The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

Abstract. In this study, we have investigated sedimentation velocity ultracentrifugation (AUC-SV), size exclusion chromatography (SEC), and circular dichroism (CD) methods for the detection and quantitation of protein aggregates using recombinant acid alpha-glucosidase (rhGAA) as a model. The results of this study showed that the formation and molecular weight distribution of rhGAA aggregated species were dependent upon the formulation conditions as well as the storage or stress conditions used to induce aggregation. The utility of CD as a probe for non-native, aggregated species was affirmed, as this method was sensitive to rhGAA aggregation levels of ≤4%. An extensive evaluation of AUC-SV variability was performed using nine levels of spiked rhGAA aggregate that were analyzed on six occasions. Based on our data, the precision of the AUC-SV results increased with increasing levels of aggregate, with a mean RSD of 37.2%. The limit of quantitation (LOQ) for the AUC-SV method, which was based on a Precision criterion of RSD <20%, was determined to be ≥3% aggregated rhGAA. The Precision and LOQ of the SEC method, determined using the same rhGAA sample set, was found to be 3.8% and ≥0.2%, respectively. In general, there was good agreement between the levels of aggregated rhGAA determined using the AUC-SV and SEC methods, with a slight positive bias noted for the AUC-SV results. These studies emphasize the value of applying multiple, well-characterized analytical tools in the evaluation of therapeutic protein aggregation.