Physiochemical characterization of the BsAbs
Biochemical and biophysical properties have been shown to have an
influence on mAb clearance in vivo .12, 19-23For the BsAbs, we evaluated these factors, which included solubility,
isoelectric point (pI), thermal stability (Tm), as well
as, the propensity for electrostatic (ie charge) and
hydrophobicity-related interactions using heparin and hydrophobic
interaction chromatography, respectively. The data from these analyses
are summarized in Table 1.
Both BsAbs were readily concentrated to ~45 mg/mL in PBS
buffer without any visible precipitation. To further compare the
aggregation propensity of the two BsAbs, the samples were concentrated
to 20 mg/mL in PBS buffer and held at 5°C for 10 days, when turbidity
was measured by a micro-turbidity assay. Negligible differences were
found between the two samples and with neither showing high turbidity,
indicating comparable aggregation propensity at this condition. The
samples were further stressed by incubating at 37°C for 7 days. Again,
both samples exhibited low turbidity with negligible differences between
them.
The experimental pI values for BsAb-1 and BsAb-2 were found to be 8.69
and 8.53, respectively. These pI values indicated that at physiological
pH both BsAb molecules would be expected to have a similar weak overall
positive charge. Additionally, the data indicated that the Fab/scFv
conversion, or the ligand binding orientation switch between the two
molecules did not grossly alter the pI (Table 1).
A previously developed heparin-based column assay was used to determine
the degree of charge-based interaction for the
BsAbs.11 In this experiment, BsAb-1 and BsAb-2 were
injected over a column of heparin sepharose and then eluted with a
linear gradient of increasing ionic strength. Retention of the molecule
and elution time was then used to determine the heparin interaction
potential (HpnIP) for charge-based interactions. Due to the small
positive charge present on the BsAbs, they are somewhat retained by the
column, eluting at [NaCl] of 218.1 mM and 191.1 mM, respectively,
but the molecules displayed similar HpnIP (24.8%HpnIP versus
22.0%HpnIP) (Table 1).
Non-specific interactions of the BsAbs driven by hydrophobic association
were evaluated using a HIC-based HPLC assay in which molecules were
injected onto a solid phase hydrophobic resin pre-equilibrated in high
concentrations of salt. Neither BsAb was retained on the HIC column
indicating that both molecules are very hydrophilic (Table 1).
Thermal stability of the BsAbs were measured by differential scanning
calorimetry (DSC). Analysis of BsAb-1 and BsAb-2 indicated that the
first Tm value of BsAb-1 is much lower than that of
BsAb-2 (59.0 oC versus 67.7 oC,
respectively) (Table 1 and Supplemental Figure 1). In comparing DSC
thermograms of BsAb-1 vs the matching IgG in BsAb-1, it is evident that
the low Tm peak of BsAb-1 is due to the scFv portion of the molecule and
that fusing the scFv domain to the C-terminal end of the HC showed no
gross changes or perturbations in the thermal stability of the IgG
portions (data not shown). This is consistent with our experience with
other IgG-scFv fusion molecules that the IgG and scFv portions fold
independently.