Application note

Development and validation of

the use of anti-idiotypic Affimer®
binders to trastuzumab in a
pharmacokinetic assay

Summary

• Anti-idiotypic Affimer reagents specific to the trastuzumab antibody were generated and fully validated
within 3 months.
• Performance and sensitivity of the Affimer-based assay was not affected by a complex human serum
background, with no matrix effects observed.
• A high level of assay reproducibility was achieved for four separate batches of the Affimer binder.

Introduction

The continuing rise in the number of innovator and
biosimilar biotherapeutics approaching the market
has generated an increased need for anti-idiotypic
tools for their analysis and validation.1 Current
options for the development of bioanalytical methods
include the use of natural ligands or the generation
of specific anti-idiotypic binders as capture reagents.
Relying upon the use of recombinant protein ligands
to interact with specific biotherapeutics within pre-
clinical and clinical samples is dependent upon there
being a consistent, reproducible and affordable supply
of this protein. Yet, for many recombinant proteins
both reproducibility and supply are often variable, and
the cost of supply can prove prohibitively expensive.2

Affimer application note - Development and validation of the use of 1

anti-idiotypic Affimer binders to trastuzumab in a pharmacokinetic assay
The alternative, of using anti-idiotypic reagents to
measure biotherapeutics levels in pharmacokinetic
(PK) assays, requires that these tools be highly specific,
highly reproducible and fast to develop. Identifying a
specific biotherapeutic antibody within pre-clinical and
clinical samples requires the anti-idiotypic reagent to
specifically bind the drug antibody within a complex
background of human serum, containing up to a
million-fold excess of natural IgG.3
The need for a consistent, reproducible supply of any
anti-idiotypic reagent selected is critical to ensure
that the drug development process can continue
without the additional pressures of having to undergo
extensive batch-to-batch normalisation or developing
new reagents during this process.2 Furthermore,
as the speed to market for many biotherapeutics is
critical to their value, being able to rapidly develop
these anti-idiotypic reagents is of key importance to
avoid unnecessary delays in the development process.
Yet, many antibodies and antibody fragments offered
as reagents in this field require either immunisation
and/or affinity maturation, involving extensive
development times.
Affimer proteins are engineered affinity proteins
that present a target-binding variable region in an
analogous manner to that of the monoclonal antibody
binding site. Selected from large phage libraries
according to their desired binding properties these
proteins can be engineered to meet each of the
essential constraints in the development of anti-
idiotypic reagents, offering rapid development of
highly specific, high affinity recombinant reagents that
can be produced in simple prokaryotic systems with
a high level of consistency. Here we demonstrate the
development and characterisation of an Affimer binder
specific to the therapeutic antibody, trastuzumab,
completed within a 3-month timeframe. Trastuzumab
is a humanised monoclonal antibody directed
against the human epidermal growth factor 2 (HER2),
for the treatment of HER2-overexpressing breast
cancer.4 Our Affimer binders are able to specifically
Affimer application note - Development and validation of the use of
anti-idiotypic Affimer binders to trastuzumab in a pharmacokinetic assay
identify the trastuzumab target at clinically relevant
concentrations5, 6 while displaying no matrix effects
in human serum and without the requirement for
affinity maturation.
Identifying Anti-idiotypic
Affimer binders
Affimer binders identified from phage libraries were
uniquely specific for the therapeutic monoclonal
antibody trastuzumab. Specificity was achieved
using positive selection to the trastuzumab target
and negative selection against several alternative
therapeutic antibodies (adalimumab, bevacizumab
and rituximab) and natural human immunoglobulin G
(hIgG). The phage selection output was subsequently
screened via our high throughput, bead-based
primary screen to identify Affimer binders that
selectively bound only to the trastuzumab target.
Affimer binders that displayed any level of selectivity
for the control antibodies were discarded.
Sequencing of the trastuzumab selective Affimer
binders identified 16 unique sequences, which
were taken forward into further ELISA validation as
potential anti-idiotypic reagents. The best binder was
subsequently selected for assay development.
Affimer reagents detect
trastuzumab at clinically
relevant concentrations
Analysis of the target sensitivity and assay performance
of the selected Affimer binder was achieved by
incorporation of the reagent as a capture reagent within
an ELISA. Affimer binders were passively adsorbed
onto ELISA plates and the trastuzumab target antibody
present in solution over the concentration range 1.95-
2000 ng/mL, with detection via a horseradish peroxidase
tagged Fc-specific IgG antibody (Fig. 1).
2
 1.5 standard curve for trastuzumab over the broad
concentration range investigated, no interaction
with any of the other three therapeutic monoclonal
OD (450-630 nm)

1.0
antibodies with the Affimer capture reagent was
observed at any concentration (Fig. 2). This highlights
the unique specificity of the anti-idiotypic Affimer
0.5 Her2
Trastuzumab Affimer reagent for trastuzumab, demonstrating the lack
mlgG2b
of requirement for affinity maturation with Affimer
0.0 technology.
1 10 100 1000 10000

Trastuzumab (ng/mL)

1.5
Assay Parameters
Functional assay range 30-1600 ng/mL

OD (450-630 nm)
Intra-assay precision (CV) 5.2-10.7% 1.0

Inter-assay precision (CV) 8.7%

Bias <5% Trastuzumab
0.5 Adalilumab
Figure 1: Anti-trastuzumab Affimer binders show excellent sensitivity to Bevacizumab
the therapeutic antibody target comparable to the natural ligand, HER2. Rituximab
(a) Capture ELISA data using anti-trastuzumab Affimer reagents passively
adsorbed onto ELISA plate (at a concentration of 1ug/mL) showed detection
of the trastuzumab antibody target over the clinically relevant concentration 0.0
range, performing in line with the natural HER2 ligand. (b) The parameters of 1 10 100 1000 10000
the Affimer-based ELISA assay for trastuzumab demonstrate a broader dynamic
Antibody conc (ng/mL)
range than for current commercially available antibody-based assays.5,6

Antibody target Limit of detection

The Affimer binder coated surface was able to
Trastuzumab 30 ng/mL
successfully capture the trastuzumab target in
Adalilumab n.d.
line with the performance of the HER2 positive
Bevacizumab n.d.
control, down to a lower concentration of 30 ng/mL,
Rituximab n.d.
demonstrating that trastuzumab can be accurately
and precisely quantified using the selected Affimer Figure 2: The anti-trastuzumab Affimer reagent is highly specific for the
trastuzumab therapeutic antibody target. Across a broad target concentration
reagent. As expected, the mouse IgG2b negative range the trastuzumab antibody was accurately detected by the Affimer
reagent, but no interaction was observed with any of the three alternative
control showed no interaction with trastuzumab. therapeutic antibodies analysed.

The observed assay range for the Affimer-based assay

shows a much broader dynamic range compared to
the majority of available trastuzumab ELISA assays.5, 6
This allows a wider range of samples to be analysed
within a single assay, increasing convenience and
reducing the need for repetition, thus reducing cost.
The degree of specificity of the Affimer reagent for
the trastuzumab target was investigated by assessing
its binding to trastuzumab and the three alternative
therapeutic antibodies used as part of the screening
process. Evaluation of binding via the ELISA assay
demonstrated that whilst the Affimer showed a typical
The sample environment does
not impact upon Affimer-based
assay performance
While the anti-idiotypic Affimer reagents were able to
readily identify trastuzumab against the background
of PBS buffer, for use in PK applications it is important
that any anti-idiotypic binder is able to specifically rec-
ognise the biotherapeutic target within human serum
samples. To assess the performance of our Affimer

Affimer application note - Development and validation of the use of 3

anti-idiotypic Affimer binders to trastuzumab in a pharmacokinetic assay
 4
binder within such a complex sample background we
performed a capture ELISA with the trastuzumab target
3
present in 10% human serum (Fig. 3).

OD (450-630 nm)
2
ELISA plates coated with anti-trastuzumab Affimer
reagents were able to detect the trastuzumab 10% serum
antibody in solution over the clinically relevant 1 No serum

concentration range 1.95-2000 ng/mL,5, 6 against a

background of 10% human serum with no matrix 0
1 10 100 1000 10000
effects observed. Indeed, the sensitivity of the Affimer Trastuzumab (ng/mL)
reagents for the trastuzumab target did not show
Figure 3: Capture ELISAs demonstrate the anti-trastuzumab Affimer
any significant difference between the buffer and reagent specifically identifies the therapeutic antibody at clinically relevant
concentrations against a complex human serum background. No significant
human serum matrices analysed, demonstrating their difference was observed in target sensitivity between the buffer and
serum matrices.
robustness to the assay environment and overcoming
any need for the advanced preparation of samples.
2.0

Batch-to-batch reproducibility is 1.5

OD (450-630 nm)

assured with Affimer reagents

1.0

Low batch-to-batch variability is an inherent Affimer batch 1

0.5 Affimer batch 2
characteristic of Affimer reagent production due Affimer batch 3
Affimer batch 4
to their robust and simple bacterial manufacturing
0.0
process. This is key to the production of anti-idiotypic 1 10 100 1000 10000

reagents and allows any drug development process Trastuzumab (ng/mL)

to progress without the need for reagent batch-

to-batch normalisation. To illustrate this benefit of M C 1 2 3 4
188 kDa
the anti-trastuzumab binders, we generated four
98 kDa
separate batches of the anti-trastuzumab Affimer
62 kDa M. MW Marker
binder and compared their purity, concentration and 49 kDa
C. 2µg BSA Control
1. Affimer batch 1
performance in a capture ELISA against a background 38 kDa 2. Affimer batch 2
3. Affimer batch 3
4. Affimer batch 4
of 10% human serum (Fig. 4). Minimal batch-to-batch 28 kDa
17 kDa
variation was observed, demonstrating the high level
14 kDa
of reproducibility of Affimer reagents and offering the
6 kDa
necessary assurance of supply for critical reagents in
3 kDa
the bioanalysis of potential therapeutics during the
clinical trials process.
Figure 4: Anti-idiotypic Affimer reagents show high batch-to-batch reproducibility.
(a) SDS-PAGE analysis of four separate batches of the anti-trastuzmab Affimer
binder. (b) Performance of the various batches of anti-trastuzumab Affimer
binder in a capture ELISA shows minimal variation between different batches.

Affimer application note - Development and validation of the use of 4

anti-idiotypic Affimer binders to trastuzumab in a pharmacokinetic assay
Summary
Anti-idiotypic Affimer reagents that can be used
in PK assays can be developed in less than three
months. They offer a broad dynamic range and
high specificity and sensitivity with no detectable
interference from serum matrices. Additionally, they
can be produced consistently and reproducibly and
therefore are good potential critical reagents for
clinical assay development.
References
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Schleypen J, Papadimitriou A. (2011) Quality
requirements for critical assay reagents used
in bioanalysis of therapeutic proteins: what
bioanalysts should know about their reagents.
Bioanalysis. 3(5):523-534.
Affimer® is a registered trade mark of Avacta.
Avacta Unit 20, Ash Way, Thorp Arch Estate, Wetherby, LS23 7FA, United Kingdom
Tel: +44 (0) 1904 21 7070 www.avacta.com @Affimers
Affimer application note - Development and validation of the use of
anti-idiotypic Affimer binders to trastuzumab in a pharmacokinetic assay
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