Results and Discussion
Phage Biopanning-based Discovery of Peptide Affinity Ligands
for
AdP
The commercially available NEB PhD-12 phage display library was chosen
for phage biopanning to identify peptide candidates that bind to AdP. As
described in the methods section 2.2, three rounds of phage biopanning
(Figure 1) were performed against AdP. Once the biopanning rounds were
completed, 20 distinct phage clones from the final eluate were randomly
picked and sequenced.
The sequencing yielded 10 unique peptides with multiple repeats obtained
for two of the peptides (Table 1).
Fluorescence Polarization Screening for Peptide
Ligands
The 10 lead candidates were then synthesized with a 5(6)-FAM
fluorescence label and fluorescence polarization (FP) was carried out to
measure the dissociation constants (Kd) of their
interactions with AdP. The experiments yielded binding curves (Figure
2), which were fitted to a 4-parameter logistic equation. Table 2 lists
the Kd values obtained after fitting for 6 out of the 10
peptides, which showed an appreciable increase in observable signal with
increase in AdP concentration. Peptides P3, P5, P6 and P8 were either
too impure (as determined by C18-RP-UPLC) or showed a
very low change in signal with increase in AdP concentration. From the
binding curves shown in Figure 2, out of the 6 peptides, P10 exhibited
the strongest binding to AdP.
Appropriate control experiments performed with free 5(6)-FAM dye in
solution incubated with AdP indicated no non-specific binding between
the dye and AdP. The P10 peptide was also tested against AdI (a
different Adnectin provided by Bristol-Myers Squibb) and minimal binding
was observed (Kd > 100 µM) indicating that
P10 was indeed specific to AdP.
Since P10 exhibited the lowest Kd, and hence the
strongest binding (Kd = 19.5 μM), it was carried forward
for the development of a peptide-ELP based affinity precipitation
process.
Development of Peptide-ELP-based Purification of
AdP
Binding and Capture of AdP using P10-ELP
Initial binding experiments between P10-ELP and AdP were performed over
a range of molar ratios of P10-ELP to AdP at pH 7.4. It can be observed
in Figure 3 that at lower ratios, the capture of AdP increased linearly
with the ratio, but the amount of AdP captured saturated at higher
ratios, plateauing at a maximum of 85%. This could be due to the
moderate affinity of the peptide-AdP interaction, which precludes 100%
binding.
In order to improve the binding of P10-ELP to AdP, the effect of pH was
examined, using pH conditions of 3.0, 4.0 and 5.0 (Figure 4a). P10-ELP
was incubated with AdP (at a ratio of ~2:1 of P10-ELP to
AdP) and precipitated at these conditions. The results were compared
with those obtained at pH 7.4 (Figure 3) demonstrating that the capture
improved by nearly a factor of 2 with pH 4.0 resulting in the highest
capture of AdP in the precipitate. Since pH 4.0 and 5.0 are close to the
pI of AdP, these conditions could result in precipitation of AdP.
However, controls performed with 0.33 M sodium sulfate at pH 4.0 and 5.0
with 40 μM AdP (~1 mg/ml) resulted in no product
precipitation. Binding and capture experiments were repeated at pH 4.0
to examine the impact of molar ratios of P10-ELP to AdP. As can be seen
in Figure 4b, while a similar trend was observed for the effect of molar
ratio on product capture, a higher capture was obtained at pH 4.0 for
all ratios, plateauing at approximately 94% at 8:1.
Elution of AdP from
P10-ELP
A range of pH conditions were evaluated for their efficacy at eluting
AdP from the P10-ELP affinity reagent. The results indicated that while
low pH did not result in any elution, higher pH values were able to
elute up to 40% of the AdP. In order to improve the efficacy of the
elution step, mobile phase modifiers were included in the eluent. Sodium
chloride, ethylene glycol and arginine were selected due to their
different mechanisms of disrupting protein-ligand interactions. Sodium
chloride is widely employed in bioseparation techniques such as ion
exchange and affinity chromatography (Lee & Chen, 2001) and operates
via charge shielding and ion binding to reduce electrostatic
interactions (Tsumoto et al., 2007). Ethylene glycol has been shown to
weaken hydrophobic interactions while also increasing electrostatic
interactions (Hou and Cramer, 2011). In fact, Kelley and co-workers have
used ethylene glycol as an eluent in peptide-based affinity purification
of Factor VIII (Kelley et al., 2004). Arginine is widely used as an
elution modifier for several chromatographic systems. Although the
mechanism of interaction of arginine is not fully understood, molecular
dynamics simulations (Shukla et al., 2011) and experiments (Hirano et
al., 2014) have suggested that arginine interacts with both aromatic as
well as positively and negatively charged moieties leading to the
mediation of both electrostatic and hydrophobic interactions.
Applications have included both multimodal (Holstein et al., 2011) as
well as protein A systems (Ejima et al., 2005).
The use of elution buffers with modifiers in our affinity precipitation
system requires that the P10-ELP-AdP complex remain soluble during
elution while also enabling subsequent precipitation of the P10-ELP upon
addition of sodium sulfate. The three modifiers employed in this study
were examined under elution conditions and were shown to not have any
impact on these two critical constraints. Further, since the AdP product
was known to be susceptible to dimerization at higher pH, these elution
studies were carried out in the presence of 5 mM TCEP (a mild reducing
agent to prevent cysteine dimerization).
Figure 5a presents the effects of the three modifier solutions tested
independently over a pH range of 3-9. The general trend observed was
that high pH resulted in increased elution, irrespective of which
additive was used. The order of modifier effectiveness was arginine
> ethylene glycol > sodium chloride. In
addition, distinct trends were seen with each modifier as a function of
pH. While recovery with sodium chloride exhibited a linear increase in
recovery with pH, elution with ethylene glycol was only moderately
impacted by pH. In contrast, arginine was very effective as an eluent
modifier; with the recovery showing an initial increase followed by
saturation as a function of pH.
Once arginine was identified as the most effective modifier, the effect
of concentration was evaluated in the pH range of 6.0-9.0 where good
recoveries were observed at 1 M. The effect of concentration was less
pronounced at higher pH (Figure 5b). As can be seen, recoveries of
greater than 80% were attained for conditions with pH ≥ 8.0 and
arginine concentrations ≥ 300 mM. Based on this data, a pH of 8.5 and an
arginine concentration of 500 mM were selected as elution conditions for
the complex feed study described below.
Purification of AdP from Crude
Mixtures
After establishing the operating conditions for the affinity
precipitation process using pure AdP, we then evaluated the performance
of this process with a crude feed, now incorporating three intermediate
precipitate wash steps. Importantly, this process worked extremely well
with the crude feed, resulting in a final AdP purity of 88% and an
overall recovery of 81%. Figures 6 and 7 show the efficacy of this
process using SDS PAGE and RPLC analysis, respectively. As can be seen
in the gel, while the feed containing the AdP was complex (lane 5), the
final supernatant from the process (lane 8) was quite pure. The RPLC
analysis also clearly demonstrates that the final product is
significantly purified during the process.