Comparison of Adsorption Isotherm
Figure 3 shows the adsorption isotherms of Fc-fusion protein A at pH 7.0 for all the three resins. Table 1 lists the thermodynamic
max and K). It shows that amongst the three resins, MabSelect had the highest Q
max value, which was consistent with DBC results. On the other hand, Qmax for ProSep-vA was slightly lower than IgSelect even though their relative trend for dynamic binding capacity was opposite.
This can be explained by improved flow properties for the glass-bead–based ProSep-vA resin, which causes its DBC to be closer
to its equilibrium capacity. Furthermore, K values for all three resins were very similar, indicating that the binding strength for Protein A and this new ligand was
Figure 3. Comparison of adsorption isotherms on MabSelect, IgSelect, and ProSep-vA resins at pH 7.0
Comparison of Selectivity
As mentioned before, it has been shown in the literature that none of the small-molecule synthetic ligands developed as Protein
A alternatives were able to provide the lock-and-key induced fit as Protein A.24,29 The non-specific binding of host cell proteins to these ligands have made their selectivity much lower than that of Protein
A resins. On the other hand, the VHH fragment has the capability to recognize unique conformational epitope and hence has
the promise of showing greater selectivity.
Table 1. Langmuir Isotherm data at pH 7.0
To compare the selectivity of the new resin to Protein A, host cell protein clearance was compared for the four antibodies
and Fc-fusion proteins using complex cell-culture fluid. The details of these preparative experiments are outlined in the
experimental section. Protein recoveries for all of these experiments were comparable and >90%. The elution pools from these
experiments were collected and analyzed for host cell protein (Chinese hamster ovary protein, CHOP) levels. Figure 4 plots
the CHOP log reduction values (LRVs) from these experiments. The higher the LRV, the more selective is the resin. Figure 4
shows that IgSelect demonstrated comparable host cell protein clearance to the Protein A resins. In fact, for molecules A,
B, and D, the LRV values for IgSelect were slightly lower or comparable to MabSelect but higher than the other Protein A resin
ProSep-vA. ProSep resins are known to give slightly lower CHOP clearance than the agarose-based Protein A resins because of
non-specific interactions of CHOP with their silica backbone.37 On the whole, the selectivity shown by this new camelid antibody resin appears to be very promising.
Figure 4. Log reduction value (LRV) of Chinese hamster ovary host cell proteins (CHOP) under preparative conditions on MabSelect,
IgSelect, and ProSep-vA resins