicate that high alglucosidase alfa concentrations (i.e., >100 nM) are required for efficient cellular uptake in L6 myoblasts. In contrast, substantially lower ATB200 concentrations are needed for efficient cellular uptake in the same cellular assay. The extrapolated efficiency coefficient for cellular uptake (Kuptake) for ATB200 is approximately 5–15 nM and >125 nM for alglucosidase alfa. It should be noted that saturation of cellular uptake was not achieved for alglucosidase alfa with this concentration range such that an accurate Kuptake for that rhGAA ERT could not be determined. rhGAA, recombinant human acid alpha-glucosidase; ERT, enzyme replacement therapy.