Introduction
While monoclonal antibodies continue to dominate the biologics market,
there is significant activity in the discovery of non-mAb scaffolds as
biological products for several reasons such as lower half-life, smaller
size and ease of engineering. Out of the different protein scaffold
families, one is the 10th Fibronectin Type III (10Fn3)
domain, popularly known as ‘monobodies’ or ‘Adnectins™’ (Koide et al.,
2012). These molecules also possess flexible loops similar to the CDRs
in antibodies but are nearly 10-fold smaller than monoclonal antibodies
(~10-12 kDa in size compared to monoclonal antibodies
which are ~140-150 kDa in size) (Lipovsek, 2011). It is
well known that protein A targets the conserved Fc region of IgGs and is
a well-established platform affinity ligand for the purification of
monoclonal antibodies (Huse et al., 2002). However, unlike IgGs, small
scaffold proteins such as Adnectins contain only a single domain. A
70-80% homology was observed from the sequence alignment of existing
Adnectin molecules in the literature. These homologous regions can be
potentially targeted for a generalizable purification platform for
Adnectins.
Peptides have shown considerable potential as affinity ligands for the
purification of biological targets due to several favorable attributes
such as structural flexibility, stability, sequence diversity and ease
of production. While rational approaches have been used for the design
of peptide ligands against protein targets (Chandra et al., 2013),
combinatorial display techniques have also resulted in the
identification of affinity peptides against protein targets.
Peptide-based phage display has been widely used for the identification
of peptide affinity ligands for drug discovery (Nixon et al., 2014),
biosensing (Wu et al., 2011), as well as in the affinity purification of
biologics (Kelley et al., 2004). Phage display-aided peptide selection
employs a procedure known as biopanning which involves challenging a
library of bacteriophage against an immobilized target. Strategic
washes, negative selections and multiple biopanning rounds result in the
discovery of affinity peptide ligands specific to the target. Peptides
have been previously employed for the chromatographic purification of
mAbs (Yang et al., 2008) as well as various non-mAb targets such as
Factor VIII (Kelley et al., 2004), human growth hormone (Chandra et al.,
2019), erythropoietin (Kish et al., 2018), Fab fragments (Nascimento et
al., 2019) and viruses (Heldt et al., 2008). The moderate affinities of
peptide affinity ligands to their corresponding target result in both
effective purification as well as relatively milder conditions for
elution and subsequent target recovery.
The class of phase transitioning smart biopolymers – elastin-like
polypeptides (ELPs) – has been investigated over the last two decades
for the downstream processing of proteins (Yeboah et al., 2016). ELPs
are typically composed of multiple repeats of the pentapeptide unit
V-P-G-X-G (where guest residue X can be any amino acid except proline)
and can undergo precipitation upon the change in stimuli such as
temperature and salt concentration (Meyer & Chilkoti, 1999). Different
approaches involving ELP-protein fusion systems have been effectively
employed for protein purification (MacEwan et al., 2014). However, these
often require proteolytic cleavage or the introduction of self-splicing
inteins (Fong et al., 2009) to dissociate the ELP-tag from the product,
resulting in the requirement of additional purification steps.
Therefore, an alternate strategy – affinity precipitation – involving
fusions of ELPs and affinity ligands have been employed for the tag-free
purification of proteins (Madan et al., 2013). Previous work by Cramer
and Chen has successfully demonstrated the purification of monoclonal
antibodies from complex mixtures using ELP-Z-based affinity
precipitation (Sheth et al., 2013). This process was found to be
comparable to protein A purification and also readily scalable when
performed with membrane filtration steps (Sheth, Bhut, et al., 2014;
Sheth, Jin, et al., 2014). The Cramer and Karande labs have also
recently demonstrated the purification of model proteins such as
Anti-Flag Antibody M2 and streptavidin using peptide-ELP fusions
(Mullerpatan et al., 2020).
In this work, we specifically focused on an industrially relevant small
non-mAb scaffold protein, P-Adnectin (AdP). At present the purification
of AdP has been carried out using affinity tag fusions (Mitchell et al.,
2014) or with a series of chromatographic steps. In the current work, we
first employed phage panning to identify peptide affinity ligands
against AdP. Lead candidates were then synthesized and their binding
behavior to the product was evaluated using fluorescence polarization. A
peptide-ELP fusion of the best performing candidate was then produced inE. coli and various conditions of stoichiometry and pH were
examined to establish appropriate conditions for binding and
precipitation of the AdP. Elution conditions were then established using
a combination of pH and fluid phase modifiers. Finally, the optimized
capture and elution conditions were employed in a proof of concept
experiment demonstrating the utility of a single-step affinity
precipitation process that was capable of capturing
~80% of AdP from a crude E. coli lysate at a
purity of ~90%.