TABLE 1

In vitro analytical data for heparin binding variants of FS315-hFc

The binding of recombinant FS315-hFc variants to heparin, myostatin, or hFcRn was determined by SPR. The binding affinities were measured and reported as the KD. The charge heterogeneity of the variants was determined by cIEF and was shown as a range of pI values. The statistical parameters and analysis, including association constant (ka) with S.E., dissociation constant (kd) with S.E., and the average KD values with S.D. (n = 4 independent batches) for an internal control used in all SPR assays, are reported in Supplemental Table 3. ND*: No detectable heparin binding in tested concentration ranges of 0.019–25 nM FS315-hFc variants.

FS315-hFc VariantHeparin Binding KDMyostatin Binding KDhFcRn Binding KDcIEF
nMpMnMpI
ControlsWild type0.220.231.45.07–5.89
ΔHBSND*17.448.04.82–5.72
del75-86ND*57.138.84.83–5.26
One–amino acid substitutionK84E0.99.934.95.07–6.01
K82E1.511.910.55.48–6.09
Two–amino acid substitutionK(76,84)E0.89.033.04.87–5.95
R78E/K84E0.87.245.55.06–5.96
K(75,76)E1.111.734.25.05–5.26
K(82,84)E1.19.045.14.86–5.95
R78E/K82E1.33.953.34.96–5.96
K(81,82)A1.511.938.55.31–5.96
K(76,82)E3.910.538.24.89–5.26
K(81,82)E10.79.940.84.83–5.25
K(81,82)D20.67.124.74.88–5.59
Three–amino acid substitutionK(76,81,82)A9.411.341.65.24–5.93
K(76,82,84)E13.84.750.84.85–5.80
K(76,81,82)END*4.244.04.87–5.80
K(76,81,82)DND*5.959.94.82–5.67