Download Summary Affimer reagents in affinity resins Specific target binding

Affimer®reagentsfacilitateaffinity
chromatographypurificationofIgG.
Geoffrey Platt, Chris Miller
Affimer reagents in affinity resins
Stability and reproducibility
Affimer reagents are proteins which present high affinity binding surfaces
for specific interactions with a wide range of targets. The scaffold is based
on the cystatin fold and permits the presentation of two variable binding
loops (Figure 1). The Affimer reagents are small (~12-14 kDa), easy to
manufacture in bacterial expression systems and biophysically stable over a
wide range of pH conditions.
The reproducibility and stability of an Affimer-based resin was measured by
repeated injection and elution of pure hIgG (81 runs), including 19 clean-inplace (CIP) cycles (100 mM NaOH, 10 min). The data indicate consistent
capture performance over the course of this study (Figure 4A). No leaching
from Affimer columns was detected by western blotting of concentrated
elution fractions (Figure 4B).
A
As both the scaffold and the randomised binding loops are engineered to
lack cysteines, the introduction of this residue using basic molecular biology
methods provides a straightforward route for immobilising the protein to
surfaces in a defined orientation via thiol chemistry (Figure 1).
A
C
b-turn 3
Binding loops
B
B
C-terminus
10 atom spacer
agarose
Figure 1A. Structure of
the Affimer scaffold
protein with the two
regions
containing
variable amino acid
loops in grey.
1B. Schematic showing
conjugation of Affimer
protein to agarose
resin.
1C. Column loaded
with affinity resin.
Figure 4A. The D11 Affimer column demonstrated high levels of reproducibility (as
judged by amount of protein in the eluted peak) over 81 consecutive purification cycles,
the capacity increased after CIP (red points) due to elution of residual target under
these conditions. Inset: Example traces for a section of the experiment. 4B. Example
western blot indicating no detectable leaching from Affimer-based resins during
elution, pure D11 Affimer protein is included for reference.
Binding capacity
Two Affimer clones (D11 and G1) that bind IgG molecules were immobilised
on highly cross-linked 4 % agarose beads via iodoacetyl chemistry and the
resin packed on columns (bed height 31 mm, column volume 0.6 mL). The
characteristics of the affinity resins were studied for target specificity,
reproducibility, stability and binding capacity.
The G1 Affimer resin displayed a broader range of immunoglobulin binding
than the D11 resin, in common with many of the affinity resins currently
used to purify therapeutic antibodies. To assess the potential of the G1
clone for this application the binding capacity of this column was studied by
injecting increased amounts of target protein (Figure 5).
Specific target binding and elution
Target protein (human IgG, hIgG) was injected on to the column in PBS (pH
7.4) and eluted in low pH conditions (glycine pH 3.0). Each resin binds hIgG
even when loaded in the presence of a complex mixture (Figure 2).
Figure 5. Injection of up to 12 mg
pure hIgG onto a 0.6 mL G1 Affimer
resin demonstrated that > 96 % of
the sample is captured and eluted
(red bars).
Unbound (UB)
Eluted (El)
Figure 2. Traces produced
upon loading IgG-depleted
human serum spiked with
known concentrations of hIgG
onto a D11 Affimer resin.
Elution, as measured by
A280 nm, is achieved by flushing
the column with low pH
buffer.
Inset:
SDS-PAGE
(under reducing conditions)
confirms specificity of binding
and elution.
In addition to displaying specificity for IgG over IgM and IgA from human
serum, the D11 Affimer resin demonstrated selectivity for human IgG over
other mammalian homologues. This indicates the high specificity possible
with Affimer-based resins and suggests a potential application in
pharmacokinetic studies performed in plasma from these animal models
(Figure 3).
The G1 Affimer column demonstrated a dynamic binding capacity of
14.8 mg mL-1 at 10 % breakthrough (3 min residence time) and a recovery
of 17.4 mg mL-1 in the eluted fraction (Figure 6).
A
B
Elution
Injection
Wash
Figure 6. Dynamic binding for injection of 0.5 mg mL-1 pure hIgG at a range of flow rates
over G1 Affimer resin - column residence times are displayed in the key. 6A. Full traces
showing injection, wash and elution phases. 6B. Breakthrough curves from this data.
Summary
Figure 3. % Area of elution
peak after injection of 1 mg of
pure immunoglobulins.
The results for these two Affimer clones establish potential research and
industrial applications for Affimer protein-based affinity resins. Of particular
importance in each case is their considerable robustness to a wide range of
pH conditions that enables a high level of reproducibility and ability to
endure cleaning routines. Further improvements can be achieved by
optimisation of chemical coupling and matrix selection.
Affimer reagents have the capacity to facilitate purification in different
applications as they bind a range of targets with high specificity and affinity.
For further information regarding Affimer technology, please contact [email protected] or visit www.avactalifesciences.com