RESULTS AND DISCUSSIONS
Comparison of Binding Affinity and Elution pH
Both the ligands evaluated in this study (Protein A and the camelid antibody) use a similar operating scheme—the protein is
bound at close to neutral pH and eluted with a lower pH. At lower pH values, the electrostatic repulsion between the ligand
and the bound protein helps to overcome the attractive forces and hence cause elution.4,33 The pH of elution in a way can be used as a measure of the strength of interaction with the ligand and is a useful parameter
to compare the affinity of various resins. Figure 1 plots the elution pH at peak maxima under linear gradient conditions using
a wide variety of proteins. These proteins were carefully chosen to test a variety of possible protein–ligand interactions
and included model proteins of varying hydrophobicity (Horse cytochrome c and HSA), MAbs (molecules C and D), as well as Fc-fusion
proteins (molecules A and B). In an ideal case, a resin that is selective for IgG-based molecules should only bind those and
not any other non-IgG–based model protein.
Figure 1. Elution pH of model proteins and IgGs on MabSelect, IgSelect, and ProSep-vA resins
As shown in Figure 1, the model proteins cytochrome c and HSA had an elution pH of around 6.5 (binding condition as described
in the experimental section) on all three resins clearly indicating that they were not retained under these conditions. This
is in contrast to results shown for other Protein A alternatives such as HCIC and mimetic ligands that bound model proteins
as well as Ig-based molecules.24 The non-binding of the model protein to the IgSelect resin (similar to what is seen for Protein A resins) is indicative
of its selectivity. Moreover, the elution pHs for all the IgG-based molecules (antibodies and Fc-fusion proteins) were somewhat
comparable across the three resins and fell in the pH range of 3.5–3.9. Thus, it can be said that the new camelid antibody
ligand had very similar affinity to IgG molecules as a conventional Protein A ligand under the conditions tested.
Comparison of Dynamic Binding Capacity
Dynamic binding capacity (DBC) is one of the most important performance parameters for chromatography resins. This is particularly
true for Protein-A–based resins, which are very expensive, and are used as a capture step in antibody purification. DBC was
calculated for all four antibodies and Fc-fusion proteins using the methodology described in the experimental section. Figure
2 plots the 1% breakthrough capacity on all three resins.
Figure 2. Dynamic biniding capacity (DBC, 1%) results of the four IgG molecules on MabSelect, IgSelect, and ProSep-vA resins.
The tests were carried out at pH 7.0 and six minutes residence time.
From Figure 2, it can be seen that the two Fc-fusion proteins (molecules A and B) had very similar capacities on all three
resins. Furthermore, the Fc-fusion proteins had a lower binding capacity than the two antibodies (C and D), particularly on
MabSelect. This has been noted in the literature and has been explained by the differences in the steric hindrance exerted
by Fc-fusion proteins versus MAbs.34 On the other hand, a difference in DBC was seen amongst the three resins for the two antibodies, molecules C and D. Both
antibodies showed the highest capacity on MabSelect. The capacity of the new resin IgSelect was less than MabSelect but comparable
(or even slightly higher for D) to ProSep-vA. Controlled pore glass resins such as ProSep-vA have been shown to have lower
binding capacities but improved mass transport properties.35,36 The performance of the new resin was in-between the two Protein A resins with respect to DBC. The dynamic binding capacity
on all these resins were compared at 6 minutes residence time. These resins might have different responses with varying residence
time based on the transport properties of their backbone. However, the response of MabSelect and IgSelect would be very similar
because they are based on a similar cross-linked agarose backbone.
It has been conjectured in the literature that the monomeric VHH fragments might have a lower binding capacity because they
do not have the capability to bind multiple IgGs like Protein A, which has multiple binding domains.29 This was found to be true by our experimental results with antibodies, particularly on MabSelect and IgSelect because their
backbone and ligand densities are comparable. Interestingly, the single-binding domain did not seem to have any negative effect
on Fc-fusion proteins because they have a larger steric hindrance and thereby cannot optimally access the multiple binding
domains of the Protein A ligand.