development of a hghab-based surface acoustic wave …
TRANSCRIPT
UNIVERSITY GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Department Pharmaceutical Analysis
Laboratory for Drug Quality and Registration
Academic year 2013-2014
DEVELOPMENT OF A HGHAB-BASED SURFACE ACOUSTIC
WAVE METHOD FOR THE FUNCTIONAL QUALITY
CHARACTERIZATION OF NOTA-MODIFIED SOMATROPINS
Sophia BARHDADI
Master of Industrial Pharmacy
Promoter:
Prof. Dr. B. De Spiegeleer
COPYRIGHT
"The author and the promoters give the authorization to consult and to copy parts of this
thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited.”
Promoter Author
Prof. Dr. Bart De Spiegeleer Sophia Barhdadi
ACKNOWLEDGEMENTS
I would like to make use of this opportunity to thank all the people who helped me to bring
this thesis to a successful end.
First of all, I would like to thank Prof. Bart De Spiegeleer for giving me the opportunity to do
my research internship in the DruQuar laboratory, but especially for his guidance and for
pushing me in the right direction for my thesis.
I am also very grateful to Nathalie Bracke for supervising me throughout this past year, for
her endless patience and for kindly answering my many questions. Without your time and
energy, I never would have been able to finish this work. It was a pleasure to work with you
and I wish you all the best with the rest of your PhD.
Furthermore, I want to thank all the lovely people from the lab. They were always there for
support or just a simple talk. These little things helped to create a pleasant work environment.
Thank you to Sarah and Antoon, for your friendship and for the unforgettable moments we
shared in DruQuar.
Last but not least, I would like to thank all my friends and family for their continuous support.
A special thank goes to my parents, who gave me the chance to obtain an additional Master
degree and for always believing in me.
ABSTRACT
Introduction
Somatropin (i.e. recombinant human growth hormone, rhGH) is a biologic drug, currently
approved to treat growth hormone deficiencies. Recently, studies indicate a possible role of
hGH in certain cancers; hence, the oncologic potential of somatropin and analogues/modified
proteins are currently under investigation. Modifications with chelating agents like 1,4,7-
triazacyclononane-1,4,7-triacetic acid (NOTA) allows the incorporation of radiometals for
SPECT/PET-diagnostic (67Ga, 68Ga, 111In) or therapeutic (90Y) purposes. Somatropin has nine
potential binding sites (Lys) for NOTA. Therefore, it is important to characterize the obtained
product under different specified synthesis procedures like the excess of NOTA (1:1, 3:1 and
10:1 molar based) added to somatropin.
Objective
The main objective of this paper is to develop a hGHAb-based SAW method in order to
perform a functional quality characterization of NOTA-modified somatropins.
Methods and Results
Five different ligand immobilisation techniques were explored and compared in terms of
ligand immobilisation density, binding capability and specificity. The covalent amine
immobilisation of somatratopin was found the best immobilisation method (59 pg/mm2
surface-bound ligand, 66.7% RR% and 1.5% NSB%). An appropriate regeneration procedure
was also developed: a mixture of 50 nM NaOH and 0.1% SDS completely removed the bound
hGHAb from the immobilised somatropin surface, while preserving the ligand ‘s activity. The
developed SAW method was used to determine the binding affinities and kinetics of NOTA-
modified somatropins for hGHAb. A KD in sub-nanomolar range was observed for
somatropin and NOTA-modified somatropins.
Conclusion
The NOTA-modifications do not alter the binding affinity of somatropin toward hGHAb. The
hGHAb-based SAW method was successfully used as a system to investigate at least part of
the functional quality of NOTA-modified somatropins. Binding to hGHAb does not
automatically correlate with the binding potency to hGHR expressing tumour cells. These
questions are currently further being explored.
SAMENVATTING
Inleiding
Somatropine (recombinant humaan groeihormoon, rhGH) is een biologisch geneesmiddel, dat
goedgekeurd is voor de behandeling van groeihormoon deficiënties. Recente studies tonen
aan dat humaan groeihormoon een belangrijke rol kan spelen in de ontwikkeling van bepaalde
tumoren. Vandaar dat het oncologische potentieel van somatropine en analogen momenteel
wordt onderzocht. Somatropine werd geconjugeerd met een chelator zoals 1,4,7-
triazacyclononaan-1,4,7-triazijnzuur (NOTA) zodat het radioactief gelabeld kan worden voor
SPECT/PET-diagnostische (67Ga, 68Ga, 111In) en therapeutische (90Y) doeleinden. Het is
daarom belangrijk om de verkregen eindproducten, die gesynthetiseerd werden onder
verschillende omstandigheden (1:1, 3:1 en 10:1 NOTA overmaat), te karakteriseren
Objectieven
Het doel van deze thesis is het ontwikkelen van een hGHAb-gebaseerd SAW methode, die
gebruikt kan worden voor de functionele kwaliteit karakterisatie van NOTA-gemodificeerde
somatropines.
Methoden en Resultaten
Er werden vijf verschillende immobilisatietechnieken onderzocht en met elkaar vergeleken
voor de ligand immobilisatie densiteit, de binding capaciteit en de specificiteit. De
immobilisatie met de aminekoppeling van somatropine gaf de beste resultaten (59 pg/mm2
oppervlakte-gebonden ligand, 66.7% RR% en 1.5% NSB%). Er werd een regeneratie
procedure ontwikkeld voor de gevonden immobilisatietechniek. De meeste geschikte conditie
was een 1:1 mengel van 50 nM NaOH en 0.1% SDS die zorgde voor het complete wegwassen
van gebonden hGHAb van het geimmobiliseerde somatropine oppervlak, waarbij de activiteit
van het ligand werd bewaard. Tot slot werd de ontwikkelde methode toegepast om de
bindingaffiniteit en kinetiek te bepalen van NOTA-gemodificeerde somatropines voor
hGHAb. De gevonden KD (sub-nanomolair) waren equivalent voor somatropine en NOTA-
gemodificeerde somatropines.
Conclusie
De hGHAb-gebaseerd SAW methode werd succesvol toegepast voor karakterisatie van
NOTA-gemodificeerde somatropines. Er werd gevonden dat de NOTA-modificaties bij
somatropine geen verandering veroorzaken in de bindingaffiniteit tegenover hGHAb.
Momenteel wordt er verder onderzoek verricht naar de functionele kwaliteit van humaan
groeihormoon receptor (hGHR) op tumorcellen.
TABLE OF CONTENT
1! INTRODUCTION* 1!
1.1! ANALYTICAL*TOOLS*FOR*THE*FUNCTIONAL*CHARACTERIZATION*OF*BIOPHARMACEUTICALS* 1!1.1.1! LABELLED)LIGAND!BINDING!ASSAYS! 2!1.1.2! ISOTHERMAL!TITRATION!CALORIMETRY! 2!1.1.3! OTHER!BINDING!ASSAYS! 2!1.2! BIOSENSORS* 3!1.2.1! SURFACE!PLASMON!RESONANCE!BIOSENSOR! 3!1.2.2! QUARTZ!CRYSTAL!MICROBALANCE!BIOSENSOR! 5!1.2.3! SURFACE!ACOUSTIC!WAVE!BIOSENSOR! 5!1.2.4! APPLICATIONS! 7!1.3! THE*HUMAN*GROWTH*HORMONE*SYSTEM* 9!1.3.1! SOMATROPIN:!HISTORY,!USE!AND!ABUSE! 9!1.3.2! PHYSIOPATHOLOGY!OF!HUMAN!GROWTH!HORMONE!IN!CANCER! 10!1.4! PEPTIDE=*AND*PROTEIN=BASED*RADIOPHARMACEUTICALS* 13!
2! OBJECTIVES* 15!
3! MATERIALS*AND*METHODS* 16!
3.1! LITERATURE*REVIEW*=*APPLICATIONS*AND*METHOD*VARIABILITY* 16!3.2! REAGENTS*AND*MATERIALS* 16!3.3! SENSOR*CHIP*PREPARATION*FOR*LIGAND*IMMOBILISATION* 17!3.3.1! CLEANING!AND!CHEMICAL!ETCHING!OF!THE!SENSOR!CHIP! 17!3.3.2! PREPARATION!OF!A!CARBOXYMETHYL!DEXTRAN!HYDROGEL!SENSOR!CHIP! 17!3.4! DEVELOPMENT*OF*A*HGHAB=BASED*SAW*METHOD* 18!3.4.1! LIGAND!IMMOBILIZATION! 18!3.4.2! SCREENING!FOR!A!REGENERATION!CONDITION! 19!3.4.3! ANALYTE!INTERACTION!WITH!IMMOBILISED!LIGAND! 20!3.4.4! NON)SPECIFIC!BINDING!OF!ANALYTE!TO!IMMOBILISED!LIGAND!SURFACE! 20!3.4.5! NON)SPECIFIC!BINDING!OF!ANALYTE!TO!DEXTRAN!HYDROGEL!SENSOR!CHIP! 20!3.5! FUNCTIONAL*CHARACTERIZATION*OF*NOTA=MODIFIED*SOMATROPINS* 21!3.5.1! IMMOBILISATION!OF!SOMATROPIN!(DERIVATIVES)!VIA!AMINE!COUPLING! 21!3.5.2! BINDING!EXPERIMENTS!BETWEEN!HGHAB!AND!NOTA)MODIFIED!SOMATROPINS! 21!3.6! DATA=ANALYSIS* 22!3.6.1! FEASIBILITY TEST FOR SAW BINDING EXPERIMENTS! 22!3.6.2! EVALUATION!OF!THE!IMMOBILIZATION!STRATEGIES! 23!
3.6.3! EVALUATION OF THE REGENERATION CONDITIONS! 23!3.6.4! DETERMINATION!OF!DISSOCIATION!CONSTANT! 24!
4! RESULTS* 27!
4.1! LITERATURE*REVIEW*=*APPLICATIONS*AND*METHOD*VARIABILITY* 27!4.1.1! RANDOM!VARIABILITY!IN!KD!VALUES! 27!4.1.2! MODEL!VARIABILITY! 28!4.2! DEVELOPMENT*OF*A*HGHAB=BASED*SAW*METHOD* 29!4.2.1! EXPLORING!LIGAND!IMMOBILISATION!STRATEGIES! 29!4.2.2! EVALUATION!OF!THE!NON)SPECIFIC!BINDING!OF!ANALYTE!TO!DEXTRAN!HYDROGEL!SENSOR!CHIP! 34!4.2.3! SCREENING!FOR!REGENERATION!OF!SOMATROPIN!LIGAND! 35!4.3! FUNCTIONAL*CHARACTERIZATION*OF*NOTA=MODIFIED*SOMATROPINS* 35!4.3.1! IMMOBILIZATION!OF!SOMATROPIN!AND!DERIVATIVES!USING!AMINE!COUPLING! 35!4.3.2! BINDING!EXPERIMENTS!BETWEEN!IMMOBILIZED!SOMATROPIN!(DERIVATIVES)!AND!HGHAB! 37!4.3.3! SPECIFICITY!OF!THE!METHOD! 39!
5! DISCUSSION* 41!
5.1! LITERATURE*REVIEW*=*APPLICATIONS*AND*METHOD*VARIABILITY* 41!5.1.1! RANDOM!VARIABILITY!KD!VALUES! 41!5.1.2! MODEL!VARIABILITY! 42!5.2! DEVELOPMENT*OF*A*HGHAB=BASED*SAW*METHOD* 42!5.2.1! EXPLORING!IMMOBILISATION!PROCEDURES!FOR!THE!IMMOBILISATION!OF!ANTIBODIES! 43!5.2.2! SCREENING!FOR!OPTIMAL!REGENERATION!CONDITIONS! 46!5.3! FUNCTIONAL*CHARACTERIZATION*OF*NOTA=MODIFIED*SOMATROPINS* 46!5.3.1! IMMOBILIZATION!OF!SOMATROPIN!AND!DERIVATIVES!USING!AMINE!COUPLING! 46!5.3.2! BINDING!EXPERIMENTS!BETWEEN!IMMOBILIZED!SOMATROPIN!()DERIVATIVES)!AND!HGHAB! 47!
6! CONCLUSION* 50!
7! REFERENCES* 51!
8! LIST*OF*ATTACHMENTS* 62!
LIST OF USED ABBREVIATIONS
BSA Bovine serum albumin
cGH Cadaveric growth hormone
CM carboxymethylated
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
DruQuaR Drug Quality and Registration
EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
ELISA Enzyme-linked immunosorbent assay
EMA European Medicines Agency
FDA Food and Drug Administration
FIA Fluorescence immunoassay
GAG Glycosaminoglycan
GH Growth hormone
GHD Growth hormone deficiency
HBS HEPES buffered saline
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hGH Human growth hormone
hGHAb Human growth hormone antibody
hGHR Human growth hormone receptor
HTS High throughput screening
IDT Interdigital transducer
IGF Insulin growth factor
ITC Isothermal titration calorimetry
mAb Monoclonal antibody
MUD 11-mercapto-1-undecanol
NET Neuroendocrine tumour
NHS N-Hydroxysuccinimide
NOTA 1, 4, 7-triazacyclononane-1,4,7-triacetic acid
QC Quality control
QCM Quartz crystal microbalance
rhGH Recombinant human growth hormone
RIA Radio immunoassay
SAM Self-assembling monolayer
SAW Surface acoustic wave
SDS Sodium dodecyl sulphate
SPR Surface plasmon resonance
TETA 1,4,8,11-tetraazacyl cotetradecane-N,N',N ",N"'-tetraacetic acid
WADA World Anti-Doping Agency
1
1 INTRODUCTION
1.1 ANALYTICAL TOOLS FOR THE FUNCTIONAL CHARACTERIZATION OF
BIOPHARMACEUTICALS
The biopharmaceuticals or biotechnological medicinal products (“biotech drugs”) are
an increasing success within the pharmaceutical industry. It is expected that by 2015, 50% of
the newly introduced drugs will be biotech drugs [1]. Because the patents of many
biopharmaceuticals are expired or are about to, the opportunity opens for the development of
biosimilars, i.e. copy-versions of an already authorized biopharmaceutical, which are not
identical but highly similar. Many challenges come with the development and regulatory
approval of biosimilars, due to their magnitude (i.e. large primary structure with small
variations possible), post-translational modification (e.g. glycosylation), three dimensional
structure (e.g. T-shape vs. Y-shape antibodies) and protein aggregation. The manufacturers of
the biosimilars have to prove that their product is comparable to the authorized reference
medicinal product at the level of quality, efficacy and safety [2] (Figure 1.1). The complexity
of the authorization requirements of biosimilars illustrates the emerging role of analytical
technologies in the regulatory approval of biosimilars [3].
Figure 1.1: Analytical characterization of biosimilar products to establish biosimilarity - a step-by-step approach. Adapted from reference [4].
A critical aspect of the regulatory approval of biosimilars is the side-by-side
comparison of the functional activity, which is basically achieved by in vitro cell-based assays
[5]. One of the parameters to be evaluated is the binding affinity, which is quantitatively
2
measured as the dissociation constant (KD). Moreover, the European Medicine Agency
(EMA) recommends the use of emerging technologies such as real time binding assays [6].
1.1.1 Labelled-ligand binding assays
In labelled-ligand binding assays radio- and fluorescent-labelled ligands are used for
the measurement of the binding affinity. A radioactive labelled (with radioisotopes such as 3H, 125I, 35S) or fluorescent-labelled ligand is applied to detect and quantify the binding to a
specific target (enzymes, receptors in tissues or cell cultures). The drawback with labelled-
ligand binding assays is the labelling itself, because this can lead to potential alterations in the
binding characteristics. Also the high cost and hazards for the removal of radioactive waste,
encourages the development of label-free (non-radioactive) techniques [7].
1.1.2 Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) determines the binding affinity, enthalpy,
entropy and reaction stoichiometry in a label-free thermodynamic assay by sensing heat
changes caused by the binding reaction [8]. ITC involves two identical cells, a reference cell
(contains buffer or water) and a sample cell (contains the ligand solution). The analyte is
added to the sample cell, which results in enthalpic changes induced by the ligand-analyte
interaction. These changes are measured as the input of power per time required to retain an
equal temperature between both cells [9]. ITC does not require any labelling or
immobilisation, which allows us to study the binding interaction in their native state.
However, large sample volume (>ml) and sample concentration (>µM) are required for a
single experiment [10]. ITC is used to investigate protein-protein interactions, protein-
DNA/RNA interactions, protein-small molecule interactions and enzyme kinetics [11]. ITC is
less suitable for the evaluation of the binding affinity with receptors presented on cells in
culture and tissues as the cells will die because of the lack of oxygen during the experiment
[12].
1.1.3 Other binding assays
Other techniques used for the investigation of ligand-receptor interaction are structure-
based ligand binding assays. Spectroscopic techniques such as NMR and X-ray
3
crystallography are able to monitor changes in the molecular structure that occur upon
interaction [13, 14].
Biosensors are an emerging method for the label-free determination of binding
characteristics in real time. The different techniques and applications will be further discussed
in chapter 1.2.
1.2 BIOSENSORS
A biosensor is an analytical device, which employs immobilized biological
compounds or ligands for detection of analytes in samples. Biosensors can be divided in
different classes according to the mode of transduction: optical, mass, electrochemical and
thermal [15]. They are able to investigate molecular interactions in real time without labelling
of the analyte. Binding affinity and kinetics of an interaction are determined based on the
observed binding rates. As previously mentioned, label-free methods have quite some
advantages over fluorescent-labelling and radiolabelling, e.g. cost and time saving, less
harmful and less false conclusions due to interferences of the labelling agent [16].
Three biosensor systems are currently available on the market; the Surface Plasmon
Resonance (SPR), the Quartz Crystal Microbalance (QCM) and Surface Acoustic Wave
technology (SAW), each is based on different detection platforms.
1.2.1 Surface Plasmon Resonance biosensor
SPR biosensor is the most popular technique among the biosensors. It is the most
commonly used biosensor as it is the most frequently published in biosensor literature (Figure
1.2).
Figure 1.2: A summary of the number of publications per year referencing “surface acoustic wave”, “quartz crystal microbalance” or “surface plasmon resonance” based on the ISI citation database.
4
SPR technology is an optical biosensor that detects changes in the refractive index
near the surface layer of the sensor chip. Surface plasmon resonance is an optical
phenomenon that occurs under the conditions of total internal reflection (TIR): all the incident
light is reflected at a surface-solution interface. Although no light is coming out at the
interface of the solution, an electromagnetic field called ‘evanescent waves field’, extends a
short distance into the medium of lower refractive index.
In the setup of an SPR biosensor, monochromatic and plane-polarized light passes
through a prism, reflects on the backside of the sensor chip surface into a detector (Figure 1.3
top). At a certain angle of incident light, known as the resonance angle, the photons will
interact and be absorbed by oscillating electrons in the metal film (i.e. surface plasmons). This
phenomenon, surface plasmon resonance, causes a reduction in the intensity of reflected light
and can be seen as a dip in the SPR reflection intensity curve (Figure 1.3 bottom).
The surface plasmons are sensitive to fluctuations of electron density at the interface of
two materials. Thus, the resonance angle is dependent on the refractive index of the solution
close to the gold layer. This means that when analyte and ligand interact at the interface, the
resonance angle is modified. The shift in resonance angle is proportional to the mass of the
bound molecules [17].
Figure 1.3: Mechanism of SPR biosensor. Interaction between ligand and analyte can be monitored in real time by analysis of the reflected light. The resonance angle shifts when a molecular binding event takes place that changes the refractive index on the sensor chip surface. Figure adapted from reference [18].
5
1.2.2 Quartz Crystal Microbalance biosensor
In contrast to SPR, where binding processes are measured by changes in refractive
index, the QCM is based on the properties of piezoelectric materials. The principles are
similar to the SAW biosensor, which is discussed further below (1.2.3.1). The difference with
SAW biosensor is that the acoustic waves are propagated along the whole substrate, thus are
not limited to the surface of the quartz crystal.
1.2.3 Surface Acoustic Wave biosensor
Surface acoustic wave biosensors are devices that utilize a high frequency acoustic
wave (100 MHz - 300 MHz), as the name implies [15]. They were first used as filters and
resonators in the telecommunication industry [19]. Later, they were picked up by the bio-
chemical scientific community for sensing applications. These types of biosensors are highly
sensitive towards mass changes and viscoelastic changes on the sensor surface.
1.2.3.1 Principle of SAW sensor technology
Basically, a SAW sensor generates and detects acoustic waves on the surface of a
piezoelectric substrate. When an electric field is applied to a piezoelectric material, this
creates a mechanical stress; and the other way around, imposing an appropriate mechanical
stress generates an electric field. This mechanical property of piezoelectric materials is used
in the setup of the SAW biosensor. Two interdigital transducers (IDT) are placed on the top of
the substrate. The input IDT is used to provide an electric field that acts on the piezoelectric
substrate to form an acoustic wave. The generated wave propagates through the substrate,
where the biological detection occurs and is finally reconverted at the receiving IDT into
electrical signals [20]. Most common piezoelectric material used for acoustic wave devices
are quartz, lithiumtantalate, lithiumniobate and zinc oxide [21].
SAW sensors are capable to detect mass and viscoelastic changes on the sensitive
surface by measuring wave characteristics. When the surface layer experience changes in
bound mass e.g. upon interaction between ligand and analyte, it will perturb the wave
propagation and accordingly cause shifting of the phase. Viscosity changes of the ligand on
the other hand are indicated by a change in wave amplitude [19].
6
Figure 1.4: Basics of a SAW biosensor A. Elements of a SAW sensor chip. The basis of a sensor chip is a piezoelectric crystal such as quartz (=substrate) with IDTs at both ends. On the top the substrate a waveguide layer is applied, which propagates the acoustic wave in the surface layers. The chip is covered with plain gold to allow immobilisation of ligands. B. Detail of gold surface. The interaction between analyte (green) and ligand (blue) is studied at the surface of the chip.
1.2.3.2 Love-wave mode SAW biosensors
Currently the most sensitive SAW sensors are the sensors in Love mode configuration,
where the acoustic wave is guided through a single surface. This physical effect was
discovered by Love in 1911, who observed effects of an earthquake far from the epicentre
because the waves were guided through stratified geological plates. The same principle is
applicable to SAW devices where the wave is propagated in a guiding layer at the top of the
substrate rather than in the bulk of the piezoelectric material. Therefore a maximal amount of
acoustic energy is trapped close to the surface, where it will be strongly susceptible for
alternations, yielding a higher sensitivity of the sensor. The restriction of the energy in the
guiding layer is due to a lower wave propagation velocity in the waveguide layer than in the
substrate [15].
1.2.3.3 SAW biosensors for molecular interaction analysis
SAW technology can be used to characterize interactions between ligands
(immobilized on the sensor chip) and analytes (solution flowing over the ligands). Binding of
the analyte to the ligands alters the phase velocity of the acoustic wave. This change can be
monitored in real time as shown in Figure 1.5. As the analyte binds to the surface, a positive
phase shift is observed, which leads to an increase of the response (= association). At a certain
point an equilibrium is reached, where equal amounts of analyte are associating and
7
dissociating with the ligand (= steady state). When the analyte solution is then replaced by
buffer, a decrease in signal is observed, because of the separation of the ligand-analyte
complex and loss of bound analyte (= dissociation). Binding affinities and kinetics can be
determined by fitting the curves (i.e. the sensorgram) to an appropriate binding model [20].
Figure 1.5: Phase shift increases as the analyte binds to the ligands resulting in the association phase. After equilibrium, the analyte injection is stopped and replaced by buffer flow. The phase signal decreases, which indicates the dissociation phase. The remaining signal after the dissociation phase comes from the remaining associated ligand-analyte complexes
1.2.4 Applications
SAW biosensors are currently been applied in divergent areas such as food analysis
[22], genomics [23], microbiology [24], environmental studies [25], chemical warfare
detection [26] and the medical diagnostics field [27]. The recent evolution in the biosensor
technology has expanded the capabilities of the biosensors, which allows the successful use of
biosensors in the drug development process [28]. In the next paragraphs, examples are given
of the biosensor application in the drug discovery, preclinical and toxicological studies,
formulation development studies and quality control environment.
1.2.4.1 Application of biosensors in drug development
Biosensors can be applied in many areas of the drug development process. Optical
biosensors have found their way into the pharmaceutical industry for ligand fishing, high
throughput screening (HTS), hit confirmation and lead optimization (Figure 1.6) [18, 29].
During hit confirmation, it is often desirable that the KD is within a specific concentration
Associa'on)
Dissocia'on)
Equilibrium)
PHAS
E)SH
IFT)(°))
TIME)(SEC)))
Injec'on)
8
range. The binding constants of receptor-drug complexes are generally situated in the
micromolar to nanomolar range [30].
Further applications in the pharmaceutical field are for example interactions studies
between a new lead compounds and biological membranes. Permeation of biologically active
compounds would result in changes of the viscoelastic properties of the lipid bilayers in the
immobilized model membranes [31]. Another example is the application of a SPR-based
method to predict the binding affinity of new drug compound for serum proteins, using
immobilized albumin or alpha-1-glycoprotein [32].
In addition, these devices have the potential to be used in toxicity studies. Cell toxicity
of new drug compounds can easily be analysed with whole cell systems; cytoskeletal changes
resulting in cellular volume retribution are detected with biosensors in a reproducible manner
[33].
Biosensors could also be useful in the formulation development. A biocompatibility
study between protein pharmaceuticals and a silicon-oil coated surface was performed with a
QCM. This information was used to determine potential interactions and/or adsorption of
proteins to silicon-oil coated packaging [34].
With the development of process analytical monitoring (PAT) within the
biopharmaceutical industry, bioprocess monitoring using biosensors may permit automated,
real time monitoring of critical performance quality attributes. An automated at-line SPR
detection system has recently been described to monitor secreted protein in a bioreactor
culture of transiently infected embryonic kidney cells [35] and automated SPR methods have
also been used to quantify levels of bioactive monoclonal antibody produced by a hybridoma
cell line.
Finally, biosensor assays have a number of advantages over alternative bioassay
techniques such as ELISA, FIA or RIA which are traditionally used for biopharmaceutical
process development and quality control (QC): (i) no need for reporter molecules such as
enzymes/substrates, (ii) monitoring in real time, (iii) reactants retain conformational integrity
and (iv) use of low concentrations of products within a crude sample [36, 37].
9
Figure 1.6: Overview of the application areas for biosensors in drug development process. Figure adapted from reference [18].
1.3 THE HUMAN GROWTH HORMONE SYSTEM
1.3.1 Somatropin: history, use and abuse
Somatropin or recombinant human growth hormone (rhGH) is widely used for the treatment
of growth hormone deficiencies (GHD) in both children and adults [38]. The first
recombinant hGH was produced in 1982 by Genentech Inc. Before the area of recombinant
DNA technology, children with GHD were treated with GH extracted from pituitary glands of
human cadavers (cGH) because bovine or porcine GH was not effective in humans because of
the major differences in molecular structure. When in 1985, several cases of Creutzfeldt-
Jacob disease were reported with patients who had received cGH [39], regulatory authorities
removed cGH from the market and accelerated the approval of rhGH.
The hGH is a single chain protein of 191 amino acid residues, with a molecular weight
of 22 kDa. The protein structure includes four alpha-helices and two disulphide bonds. The
hGH is a hormone secreted in the anterior pituitary gland. The main function of hGH is to
stimulate the synthesis and secretion of insulin-like growth factors (IGFs) in the liver, skeletal
muscle and other tissues. IGFs stimulate the proportional body growth and regulate several
aspects of metabolism. The actions of hGH are characterized as anabolic and lipolytic [40].
Today seven different somatropin formulations have been authorized in the EU
including two as biosimilars [38]. Moreover, somatropin was the first biosimilar approved in
the EU in 2006. This requires standardization of both protein content and specific activity of
10
rhGH to assure the similar efficacy of the different preparations [41]. With the introduction of
biosimilars, questions were raised about the criteria to demonstrate the comparability of these
biotherapeutics with their originator reference products and the development of new
analytical tools to support the required comparability studies [3].
Somatropin is currently only indicated for the treatment of GHD. In children, GHD are
manifested by short stature and growth retardation. The most common cause are congenital
disorders e.g. GHD resulting from mutations in GH or the growth hormone releasing hormone
receptor genes.
In adults, GHD rarely occurs and is often acquired by inflammation, trauma or tumour
of the hypothalamus or pituitary [42]. In addition to the use of rhGH as hormone replacement
therapy, somatropin is approved by the European Medicines Agency (EMA) to correct short
stature in children with Turner syndrome, Prader-Willi syndrome and chronic renal failure
[43]. Somatropin has also been approved by the FDA for the treatment of AIDS-related
cachexia, but this indication was however rejected by the EMA. Therefore, the use of
somatropin for AIDS wasting is considered off-label in Europe.
Somatropin is included in the prohibited list of the World Anti-Doping Agency
(WADA) [44]. rhGH is very popular among athletes as a performance-enhancing substance
because of its anabolic effects (increases lean body mass, reduces body fat) [45-47].
Somatropin is also illegitimately prescribed and/or delivered by doctors as an anti-aging
product to increase vitality [48]. The abuse of somatropin in sports and as an anti-aging
compound resulted in an increase of spurious/falsely-labelled/falsified/counterfeit (SFFC)
formulations [49].
1.3.2 Physiopathology of human growth hormone in cancer
Recent research showed that hGH and human growth hormone receptor (hGHR) might
play a role in the development of several tumours [50]. Acromegaly and other conditions
characterized with high levels of hGH have been associated with an increased risk for colonic
adenomas. However, rhGH was not found to be oncogenic when applied in patients to correct
for low hGH levels: patients with GHD who received hGH replacement therapy are not
associated with an increased risk of carcinomas [50, 51]. Moreover, the up-regulated
11
expression of hGHR has been widely reported in several malignancies e.g. prostate [52, 53],
breast [54], colon [55-57] and gastric cancers [58], indicating the involvement of hGH-system
in these tumours.
1.3.2.1 Prostate cancer
Androgens like testosterone play a key role in the progression of prostate cancer. This
explains the treatment of early-diagnosed Prostate cancer with anti-androgens and in some
cases castration. In the late stage, therapies like radiotherapy and surgery are necessary [59].
Recent studies have suggested the involvement of hGH and hGHR in the
pathophysiology of prostate cancer: (i) the expression of hGHR is increased in prostate cancer
cell lines and in human tissues from patient diagnosed with prostate cancer [53], (ii) the
hGHR expression in prostate cancer cell lines is regulated by steroids and hormones
associated with the development and progression of prostate diseases [60], (iii) the hGH-
controlled proliferation of prostate cancer cells, (iv) stimulation of IGF and the expression of
bèta-oestrogen receptors in prostate cancer cell lines by hGH, and (v) hGH interaction with
IGF and oestrogen to stimulate proliferation of androgen dependent prostate cancer cells [61].
These studies indicate the involvement of hGH and hGHR in prostate cancers, although
aspects of the exact mechanism remain unclear. hGHR may be considered as a target for the
development of new therapeutics against prostate cancer [52].
1.3.2.2 Breast cancer
The current paradigm in oncology state that tumour growth is a pathological
reproduction of the developmental processes. The development of the mammary glands
involves complex interactions between oestrogen, hGH and prolactin. hGH is necessary for
the formation of rapidly proliferating terminal end bud structures in the developing mammary
gland [62]. The involvement of ovarian hormones in the development and progression of
breast cancer is well known [63, 64]. In the last decade, more evidence suggests the same for
(autocrine) hGH (Figure 1.7).
First indication of the association between hGH and breast cancer is the increased
level of hGHR expression in breast cancer tissues [54]. Another indication is the presence of
locally produced hGH. Autocrine hGH promotes cell proliferation and cell survival in
12
mammary cancer cells. In addition, studies have showed that hGH increases the telomerase
activity, which could contribute to the unlimited replication capacity of these cells [62]. More
recent findings suggest that autocrine hGH act as a potential regulator, which promotes
angiogenesis and induces resistance to chemotherapeutic drugs [65, 66].
hGHR antagonism is considered as a potential approach for the treatment of breast
cancer. However, it appears that the hGH signalling in breast cancer cell lines is also
mediated by the association of hGH to prolactin receptor. Therefore, both hGHR and
prolactin receptor should be considered as potential targets in the development for new
therapeutics for breast cancer [67].
Figure 1.7: Role of autocrine hGH in mammary cancer. Figure adapted from reference [62]
1.3.2.3 Neuroendocrine tumours
Neuroendocrine tumours (NET’s) are a heterogeneous group of neoplasms from cells
of the neuroendocrine system. NETs can occur anywhere in the body, but are mostly found in
the gastrointestinal tract (∼70%) and the bronchopulmonary system (∼25%). Some do not
cause symptoms, while other NETs are associated with a wide variety of symptoms due to the
secretion of diverse peptide hormones and bioactive amines [68].
Most NETs overexpress receptors for somatotropin release inhibiting factor or
somatostatin. The cornerstone of the symptomatic treatment for NETs is somatostatin (and
analogues), which inhibit hormone secretion and therefore reduce the hormone-related
13
symptoms as well as tumour progression [69]. A radiolabelled somatostatin analogue is
currently under investigation (phase II) as therapeutic approach for certain types of NETs
(stage IVc with visual tumour uptake of 111In-Octreoscan) [70]. This approach is based on
the linkage of high-energy radioisotope (90Y or 177Lu) to a specific peptide-ligand
(edotreotide) of which receptors (somatostatin receptor subtype 2) are overexpressed on the
tumour cell’s surface [71].
Recently overexpression of hGH and hGHR was found in LCNEC (large cell
neuroendocrine carcinomas) patients suggesting a role for hGH and hGHR in the progression
of NETs [72]. This information could be valuable for the development of peptide- or protein-
based radiopharmaceuticals.
1.4 PEPTIDE- AND PROTEIN-BASED RADIOPHARMACEUTICALS
The radiolabeled somatostatin analogue is one of the many examples of protein-based
radiopharmaceuticals used for the diagnostic and/or treatment of certain cancers. In the past
decades, nuclear medicine has played an important role in the targeting and treatment of
cancers. Most research is focussed on radiolabelled antibody conjugates, i.e. radionuclides
(111In, 68Ga, 99mTC) combined with monoclonal antibodies. The antigen-binding specificity
provides the capability to localize the tumour, while the radionuclide allows the visualization
(diagnostic function) and/or the killing of the cancer cells [73].
In recent years, radiolabelled (small) peptides have gained enormous interest within
the scientific community [74]. The overexpression of peptide receptors in tumour cells (e.g.
somatostatin receptor in NETs) is the basis for the use of peptide-based radiopharmaceuticals.
The receptors on the surface of the tumour cells are potential targets. The most widely used
radiolabelled peptide is 111In-DPTA-octreotide, a radiolabelled analogue of somatostatin used
for scintigraphy of NETs. For the development of a radiolabelled peptide, it is important that
the high binding affinity of the peptide with its receptor remains the same after radiolabelling,
i.e. the chemical modifications do not influence the binding affinity [75].
There are two different methods to link radioisotopes to the ligand. In a direct
labelling process, the radioisotope is directly attached to a functional group of the ligand (e.g. 123I via iodination of tyrosine residues) [74]. Indirect methods involve the use of chemical
14
spacers to link the radionuclide to the ligand. These organic chemical spacers, called
bifunctional chelating agents, contain a reactive group for coupling to the ligand as well as a
metal-chelating group for the complexation of the radionuclide. A wide variety of
bifunctional chelating agents are available e.g. DOTA, NOTA (Figure 1.8) and TETA [76].
For the therapeutic application of a radiolabelled peptide, the internalization of the
peptide ligand – receptor complex into the tumour cells is desirable [75] and studies have
reported the internalization of hGH-hGHR complex [77]. This finding and the overexpression
of hGHR on the surface of various types of cancer cells make hGHR a potential candidate for
therapeutic tumour targeting with radiolabelled somatropin.
Figure 1.8: p-SCN-Bn-NOTA. NOTA is a nine-member cyclic compound and has three carboxylated groups and nitrogens, for six dative bonds with metal ions (e.g. Ga(III)). The thiocyanate groups forms the reactive site for the covalent attachment to free amines (e.g. lysine residues
15
2 OBJECTIVES
Somatropin (i.e. recombinant human growth hormone, rhGH) is a biologic drug,
currently only approved to treat growth hormone deficiencies. Recently, studies indicate a
possible role of hGH in certain cancers; hence, the oncologic potential of somatropin and
analogues/modified proteins is currently under investigation. Modifications with chelating
agents like 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) allows the incorporation of
radiometals for SPECT/PET-diagnostic (67Ga, 68Ga, 111In) or therapeutic (90Y) purposes.
Somatropin has nine potential binding sites (Lys) for NOTA. Therefore, it is important to
characterize the obtained product under different specified synthesis procedures.
The main objective of this paper is to develop a hGHAb-based SAW method in order to
perform a functional quality characterization of NOTA-modified somatropins.
(i) The first part of this master dissertation will discuss whether or not SAW biosensors
are applicable tools in the functional quality control. A thorough literature search
will be performed.
• What is the current status of biosensors in drug development and quality control?
• What are analytical patterns among study results, such as error on the KD
determinations in SAW biosensor experiments?
• Can biosensors be used for the determination of binding differences of chemical
modified proteins? When do authors talk of a significant change in the binding
affinity?
(ii) The second part of this master dissertation will focus on the further development of the
hGHAb-based SAW method (Figure 2.1). More specifically, we will investigate
which immobilisation strategy leads to the most optimal conditions (ligand
immobilisation density, binding capability and specificity). Furthermore, an appropriate
regeneration procedure is required.
(iii) In the third part of this master dissertation we will apply the developed SAW method to
investigate if NOTA-modified somatropins, prepared under different conditions, result
in changes of the binding properties towards hGHAb.
16
3 MATERIALS AND METHODS
3.1 LITERATURE REVIEW - APPLICATIONS AND METHOD VARIABILITY
Relevant research papers concerning SAW biosensor technology were collected by a
thorough literature search in Web of Science and Pubmed databases. Publications were
selected from 2008 until present (2013) with search terms "love wave” or “surface acoustic
wave", and "biosensor” or “immunosensor" (in the title or abstract). Publications were found
suitable for consideration in this review if: (i) the device used in the study was a surface
acoustic wave biosensor, and (ii) the application of the study was a binding assay. Finally,
scientific articles wherein SAM® 5 Blue or S-Sens K5 instrument was mentioned, were
included as well.
The next step was a detailed examination of the articles, for further selection. Criteria
were introduced based on the technical specifications for the love wave biosensor similar to
the SAM®5 or S-Sens K5 instrument: 1) a working frequency within 100 MHz to 300 MHz
and 2) exclusion of studies in the gaseous phase. These criteria especially apply to in-house
assembled biosensors.
3.2 REAGENTS AND MATERIALS
Ammonium hydroxide (28% w/v), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic
acid (HEPES), ethanol absolute, 2-propanol, 11-mercapto-1-undecanol, epichlorhydrin,
diglyme, NaOH, dextran from Leuconostoc spp., bromoacetic acid, NaCl, NaIO4, acetic acid,
N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS),
ethanolamine, protein G’ from streptococcus sp. (recombinant, expressed in Escherichia coli),
carbohydrazide and sodium dodecylsulfate (SDS) were purchased from Sigma-Aldrich
(Diegem, Belgium). Sodium acetate trihydrate, albumin fraction V (from bovine serum)
(BSA), sodium borohydride and H2O2 (30%, v/v) were procured from Merck (Overijse,
Belgium). Zomacton® 4 mg, (Ferring, somatropin Ph. Eur.) was obtained from the Ghent
University Hospital (Ghent, Belgium). Lyophilized NOTA-modified somatropin was
synthetized at DruQuaR (Ghent University). The monoclonal mouse antibodies against hGH
were purchased from Thermo Fisher Scientific (Aalst, Belgium). All aqueous solutions were
prepared with 18.2 MΩ × cm deionized water, purified in an Arium 611 purification system
(Sartorius, Göttingen, Germany).
17
SAW experiments were performed with the SAM®5 Blue from SAW Instruments
GmbH (Bonn, Germany). Sensor chip, covered with a thin layer of gold, were also purchased
from SAW Instruments. Each chip received a unique number, to record its use-history. All
SAW experiments were performed at 20°C using HBS running buffer (10 mM HEPES (pH
7.4) and 150 mM NaCl) unless otherwise specified. The solutions intended for injection in the
SAW biosensor were prepared in LoBind Eppendorf® tubes or in glass vials.
3.3 SENSOR CHIP PREPARATION FOR LIGAND IMMOBILISATION
Before immobilisation of ligands, cleaning and chemical etching (i.e. removal of all
organic residues incl. immobilised ligand, up to the gold surface) of the sensor chip were
performed. These steps enabled the repeated use of the sensor chips [78].
3.3.1 Cleaning and chemical etching of the sensor chip
The chip was first pre-cleaned by 3 minutes ultrasonification in deionized water to
remove salts and polar compounds (e.g. carbohydrates). Then the chip was ultrasonicated 3
minutes in aceton followed by 3 minutes in isopropanol for the removal of lipophilic and non-
polar compounds (e.g. lipids, proteins, sugars). The surface was dried in a stream of N2. To
remove all organic residues from the gold layer, a chemical etching procedure was performed.
The chip was immersed for 3 minutes in a freshly prepared boiling solution of 5:1:1 deionized
water, ammonia and hydrogen peroxide at 70°C. Next the chip was rinsed in deionized water
and dried in a stream of N2.
3.3.2 Preparation of a carboxymethyl dextran hydrogel sensor chip
A self-assembled monolayer with end-standing hydroxyl groups was formed by
incubating the sensor chip in a 2 mM 11-mercapto-1-undecanol (MUD) solution in ethanol
over night. Afterwards the MUD modified chip was sonicated three times in absolute ethanol.
Next step was the formation of carboxymethyl (CM) dextran hydrogel (Figure 3.1). This was
achieved by the initial activation of the –OH groups with 0.6 M epichlorhydrin in a 1:1
mixture of diglyme and 0.4 M NaOH solution. After an incubation period of 4 hours, the chip
was covered with a 0.303 g/ml dextran solution in 0.1 M NaOH. Finally the dextran hydrogel
was carboxylated by covering the chip in a 1 M bromoacetic solution in 2 M NaOH for at
18
least 20 hours. The chip was rinsed with deionized water and air-dried. The chip was stored at
4°C to prevent microbial growth. Before use, the chip was inserted in the SAM®5 instrument
and conditioned with HBS running buffer (pH 7.4) for approximately 20 min.
Figure 3.1: Scheme of CM-dextran hydrogel modification of the golden surface. Adapted from reference [79].
3.4 DEVELOPMENT OF A HGHAB-BASED SAW METHOD
3.4.1 Ligand immobilization
3.4.1.1 Immobilisation of hGHAb via amine coupling
Prior to the immobilisation of hGHAb on a carboxymethylated (CM)-dextran
modified sensor chip, 10 µl of 0.1 % SDS was injected to wash away all non-specific bound
material on the chip. The chip was then equilibrated for 20 min in HBS running buffer (pH
7.4) in order to get a stable baseline. Next the chip was activated by injection of 130 µl of a
1:1 EDC:NHS mixture at a flow rate of 12.5 µl/min. Three successive injections of 25 µg/ml
hGHAb in 10 mM sodium actetate buffer pH 4.5, were carried out for 670 s to obtain an
effective immobilisation of the antibody. Finally, 130 µl of 1 M ethanolamine (pH 8.5) was
injected to block the remaining active sites, followed by an equilibration time of 30 min to
stabilize the baseline.
3.4.1.2 Immobilisation of hGHAb as somatropin:hGHAb-complex via amine coupling
A 1:1 somatropin:hGHAb solution was prepared to be immobilised directly to the
surface. In a LoBind Eppendorf® tube, equimolar quantities of hGHAb and somatropin in
HBS buffer were incubated for 10 min at room temperature. The hGHAb:somatropin solution
was further diluted to a final concentration of 25 µg/ml hGHAb with 10 mM sodium acetate
buffer (pH 4.5). The immobilisation of the complex was performed according to the protocol
in 3.3.1.1. After the deactivation step with 1 M ethanolamine, a 20 µl 0.1 % SDS injection
19
was performed to dissociate the complex, followed by an equilibration time of 20 min. to
stabilize the baseline.
3.4.1.3 Immobilisation of somatropin via amine coupling
Somatropin was also immobilised to the CM-dextran sensor chip using amine-
coupling chemistry. After the equilibration of the sensor chip, 50 µg/ml somatropin in acetate
buffer pH 4.5 was immobilised to the surface at the same conditions as 3.3.1.1.
3.4.1.4 Immobilisation of hGHAb via aldehyde coupling
The cis-diols of the sialic acids of the glycoproteins on hGHAb were oxidized to
aldehyde functions on ice using 4 µl of a 0.5 mg/ml hGHAb solution mixed with 0.8 µl of a
50 mM sodium m-periodate in 100 mM sodium acetate (pH 5.5) in a LoBind Eppendorf® tube
(final volume: 41.6 µl). The reaction was stopped after 20 min by desalting the mixture on a
desalting-column with 10 mM sodium acetate buffer (pH 4.0). The oxidized antibody was
eluated in 3.5 ml. The sensor chip was activated with a 1:1 NHS:EDC solution according to
3.3.1.1, followed by hydrazide modification using a 87.5 µl-injection of 5 mM
carbohydrazide in water. Any remaining active esters were inactivated with 130 µl injection
of 1 M ethanolamine (pH 8.5). Multiple 140 µl injections of oxidized-hGHAb were
performed in order to achieve an efficient immobilisation. Finally, 0.1 M sodium borohydride
in 0.1 M sodium acetate buffer (pH 4.0) was injected for 20 min to stabilize the hGHAb-
modified surface.
3.4.1.5 Immobilisation of hGHAb via protein G
50 µg/ml protein G (in 10 mM sodium actetate buffer pH 4.5) was immobilised to the
surface following the same protocol as used in 3.3.1.1. After the deactivation of the remaining
active esters, hGHAb was captured to the protein G. Immobilisation of 25 µg/ml hGHAb in
HBS buffer (pH 7.4) was performed by three 140 µl injections.
3.4.2 Screening for a regeneration condition
0.1% SDS, as used in the pilot studies, was not able to fully regenerate (i.e. removal of
the remaining bound analyte without affecting the immobilised ligands) the somatropin-
immobilised surface. Therefore, a screening with different regeneration conditions was
20
performed: 0.5% SDS, acetate buffer pH 4.5, 0.5 NaCl, 5% ethyleenglycol, 1 mM NaOH and
a 25 mM NaOH and 0.05% SDS solution. After 200 µl injection of 100 nM hGHAb, each
regeneration solution was injected over the sensor chip surface for 60s, followed by a
equilibration time of 5 min.
3.4.3 Analyte interaction with immobilised ligand
The interaction between analyte and immobilised ligand was studied. The hGHAb-
immobilised surfaces were investigated with 200 nM somatropin injections. The somatropin-
immobilised surface was studied with 100 nM hGHAb injections. The samples were injected
through the five channels for 600 s at a continuous flow rate of 20 µl/min. All injections were
performed with an initial burst i.e. a brief increase of the flow rate to 150 µl/min at the
beginning of the injection. Changes in phase shift were measured. The dissociation was
observed for 30 min, during which only HBS buffer was passed over the surface. After each
binding cycle, regeneration was carried out for 60 s. A blank injection (running buffer) was
performed prior to each interaction analysis. Subtraction of the response of the blank injection
from the analyte injections removes small but systematic imperfections in the response
curves. Data represent the mean ± SEM of at least two measurements (n ≥ 2).
3.4.4 Non-specific binding of analyte to immobilised ligand surface
The specificity of the somatropin- and hGHAb immobilized surface was studied with
a 100 nM and 200 nM BSA injection, respectively. The samples were injected through the
five channels for 600 s at a continuous flow rate of 20 µl/min. Changes in phase shift were
measured. The dissociation was observed for 30 min, during which only HBS buffer was
passed over the surface. After each binding cycle, regeneration was carried out for 60 s.
3.4.5 Non-specific binding of analyte to dextran hydrogel sensor chip
The non-specific binding of somatropin, hGHAb and BSA to the dextran hydrogel
surface (i.e. without immobilised ligand) was evaluated and compared with each other. The
carboxymethylated dextran chip was first equilibrated for 20 min in HBS running buffer (pH
7.4) in order to get a stable baseline. The chip was then activated by injection of 130 µl of a
1:1 EDC:NHS mixture at a flow rate of 12.5 µl/min. Next, 130 µl of 1 M ethanolamine (pH
8.5) was injected to block the active sites, followed by an equilibration time of 30 min to
21
stabilize the baseline. On each chip, 200 µl somatropin, hGHAb and BSA were injected at a
flow rate of 20 µl/min with HBS running buffer, followed by 800 s dissociation.
3.5 FUNCTIONAL CHARACTERIZATION OF NOTA-MODIFIED SOMATROPINS
3.5.1 Immobilisation of somatropin (derivatives) via amine coupling
Somatropin was immobilised using amine-coupling chemistry. Prior to the
immobilisation of somatropin on CM-dextran modified sensor chip, 10 µl of 0.1% SDS was
injected to remove all non-specific bound material on the chip. The chip was equilibrated for
20 min in HEPES running buffer (pH 7.4) in order to get a stable baseline. Next the chip was
activated by injection of 130 µl of a 1:1 mixture of 0.4 M EDC and 0.1 M NHS at a flow rate
of 12.5 µl/min. Three successive injections of 50 µg/ml somatropin (in 10 mM sodium
actetate buffer pH 4.5) were carried out for 670 s to obtain an effective immobilisation. After
immobilisation of somatropin, 130 µl of 1 M ethanolamine (pH 8.5) was injected to block the
remaining active sites, followed by an equilibration time of 30 min. to stabilize the baseline.
The same protocol was used for the immobilisation of the somatropin derivatives.
3.5.2 Binding experiments between hGHAb and NOTA-modified somatropins
For the binding assay, hGHAb was diluted with HBS buffer covering a concentration
range between 25 nM - 100 nM. The dilutions were prepared in glass vials to prevent leaching
from plastic vials. The samples were injected through the five channels for 600s at a
continuous flow rate of 20 µl/min. All injections were performed with an initial burst, i.e. a
brief increase of the flow rate to 150 µl/min at the beginning of the injection. The dissociation
was observed for 30 min, during which only HBS buffer was passed over the surface. After
the dissociation, regeneration was carried out for 60 s with a regeneration solution consisting
of 25 mM NaOH and 0.05% SDS, followed by two 30 µl buffer injections. For the control
experiments 100 nM BSA was injected under the same conditions, except for the regeneration
step, which was carried out with 0.1% SDS. In addition, a blank injection (running buffer
without analyte) was performed prior to each bindings experiment and its response was
substracted from the analyte injections. Data represent the mean ± SEM of at least three
measurements (n ≥ 3).
22
3.6 DATA-ANALYSIS
3.6.1 Feasibility test for SAW binding experiments
The signal obtained from a SAW biosensor is related to the mass changes caused by the
investigated molecules. A prediction of whether a certain molecule will cause a sufficient
mass change to obtain a detectable signal is useful to estimate the feasibility of the method.
This is mainly determined by the sensitivity of the equipment.
A theoretical formula was previously proposed to evaluate the feasibility of the SAW
biosensor method for the application under study [80]. For proteins, peptides or small
molecules as ligand with known molecular weight following formula was proposed:
! = !!×!"!×!!!×!!"!
!> 0.5! !"!!! (1)
Where: X: the theoretical area-concentration of bound analyte (in pg/mm2)
P: the phase shift (°) related to the bound ligand
Mw2 is the molecular weight of the analyte (in pg/pmol)
Mw1 is the molecular weight of the ligand (in pg/pmol)
S: the conversion factor, is an equipment constant (0.0515°mm2/pg)
F: binding stoichiometry
This formula determines the amount of analyte that can be detected, with the
assumption that all binding sites of the immobilised ligand are available and active for a 1-to-f
binding ratio (f = stoichiometry). In case of somatropin ligand, f is 0.5 because one
somatropin molecule binds to one of the two binding sites on the antibody. Therefore one half
of the hGHAb is occupied. If hGHAb is immobilised, f is theoretically 2, since every antibody
molecule has two binding sites. The theoretically calculated ad hoc signal value has to exceed
instrument detection limit; 0.5 pg/mm2 (area-concentration) to obtain a detectable signal with
an equipment. The amount of theoretically bound analyte will be dependent on the area-
concentration of ligand immobilised on the chip (= P/S in pg ligand/mm2), as well as the Mw
of the ligand and the Mw of the analyte.
23
3.6.2 Evaluation of the immobilization strategies
The phase shifts from the different immobilization techniques were compared using
normalized response values, calculated as follows (2):
!!% = !!!"!#$%& !!"# ×!100 (2)
Where, RR% = the relative ligand response (%)
!analyte = phase shift of analyte injection at 550s (°)
!max = maximum phase shift (°) i.e. phase shift if all immobilised
ligands were occupied calculated with formula (1).
The non-specific binding of the interactions was expressed as a ratio of the response of
BSA and analyte to the ligand immobilised surface.
!"#% = !!!"# !!"!#$%& !×!100 (3)
Where, NSB% = the relative amount of non-specific binding (%)
!BSA = phase shift of 100 nM BSA injection at 550s (°)
!analyte = phase shift of 100 nM analyte injection at 550s (°)
3.6.3 Evaluation of the regeneration conditions
In order to compare the regeneration efficiency (RE%) of the different conditions
following calculations were made:
!"! % = Δ!!!!"#"$"!%&'!"Δ!!!!"#$%&'#!(" ×100!
!!
!" % = !!!"#$%"!!!"#"$"!%&'($(!!) − !!!!"#$%!!"#"$"!%&'($!(!!)!!!!"#$%!!"#$%&!'"! !! − !!!!"#$%"!!"#$%&'#!("(!!)
×100!!!
24
Figure 3.2: Schematic representation of a sensorgram. The marked points are used for the calculation of the regeneration efficiency.
3.6.4 Determination of dissociation constant
The kinetic constants (kon and koff) and dissociation constants (KD) were determined by
fitting the obtained curves to a 1:1 binding model. The one-to-one binding model is based on
[81, 82]:
! + ! ⇌ !"
!(!")!" = ! ! !!" − (!")!!""
3.6.4.1 Association phase algorithm: kon and koff
kon is the association rate constant (units M-1s-1) and koff the dissociation rate constant
(units s-1). In biosensor experiments, the ligand L is immobilized on the sensor surface. The
concentration of complex [AL] is therefore identical to the concentration of bound analyte A.
The concentration of bound analyte is proportional to the phase P, which is detected by the
SAW biosensor. Free ligand concentration [L] is the difference between total and bound
ligand concentration. When the analyte is injected in a flow over the sensor surface, the
analyte solution is constantly replenished and hence, the free concentration of the analyte may
be considered and identical to the total analyte concentration C. The reaction between
immobilized ligand and analyte in solution can thus be assumed to follow pseudo first order
kinetics and since the concentration of complex and free ligand now can be expressed in
terms of analyte phase response P:
t1
t2 t3
25
!"!" = !!" !!"# − ! − !!""!
! ! = !!" 1− !!!!"#! !!Where,
!!!"# = !!"! + !!""
!!" =!!"!!!"#!!"#
!
Figure 3.3: One-to-one kinetic binding model according to (4) with X0: time at which the association begins (i.e. start injection [A]) and Peq: difference between plateau (response at equilibrium) and Y0 (i.e. baseline response).!
For each concentration of analyte A, an apparent rate constant (kobs) is determined
based on the association profile (0 – 550 s) (Figure 3.3). These apparent rate constants are
plotted against the concentration (nM) as illustrated in Figure 3.4. The intercept is the
dissociation rate constant. The slope is the association rate constant.
3.6.4.2 Dissociation phase algorithm: koff
The process of dissociation of the formed complexes AL can also be observed once
the analyte solution has traversed the flow cell and the system reverts to buffer flow. The rate
of dissociation of the formed complexes is described by
! ! = !!!×!!!!!""×!!
(4)
(5)
26
Figure 3.4: kobs in function of analyte concentration [A] .
Figure 3.5: An exponential one phase decay model based on formula (5) with P0: response at the beginning of the dissociation (i.e. the end of the injection and return to buffer flow) and plateau: response at the end of the dissociation.
The obtained data from the dissociation profile will be fitted in the one phase decay
model (Figure 3.5) from which the dissociation rate could be derived. Since the dissociation is
independent of analyte concentration, the koff is calculated as the mean dissociation rate
constant of the different concentrations.
The final KD-value will be calculated with the kon from the association phase and a
weighted mean of the koff values from the association and dissociation phase. The binding
constant (KD expressed in nM) is determined by:
!!""!!"
= !!
Where, koff: the off-rate, kon: the on-rate and KD: the binding constant
kobs=kon*[A]+koff+k o
bs+
27
4 RESULTS
4.1 LITERATURE REVIEW - APPLICATIONS AND METHOD VARIABILITY
The SAW applications can be classified in 9 groups (Figure 4.1), with most
publications in the medicine diagnostics field (48%). An overview of the SAW literature is
given in Attachment 1.
Figure 4.1: SAW biosensor applications.
A thorough literature search was performed to investigate the variability of a SAW
biosensor method. The variability in KD values reported in the publications as well as the
model variability i.e. the error on the fitting of the KD, were analysed.
4.1.1 Random variability in KD values
From the 13 publications which reported an affinity measurement by SAW biosensor,
only two publications performed multiple experiments (n>1) with a total of three different
interaction studies (Figure 4.2). An analysis of the reported standard deviations on the mean
KD resulted in a mean RSD of 60.43% for the three experiments. Figure 4.2 shows that the
RSD for interaction studies with membrane vesicles (RSD of 58.13% and 89.11%) is
relatively larger than the RSD of protein-protein interaction (RSD of 34.06%).
Medicine diagnostics
48%
Food analysis 7%
Biomaterials 4%
Polymer science
4%
Cell biology 3%
Rheology 3%
Microbiology 21%
Environmental science
3%
Genomics 7%
28
Figure 4.2: Variability of repeatability of SAW method. Pr = protein, MV = membrane vesicles, GAG = Glycosaminoglycan
4.1.2 Model variability
The model variability (expressed as RSE%) signifies the error on the fitting of the KD,
i.e. the variability of the goodness-of-fit (Figure 3.3). Large deviations on the fitting were
observed (mean RSE of 38.38%), especially for studies with glycosaminoglycans (Figure
4.3).
Figure 4.3 Variability of the KD fitting. Pr = protein, MV = membrane vesicles, MM = model membrane,
Pe = peptide, GAG = glycosaminoglycan.
0
20
40
60
80
100
Pr/Pr MV/Pr MV/GAG
[24] [123a] [123b]
34
89
58
RSD
(%)
Reference
n=5!
n>3!n>3!
0!10!20!30!40!50!60!70!80!90!
100!
MV
/GA
G!
MV
/GA
G!
MV
/GA
G!
MV
/GA
G!
MV
/GA
G!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
Pr/G
AG!
MV
/Pr!
Pe/P
e!
Pe/P
r!
RNA
/Pr!
MM
/Pr!
Pr/P
e!
Pe/P
r!
Pr/P
r!
[111a]![111b]![111c]![111d]![111e]![111f]![111g]![111h]![111i]![111j]![111k]![111l]![111m]![111n]![111o]![111p]![120]![121]![113]![112]![122a]![122b]![122c]!
31! 34!
58!
97!
70!
50!
1!
22!
62!
12!
71!
19!
44!49!
77!
24!
43!
32!
11!17! 13!
36!
11!
RSE
(%)!
Reference!
29
The linear association between model variability and the KD value was investigated
using the Pearson’s correlation coefficient. The dataset was first tested for homo- and
heteroscedasticity, where RSE (%) was taken as the dependent variable and KD as
independent variable. The data were found to be homoscedastic by plotting the residuals
against the predicted values (Figure 4.4B). This observation was statistically confirmed by the
Breusch-Pagan heteroscedasticity test, whereby a significance level of 0.201 (χ2=1.634) was
obtained which is higher than 0.05, thus maintaining the null hypothesis: the data is
homoscedastic.
Figure 4.4: A. Scatterplot of the model variability (RSE%) and the dissociation constant (KD). B. Scatterplot of the residuals against the predicted values.
The Pearson correlation coefficient was found to be r=0.282, indicating no linear
association between the model variability and the KD value. This was statistically confirmed
with the Student’s t-test; r<rcrit,0.05/21=0.413, the null hypothesis (no significant correlation) is
maintained. In the scatterplot, there is also no pattern observed in the relationship between the
variables in the scatterplot (Figure 4.4A).
4.2 DEVELOPMENT OF A HGHAB-BASED SAW METHOD
4.2.1 Exploring ligand immobilisation strategies
4.2.1.1 Principles of the different immobilisation techniques
Amine coupling is the most used immobilisation strategy in the biosensor field (Figure
4.5A). The carboxyl groups on the surface are first activated by reaction of EDC/NHS,
0
20
40
60
80
100
0 5000 10000 15000
RSE
(%)
KD
A!
B
30
creating reactive succinimide intermediates. The ligand is then linked to the activated surface
via nucleophilic addition of primary amine functional groups, which results in a stable amide
bond. After immobilisation, the remaining active esters are blocked with a small amine-
containing molecule i.e. ethanolamine.
Aldehyde coupling is based on the binding of aldehydes to a hydrazide-modified
surface. Antibodies, or immunoglobulins are glycoproteins; they have a branched
oligosaccharide N-linked to asparagine 297 present in the Fc region. The oligosaccharide
moiety is located away from the antigen binding sites. Therefore immobilisation to the surface
is possible without affecting the reactivity of the antigen binding site [83]. First aldehyde
groups are introduced into the glycoproteins via oxidation of oligosaccharide moieties using
periodate, which oxidize vicinal diols, such as sialic acids (e.g. acetylneuramic acid) [84]. The
carboxylated chip surface is activated with EDC/NHS, followed by an injection of
carbohydrazide to create a surface with hydrazide groups. The oxidized antibodies containing
aldehyde groups are coupled to the modified surface via condensation of aldehydes with the
nucleophile hydrazide (Figure 4.5B).
Figure 4.5: Schemes of IgG immobilisation techniques. A. amine coupling, B. aldehyde coupling and C. capturing via protein G. (R1 = CH2CH3, R2 = (CH2)3N(CH3)2). Figure adapted from [85]
Another method for site-directed immobilisation can be achieved by capturing a
monoclonal antibody (mAb) using protein G, which is a bacterial protein known for its high
affinity to immunoglobulin G (IgG) [86, 87]. The protein G interacts specifically with the Fc
B"
C"
A"
31
domain of the antibody, keeping the antigen-binding region (Fab) free for interaction. One
molecule of protein G can bind maximum two hGHAb, because of steric hindrance [88].
Protein G is first immobilised to the dextran-gold surface via amine coupling chemistry. IgG
is captured with protein G by its Fc region allowing immobilisation in a site-direct manner
with the antigen-binding sites pointing towards the analyte (Figure 4.5C).
The immobilisation of a ligand in complex with the analyte is a relatively new concept
of site-directed immobilisation [89]. The complex is immobilised using amine coupling,
whereby the analyte occupies the analyte binding sites on the ligand and hence, preventing
amine groups to participate in covalent immobilisation on the surface. At the end of the
immobilisation, a regeneration step is included to dissociate the complex. The result would be
a surface immobilised with “oriented” ligand and analyte (Figure 4.6). This immobilisation
technique was performed with a 1:1 somatropin:hGHAb complex.
Figure 4.6: Principle of the immobilisation via somatropin:hGHAb complex
4.2.1.2 Quantitative evaluation of ligand immobilisation
The immobilised ligand density was determined for the different immobilisation
techniques. The immobilisation could be followed (real-time) in a sensorgram. An example of
an immobilisation sensorgram is shown Figure 4.7. In this case somatropin is immobilised to
the dextran surface via amine coupling. The first shift in the baseline occurs after the
activation with EDC/NHS (200-1000s), indicating a chemical modification on the sensor
surface. This was followed by three somatropin injections leading to increased signals upon
immobilisation of somatropin ligand (amine coupling). The final injection of ethanolamine
was used to quench the activated carboxyl-sites. The difference in phase signal after the
activation and at the end of the immobilisation gives an indication of the total amount of
bound ligand.
Interac(on*of*somatropin*(analyte)*to*somatropin:hGHAb*complex*
Theory:*coupling*of*complex*trough*amine>coupling**
After regeneration
Dextran surface!
Ab-somatropin complex
Orientated Ab
32
Figure 4.7: Sensorgram of the online immobilisation of somatropin using amine coupling.
The total amount of surface bound ligand and the theoretical amount of analyte that
can bind, is calculated for each immobilisation technique using formula (1) (Figure 4.8). The
binding stoichiometry of the interaction used for the calculations is f = 2 for immobilised
hGHAb and f = 0.5 for immobilised somatropin (as one antibody can thoeretically bind two
somatropin molecules).
The feasibility test could not be applied to the immobilization of the complex, because
the increased phase shift is a result of immobilization of both hGHAb and somatropin. In the
case of the immobilisation with protein G, the capturing of hGHAb via protein G leaded to a
significant descending drift of the baseline over time. Therefore, the amount of hGHAb was
calculated after the baseline had stabilised. According to the feasibility test, the calculated
amount of analyte that could bind exceeded the theoretical limit of detection of 0.5 pg/mm²
for each immobilisation technique.
The aldehyde coupling however, was not further included in the method development
due to the low amount of hGHAb that was immobilized and the different steps needed in the
chemical process for oxidation of the carbohydrate functions. Also it is known that it is
difficult to control the oxidation reaction conditions [90] and excessive oxidation might not
only affect the carbohydrate moiety but also amino acid residues [91].
33
Figure 4.8: An overview of the amount of immobilised ligand (blue) and ad hoc analyte (red) (n=5).
4.2.1.3 Quantitative evaluation of the analyte-ligand interaction and the non-specific binding
The interaction between analyte and immobilised ligand was studied for the different
immobilisation techniques. The detected phase shifts upon analyte interaction and normalised
response values (RR%) for the different immobilisation techniques are given in Table 4.1.
The non-specific binding was studied with injections of analyte BSA. The phase shift from
BSA was compared to the phase shift from ligand and expressed as a percentage non-specific
binding (%NSB). The difference between NSB and specific binding is illustrated in Figure
4.9.
Table 4.1: Quantitative evaluation of analyte and non-specific binding
Analyte(1) BSA
Phase shift (°) RR% Phase shift (°) NSB%
Amine coupling hGHAb (n=5) 0.28 ± 0.04 9.61 ± 1.59 0.24 ± 0.04 84.54 ± 6.85
Protein G capturing (n=4) 0.29 ± 0.05 12.70 ± 0.63 0.01 ± 0.01 2.80 ± 1.37
Complex (n=5) (2) 0.32 ± 0.17 N/A 0.32 ± 0.22 94.43 ± 28.88 Amine coupling
somatropin (n=2) 6.88 ± 0.77 66.73 ± 9.04 0.10 ± 0.01 1.47 ± 0.37
(1) 200 nM somatropin analyte was used for hGHAb bound surface, 100 nM hGHAb ligand was used for
somatropin bound surface. Idem for BSA analyte.
(2) RR% is not calculated as the amount of immobilised hGHAb is unknown.
200
42
8
57
59
144
26
194
0 50 100 150 200 250
Amine coupling - somatropin
Protein G capturing
Aldehyde coupling
Amine coupling - hGHAb
pg/mm2
Amount bound ligand Theoretical bound analyte
hGH
Ab
ligan
d So
mat
ropi
n
ligan
d
34
The immobilisation of hGHAb using protein G and immobilisation of somatropin
using amine coupling resulted in the highest specificity (i.e. lowest NSB%). However, the
normalised response values (RR%) show that amine coupling of somatropin result in the
highest active ligand surface compared to the protein G captured surface. Therefore the
immobilization technique that will be further used in the method development is the amine
coupling of somatropin.
Figure 4.9: Sensorgram of specific and non-specific binding of different immobilisation strategies. A. hGHAb-immobilised surface (amine coupling), B. Somatropin-immobilised surface (amine coupling).
4.2.2 Evaluation of the non-specific binding of analyte to dextran hydrogel sensor chip
The non-specific binding of somatropin, hGHAb and BSA to the dextran hydrogel
surface was evaluated (Figure 4.10).
Figure 4.10: Non-specific binding of BSA, hGHAb and somatropin to dextran hydrogel surface
-0,05
0
0,05
0,1
0,15
0,2
0,25
-200 300 800 1300
Phas
e (°
)
Time (s)
Somatropin BSA
-1
0
1
2
3
4
5
6
7
-200 300 800 1300
Phas
e (°
)
Time (s)
hGHAb BSA
-0,2
0
0,2
0,4
0,6
0,8
1
0 200 400 600 800 1000 1200 1400 1600
Phas
e sh
ift (°
)
Time (s)
BSA hGHAb Somatropin
A B
35
The maximum phase shifts at 550s observed for BSA, hGHAb and somatropin were
0.37 ± 0.15°, 0.29 ± 0.11° and 0.35 ± 0.14° respectively (n=3). The phase shift from the non-
specific interaction of hGHAb with the dextran hydrogel surface is significantly lower than
the phase shift of the specific interaction to the immobilised somatropin (0.29° ± 0.11° and
6.88 ± 0.77°, respectively).
4.2.3 Screening for regeneration of somatropin ligand
An appropriate regeneration procedure is required for the chosen immobilisation
technique. The regeneration of the somatropin-immobilised surface with 0.1% SDS was not
able to remove the bound hGHAb. The dissociation after the analyte injections was not
complete (i.e. signal did not return to baseline). It is necessary to include a regeneration step
after each analyte binding cycle to remove all bound analyte from the ligand, while
maintaining the ligands’ activity. Screening for an appropriate regeneration condition was
performed with 0.5% SDS, acetate buffer pH 4.5, 0.5 M NaCl, 5% ethyleenglycol, 1 mM
NaOH. None of those conditions was able to return the signal back to baseline (Figure
4.11A). A small drop was observed with 0.5 % SDS. A 1:1 mixture of 25 mM NaOH:0.05%
SDS was used as suggested in reference [92]. This regeneration procedure was able to
regenerate the surface without loss of activity (Figure 4.11B).
4.3 FUNCTIONAL CHARACTERIZATION OF NOTA-MODIFIED SOMATROPINS
The developed SAW method was used to determine the binding affinities and kinetics
of NOTA-modified somatropins for hGHAb.
4.3.1 Immobilization of somatropin and derivatives using amine coupling
Somatropin and derivatives were immobilized to the dextran-gold surface using amine
coupling and identical experimental parameters. The total amount of surface bound
(modified)-somatropin is given in Figure 4.12. The amount immobilised ligand is higher for
unmodified somatropin and 1:1 NOTA somatropin compared to 3:1 NOTA somatropin and
10:1 NOTA somatropin.
36
Figure 4.11: Screening for regeneration conditions. A. The regeneration efficiency (%) obtained from the different regeneration conditions, B. Evaluation of ligand activity after regeneration with NaOH+SDS.
Figure 4.12: Overview of the amount of immobilised somatropin derivatives (pg/mm2)
16 1,5 1,5 0,8
7
107
-10
10
30
50
70
90
110
0.1% SDS
NaOAc NaOH NaCl 0.5% SDS
SDS + NaOH
Reg
ener
atio
n ef
ficie
ncy
(%)
Regeneration condition
59
117
10
31
0
20
40
60
80
100
120
140
Somatropin 1:1 NOTA somatropin
3:1 NOTA somatropin
10:1 NOTA somatropin
pg/m
m2
Amount surface bound ligand
A
B
37
A
4.3.2 Binding experiments between immobilized somatropin (derivatives) and hGHAb
The interaction between somatropin (ligand), the NOTA-modified somatropins
(ligand) and hGHAb (analyte) was studied (Figure 4.14). Figure 4.13 shows the sensorgram
of channel 1.
To obtain the kinetic and affinity constants, kon, koff and KD are calculated using a one-
to-one binding model. Each curve in the sensorgram was fitted using a 1:1 binding model
(with R2 of more then 0.95%). Fitting of the association phase leads to an observed rate
constant kobs for each analyte concentration (Figure 4.13B). The KD was calculated by
dividing the weighted average koff by the kon from the association phase. Table 4.2 gives an
overview of the kinetic data and calculated KD.
Table 4.2: Affinity (KD) and kinetics (kon, koff) of the interaction between somatropin and hGHAb
Channel 1 Channel 3 kon (nM-1s-1) ± SE 1.08E-04 ± 6.51E-06 9.33E-05 ± 5.41E-06
koff (s-1) ± SE 1.59E-03 ± 1,81E-05 1.57E-03 ± 1,43E-05
KD (nM) ± SD 15 ± 1 17 ± 1
Figure 4.13: Somatropin and hGHAb interaction study. A. Overlay plot of phase shifts at different hGHAb concentrations. B. Linear regression of apparent rate constant in function of analyte concentration.
0!1!2!3!4!5!6!7!
)200! 300! 800! 1300!
Phase*shift*(°)*
Time*(s)*
25!nM! 37.5!nM!50!nM! 100!nM!
y = 0,0001x + 0,0018 R² = 0,96758
0
0,002
0,004
0,006
0,008
0,01
0,012
0,014
0 50 100 150
k obs
(s-1
)
Concentration (nM)
B
38
Figure 4.14: Sensorgram of hGHAb on NOTA-modified somatropin immobilized surface (channel 5). A. 1:1 NOTA:somatropin ligand, B. 3:1 NOTA:somatropin ligand and C. 10:1 NOTA:somatropin ligand.
4.3.2.1 Binding experiment between somatropin (ligand) and hGHAb (analyte)
A average KD of 15.7 ± 1.6 nM was determined for the interaction between
somatropin and hGHAb (Table 4.2). The error of the fitting on the KD varies with a RSE
ranging from 5.8 to 6.1%. The variability (CV) between the different channels is 10.3%
(n=2).
-2
0
2
4
6
8
-200 0 200 400 600 800 1000 1200 1400
Phas
e (°
)
Time (s)
25 nM 37.5 nM 50 nM 75 nM 100 nM 100 nM
-0,1
0
0,1
0,2
0,3
0,4
-200 0 200 400 600 800 1000 1200 1400
Phas
e (°
)
Time (s) 25 nM 37.5 nM 50 nM 75 nM 100 nM 100 nM
-0,5 0
0,5 1
1,5 2
2,5
-200 0 200 400 600 800 1000 1200 1400
Phas
e (°
)
Time (s)
25 nM 37.5 nM 50 nM 75 nM 100 nM 100 nM
B
C
A
39
4.3.2.2 Binding experiment between 1:1 NOTA somatropin (ligand) and hGHAb (analyte)
The average KD is 19.0 ± 2.6 nM (n=5). The error of the fitting on the KD varies with
a RSE ranging from 7.1 to 8.4%. The variability (CV%) between the different channels is
13.5%.
4.3.2.3 Binding experiment between 3:1 NOTA somatropin (ligand) and hGHAb (analyte)
The average KD is 15.3 ± 1.1 nM (n=5). The error of the fitting on the KD varies with
a RSE ranging from 8.2 to 12.3%. The variability (CV%) between the different channels is
7.5%.
4.3.2.4 Binding experiment between 10:1 NOTA somatropin (ligand) and hGHAb (analyte)
The average KD of 15.4 ± 2.8 nM (n=5) was determined for the interaction between
10:1 NOTA somatropin and hGHAb. The error of the fitting for on the KD varies from a RSE
of 10.7 to 30.7%. The variability (CV%) between the different channels is 18.3%.
Table 4.3: Summary of binding constant and rate constants of somatropin and derivatives
KD ± SD kon ± SD koff ± SD
Unmodified somatropin (n=2) 15.7 ± 1.6 1.01E-04 ± 1.04E-05 1.58E-03 ± 1.39E-05
1:1 NOTA somatropin (n=5) 19.0 ± 2.6 3.96 E-05 ± 5.95E-06 7.50E-04 ± 1.53E-04
3:1 NOTA somatropin (n=5) 15.3 ± 1.1 8.84E-05 ± 6.85E-06 1.35E-03 ± 2.33E-05
10:1 NOTA somatropin (n=5) 15.4 ± 2.8 7.70E-05 ± 7.84E-06 1.17E-03 ± 1.15E-04
4.3.3 Specificity of the method
As a control experiment, the specificity of the binding assays was validated. The
interaction of the immobilised somatropin (derivatives) surfaces with BSA was evaluated.
The phase shift of BSA (100 nM) was compared to the phase shift of hGHAb (100 nM)
(Table 4.4) and expressed as a percentage non-specific binding (NSB%). The non-specific
interaction was less than 15% for all assays.
40
Table 4.4: Evaluation of the specificity of the method
Ligand Phase shift (°) 100 nM
BSA Phase shift (°) 100 nM
hGHAb % NSB
Somatropin (n=2) 0.10 ± 0.01 6.87 ± 0.63 1.44 ± 0.20
1:1 NOTA:somatropin (n=5) 0.23 ± 0.16 7.27 ± 0.73 3.21 ± 2.19
3:1 NOTA:somatropin (n=5)
0.05 ± 0.01 0.49 ± 0.08 10.90 ± 2.59
10:1 NOTA:somatropin (n=5) 0.18 ± 0.07 1.52 ± 0.39 11.56 ± 5.77
41
5 DISCUSSION
5.1 LITERATURE REVIEW - APPLICATIONS AND METHOD VARIABILITY
SAW biosensor literature was reviewed to gain more information about their current
status in research and to identify the analytical errors (KD variability and model variability)
among the study results. There are a few issues which must be kept in mind when considering
the research outcomes. Half of the publications from 2008 until now did not report a
dissociation constant. Especially for some experiments with in-house assembled biosensors,
no kinetic analysis was performed, hence, no KD was calculated. The aim of the affinity
determinations was mostly for qualitative purposes. The KD was determined to compare the
binding affinities of different compounds for a specific target, where conclusions were made
not statistically, but more intuitively as in ‘same order of magnitude’ or ‘nanomolar and
micromolar range’.
Secondly, an important assumption was made; when no number of experiments “n”
was reported in the publication, the reported standard error of the KD implied the statistics on
the fitting.
5.1.1 Random variability KD values
The investigation of the KD values variability generated a dataset that included only 3
experiments. As a result of this small set of data, no conclusions could be taken for the
variability of SAW biosensor experiments. More data are needed in order to take a
statistically conclusion about the variability of KD values as obtained by SAW methods. The
information about the variability of a SAW method would be valuable to set specifications
and to compare different KD values including answers to questions e.g. is the difference
between two or more KD values significant or caused by random errors in the measurements?
To give an idea about the variability of other label free binding assays; global
benchmark studies, investigating the variability of SPR biosensors, showed that the variability
was approximately 20% [93-95].
42
5.1.2 Model variability
The model variability (RSE%) was also investigated. There was no association was
found between the model variability and the size of KD value (strength of the binding
affinity). The model variability was found significantly large for some KD values (especially
for glycosaminoglycan interactions). The systematic large model variability could be the
result of an incorrect used fitting model. It is important to apply a suited binding model for
the interaction under study, as different models are possible [96]. In all assessed publications,
the 1:1 binding model was (in some cases wrongfully) applied using the Fitmaster® software
(SAW Instruments, Bonn, Germany).
5.2 DEVELOPMENT OF A HGHAB-BASED SAW METHOD
The choice of ligand immobilisation plays an important role in the analytical
performance of label-free biosensor assays. The signal magnitude, regenerability and stability
of the method depend on the amount of immobilised ligand, the remaining activity after
immobilisation and the orientation on the sensor chip surface, all characterized by the
immobilisation strategy [97].
As is the case in HPLC method development, also for SAW methods we need to
quantify the endpoint of the development effort. One of the quantitative criteria is the
feasibility test, which calculates the theoretically amount of analyte (pg/mm2) that can bind to
the surface bound ligand based on their molecular weights and binding stoichiometry. This
amount of analyte should exceed the limit of 0.5 pg/mm2 [98] to obtain detectable signals.
Secondly, ligand immobilization does not always lead to active ligand: covalent
immobilization can lead to inactivation or inaccessible analyte binding sites. Therefore, the
remaining activity of the ligand can be quantitatively compared using the relative ligand
response RR%. In other words, RR% is a normalised value to compare the binding capability
of the same ligand between different immobilisation techniques using a certain analyte
concentration. This value, however, does not necessarily provide information on the absolute
amount of active ligand. The absolute amount of active ligand can be calculated using analyte
concentrations that saturate the surface. From a practical and economical point of view, it is
not always feasible to obtain high analyte concentrations. Therefore, it is sufficient to use one
43
analyte concentration to compare the RR% between different immobilisation strategies during
method development. Thirdly, we have defined the specificity of the interaction expressed as
NSB% using a negative control protein (e.g. BSA). The NSB% expresses the proportion of an
analyte signal related to non-specific binding, such as interaction with the dextran layer and
gold. The NSB% should preferably be as low as possible. For the different ligands
(somatropin and derivatives) a 99% confidence interval was calculated for the NSB% which
amounted [0.08%;13.47%]. The NSB% of the proposed method should not be higher than
15%. Moreover, the choice of chip chemistry is decisive for the NSB%. The fourth criteria
quantitatively evaluates the regeneration between different analyte injection cycles using the
regeneration efficiency (RE%) parameter and additional ligand activity testings. The RE%
should fall within 90-110%.
5.2.1 Exploring immobilisation procedures for the immobilisation of antibodies
Different immobilisation procedures have been described for proteins and antibodies,
which can be categorised in two main groups: random immobilisation and site-directed
immobilisation. The most widely used immobilisation technique is the random and covalent
immobilisation using amine coupling. The method is easy in use and leads to a high
immobilisation density. For hGHAb, a ligand density of 194 pg/mm2 is reported, which is
lower than 2.5 ng/mm2 reported for a close-packed side-on monolayer of antibodies [97, 99,
100]. Moreover, direct amine coupling of hGHAb doesn’t ensure that all the antigen-binding
sites are available for analyte binding (Figure 5.1), hence, the random orientation of the
ligands often results in the loss of antigen binding capability [101]. The specificity of the
direct immobilisation of hGHAb was significantly low. At least 84% of the response is the
result of NSB to the CM-dextran hydrogel layer, which also results in the relatively low
activity/binding capacity of the immobilised hGHAb (RR%= 9.61 ± 1.59% using 200 nM
somatropin).
The density of immobilised hGHAb by aldehyde coupling was extremely low (26.2 ±
10.3 pg/mm2). Several factors could be responsible for the low efficiency; (i) low ligand
concentration used during immobilisation (11.7 µg/ml) or, (ii) insufficient oxidation of the
ligand. The latter could be improved by increasing the time of oxidation, the oxidants
concentration or the temperature of the reaction [90]. This method was not further included in
the method development.
44
Figure 5.1: Schematic overview of random immobilisation between hGHAb and the dextran layer. (A) All antigen binding sites are still available for analyte binding, (B) no analyte binding sites are available, (C) limited antigen binding sites are available and (D) non-covalently bound antibodies with (limited) available antigen binding sites.
The site-directed immobilisation procedures that were applied for hGHAb did not
yield in higher surface densities. The density of hGHAb captured via protein G was initially
higher than the random immobilised hGHAb. However, immediately after immobilisation, a
large amount of hGHAb was released from the surface, as indicated by the descending drift in
the baseline. The phase difference between the stabilized baseline and the response directly
after the immobilisation was 5.6 ± 0.3° (i.e. 108.7 ± 11.7 pg/mm2). It is most likely that the
captured hGHAb was dissociating from the protein G as the interaction with hGHAb is a non-
covalent affinity based interaction.
The distance between the CM-dextran hydrogel surface and the sample flow increases
when a linker molecule like protein G is used. This explains the lower adsorption effects to
the sensor surface and the lower NSB (NSB% = 2.80 ± 1.37%). Moreover, the recombinant
protein G that was used is truncated (i.e. the BSA binding sites are deleted); no interaction
with BSA is theoretically possible. According to [85] and [97], this orientated immobilisation
approach results in the highest binding capacity. Yet, the binding capacity of hGHAb
captured via protein G was unexpectedly low (RR% = 12.70 ± 0.63%, using 200 nM
somatropin). Most likely this is caused by (i) the serious dissociation of the hGHAb-protein G
complex after immobilisation, and (ii) the rapid saturation of the ligand surface without use of
regeneration. The drift in the baseline after immobilisation should be controlled. In that case
more immobilised hGHAb is available for interaction. The optimal buffer condition for IgG -
protein G binding is pH 5. Therefore, a running buffer of pH 5 is recommended for binding of
the antibody to protein G. However, the stability of hGHAb at lower pH range is unknown.
45
Protein A on the other hand needs a buffer of pH 8 for optimal binding, which is more close
to the physiological conditions [102].
The amount of bound orientated hGHAb using complexation couldn’t be determined
because of the ligands’ heterogeneity. It was assumed that the amount of hGHAb will exceed
the limit of detection as immobilisation using amine coupling chemistry results in a high
concentration of immobilised ligands. The results regarding ligand binding capability (RR%)
and specificity (NSB%) are similar with amine coupling of hGHAb.
So far, the immobilisation with hGHAb did not respond to our expectations.
Somatropin has several functional groups (-NH2) that can be involved in amine coupling: nine
lysine residues (K38, K41, K70, K115, K140, K145, K158, K168 and K172) (Figure 5.3) and
the amine-terminus. This makes somatropin an ideal candidate for amine coupling.
Figure 5.2: Crystal structure of recombinant human growth hormone (PDB:3HHR). The lysine residues are indicated in red.
The mass density of immobilised somatropin was lower compared to immobilised
hGHAb. However, the molecular density of immobilised somatropin (∼ 2.7 nmol/mm2) was
twice as high as the amount of immobilised hGHAb (∼ 1.3 nmol/mm2). This could be
explained by the fact that somatropin (Mw 22 125 Da) is a smaller protein than hGHAb (Mw
±150 000 Da), and therefore more molecules will be able to occupy the same surface. The
relative response of the somatropin ligand was higher than for the hGHAb methods and the
NSB was very low (NSB% = 1.5% ± 0.4), making it an attractive method for further
development.
46
5.2.2 Screening for optimal regeneration conditions
In normal circumstances, regeneration of the surface is required to remove the bound
analytes to recover the ligand density and to avoid saturation of the surface. Important is the
maintenance of the bioactivity of the ligand. A quantitative measure for the evaluation of the
regeneration condition can be given by calculation of the regeneration efficiency (RE%).
This should approach 100% as good as possible and must be within [90-110%]. If the
efficiency is lower than 90%, the conditions are weak and were not able to remove all bound
analytes from the immobilized surface. Conversely if the efficiency is higher than 110% the
conditions are too strong, resulting in ligand dissociation.
A solution with a mixture of 50 nM NaOH and 0.1% SDS was found to be an
appropriate regeneration condition to remove all the bound hGHAb from the immobilised
somatropin surface. SDS is a detergent, which binds to proteins by hydrophobic interactions
[103]. Alkali solutions like NaOH will break the interaction between analyte and ligand. The
high pH will negatively charge the protein binding sites, which will dissociate the analyte –
ligand interaction because of electrostatic repulsion [104].
5.3 FUNCTIONAL CHARACTERIZATION OF NOTA-MODIFIED SOMATROPINS
The functional quality of NOTA-modified somatropins was quantitatively
characterized using the developed hGHAb-based SAW method.
5.3.1 Immobilization of somatropin and derivatives using amine coupling
Somatropin and the NOTA-modified somatropin were immobilised on the dextran-
gold surface using amine coupling. It is expected that the amount of immobilised NOTA-
modified somatropin will be lower compared to somatropin. NOTA-modification involves
amine groups of the lysine residues, therefore less lysines will be available for the
immobilisation via amine coupling. The immobilisation of 1:1 NOTA somatropin was
similar to the immobilisation of unmodified somatropin. The high immobilisation efficiency
is due to the significant presence of unmodified somatropin (46%) in the 1:1 NOTA
somatropin sample (Table 5.1).
47
Table 5.1: Overview of the substitution degree (%) of NOTA-modified somatropin samples [105].
Unmodified +1 NOTA +2 NOTA +3 NOTA +4 NOTA 1:1 NOTA somatropin 46 42 12 ND ND 3:1 NOTA somatropin 13 45 35 7 ND
10:1 NOTA somatropin ND 2 39 47 12 ND: Not detectable
The immobilisation of 3:1 NOTA somatropin was similar to 10:1 NOTA
somatropin. Both have a lower total phase shift after the immobilisation. This can be due to
(i) the use of different chips with different chemistry batches and (ii) a reduced amount of
lysine residues for coupling (Table 5.1).
As previously mentioned, the quality of a sensor chip is essential to obtain reliable
measurements. The chips are reused; therefore the sensor surface is chemical etched and re-
modified with a CM-dextran hydrogel layer. Heterogeneity of the CM-dextran hydrogel can
occur, not only between different chips but also on the same sensor chip, density of the
dextran hydrogel is different on different channels. This leads to variability of the data
between different channels. The state of the plain gold surface has a major impact on the
modification. One sensor chip is used more than the other and the more a chip is chemically
etched, the more likely the sensitive areas may be damaged by erosion of gold and the
physical properties (e.g. wettability) may have changed [106]. Validation of the chip quality is
important to assure reproducible measurements. Characterization of a golden surface is
achieved by the investigating the electrochemical activity and elemental composition of the
surface [107].
5.3.2 Binding experiments between immobilized somatropin (-derivatives) and hGHAb
After the immobilization, the binding experiments were performed to determine the
dissociation constants of hGHAb to somatropin and NOTA modified somatropins.
The association rate constants for typical antibody-antigen interactions are in the range
of 105 - 106 M-1s-1 [108]. Our data indicate a slightly slower association rate of the hGHAb to
the immobilized somatropin surface (kon = 1.01 × 10-4 nM−1s-1). The association rate slightly
decreases with the amount of modified somatropin (3.96 × 10-5 nM−1s-1, 8.84 × 10-5 M−1s-1
and 7.70 × 10-5 nM−1s-1 respectively for 1:1, 3:1 and 10:1 NOTA somatropin). The
dissociation rate constants of the modified somatropins were found in the same range as for
48
somatropin (from 7.50 × 10-4 s-1 to 1.17 × 10-3 s-1). The low dissociation rate constants
suggest a relatively stable complex of the immobilized somatropins and hGHAb. Both rate
constants fall in the range of most protein-protein interactions [109]. Overall, these data
demonstrate a very high affinity and specific interaction between somatropin (derivatives) and
hGHAb.
For the kinetic evaluation of the data an appropriate binding model should be chosen.
The choice should be made rationally, e.g. a 1:1 binding model would not be a logical choice
for cooperative interactions; and the model should be evaluated statistically using “goodness
of fit” parameters and standard errors. In most antigen-antibody binding experiments the 1:1
(Langmuir) model is applied [95, 110]. The antigen binding sites of the antibodies are
assumed to be independent of each other; the binding on one Fab fragment will not interact
with the binding of the other Fab fragment.
For the kinetic evaluation, a 1:1 binding model was applied for the hGHAb-
somatropin interaction and the hGHAb-NOTA modified somatropin interaction. The RSE of
the KD-values were below 10% for somatropin and 1:1 NOTA somatropin. However for 3:1
NOTA somatropin and 10:1 NOTA somatropin, the RSE was not always below 10%.
Because the large SE gave an uncertain value of the KD, the average KD could not be
determined with high precision for 10:1 NOTA somatropin.
We determined the KD of the somatropin-hGHAb interaction to be ~ 16 nM, which is
in the range of the high affinity antibodies (low nanomolar – high picomolar range). The KD
values of the somatropin derivatives are in the same order of magnitude (Figure 5.3) and are
not statistically significant different (ANOVA; p>0.05). Thus, NOTA-modification does not
alter the binding affinity toward hGHAb. No significant differences are found in the
binding properties between the products of different synthesis procedures.
49
Figure 5.3: 95% CI of the KD of the somatropin and the different NOTA-modifications of somatropin
0!
5!
10!
15!
20!
25!
Unmodified! 1-1 NOTAsomatropin! 3-1 NOTAsomatropin! 10-1 NOTAsomatropin!
50
6 CONCLUSION
SAW biosensors are currently been applied in divergent areas. SAW biosensors are
particularly used for qualitative purpose while the quantitative aspects are underutilized. A
thorough literature search was performed concerning SAW biosensor technology. Today,
there are insufficient publications that report statistical data to draw a clear conclusion about
the general repeatability of a SAW method.
In this study five different immobilisation techniques were compared and evaluated to
determine the most appropriate SAW method. We have defined several quantitative criteria
for method development including the evaluation of different immobilisation strategies (the
ligand immobilisation density, binding capability (RR%) and specificity (NSB%)), as well as
the evaluation of the regeneration conditions. The covalent amine immobilisation of
somatropin was considered as the best immobilisation method (59 pg/mm2, 66.7% RR% and
1.5% NSB%). A suited regeneration condition was determined using the regeneration
efficiency (RE%), as well as an additional ligand activity testing. The condition combining
SDS and NaOH were able to return the signal back to baseline after a hGHAb injection
without affecting the activity of the immobilised ligands.
The developed SAW method was used to characterize the functional quality of
NOTA-modified somatropins. The NOTA-modification does not alter the binding affinity of
somatropin toward hGHAb. The binding properties of the different synthesized NOTA-
somatropin (1:1, 3:1 and 10:1) were not significantly different from each other. The hGHAb
was successfully used as a system to investigate the functional quality of NOTA-modified
somatropins.
The SAW biosensor is a very promising instrument to act as a functional quality
characterization tool in the development and quality control. However, a validation of real time
binding assays is absolutely necessary to introduce the method as part of the product release
portfolio and regulatory approval of new biotechnological drugs.
51
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62
8 LIST OF ATTACHMENTS
ATTACHMENT 1: Detailed list of SAW applications in literature and the reported
dissociation constant (6 pages)
Keyw
ords Sum
mary
Biosensor
(frequency in MH
z) K
D (nM)
SD on K
D (nM
) Purpose of K
D determ
ination R
ef.
Medicine/
Diagnostics
Interaction analysis between integrin V
LA-4
and non-anticoagulant heparin derivatives
LG: V
LA-4 [m
embrane vesicles], P-and L-
selectin [Fc-chimera]
AN
: Natalizum
ab [mA
b], heparin and (size fractionated) derivatives [glycosam
inoglycan]
S-Sens K5
(n.r.)
35.1 (V
LA-4/natalizum
ab) 2.22 x 10
3
(VLA
-4/enoxaparin) 4.61 x 10
3 (V
LA-4/tinzaparin)
2.71 x 103
(VLA
-4/RO
-H)
5.89 x 103
(VLA
-4/6-O D
S H)
11.6 x 103
(VLA
-4/2-O D
S H)
5.76 x 103
(P-selectin/tinzaparin) 6.16 x 10
3 (P-selectin/6-O
DS H
) 4.65 x 10
3 (P-selectin/ 6 sach. unit)
2.17 x 103
(P-selectin/8 sach. unit) 3.88
(P-selectin/12 sach. unit) 3.61 x 10
3 (L-selectin/tinzaparin)
3.11 x 103
(L-selectin/6-O D
S H)
4.29 x 103
(L-selectin/6 sach. unit) 7.16 x 10
3 (L-selectin/8 sach. unit)
0.556 x 103
(L-selectin/12 sach. unit)
8.4 (n=1)
0.76 x 103
(n=1) 2.68 x 10
3
(n=1) 2.63 x 10
3 (n=1)
4.12 x103
(n=1) 3.6 x 10
3
(n=1) 0.03 x 10
3 (n=1)
1.35 x 103
(n=1) 2.88 x 10
3 (n=1)
0.26 x 103
(n=1) 2.75 x 10
3 (n=1)
0.70 x 103
(n=1) 1.36 x 10
3 (n=1)
2.10 x 103
(n=1) 5.54 x 10
3 (n=1)
0.277 x 103
(n=1)
• K
D was determ
ined to com
pare binding affinity of different analytes
• Ligand screening
• Q
ualitative
[111]
(n.r.): not reported
Keyw
ords Sum
mary
Biosensor
(frequency in MH
z) K
D (nM)
SD on K
D (nM
) Purpose of K
D determ
ination R
ef.
Medicine/
Diagnostics
Technique to stabilize biological model
mem
branes for SAW
application LG
: Biotinylated m
odel mem
brane (POPC
) [lipids], V
LA-4 expressed into m
odel m
embrane (D
PPC-D
GA
/NTA
) [protein-lipids] A
N: V
CA
M-1 [protein], avidin [protein]
SAM
5 Blue
(n.r.)
9.02 x 10-2
(biotin/avidin) 2.48
(VLA
-4/VC
AM
-1)
n.r. (n=1) 0.41 (n=1)
• K
D used to com
pare with
online ligand im
mobilization
• D
etection of biological interaction
• Q
ualitative
[112]
Investigation to the mode of binding
LG: R
NA
aptamer [R
NA
/DN
A]
AN
: thrombin [protein]
S-Sens K5
(n.r.) 181
20 (n=1)
• K
D used to compare
with SPR
and filter-binding studies
• Q
ualitative
[113]
Binding betw
een Affim
ed TandAb antibody and
target tumor cells
LG: A
b [protein] A
N: target tum
or cells [cells]
SamX
(n.r.)
2 n.r.
• K
D compared w
ith flow
cytometric
analyis •
Ligand screening •
Qualitative
[114]
Detection and determ
ination of cardiac troponin I in hum
an plasma
(sandwich im
munoassay)
LG: capture A
b cTnI [protein] A
N: A
b /cardiac troponin I [protein]
In-house assembled
(200) n.r.
n.r. -
[115]
Evaluation of new chip preparations w
ith sem
icarbazide for Ag/A
b interactions LG
: HA
, FLAG
Ag [protein]
AN
: anti-HA
/FLAG
Ab [protein]
In-house assembled
(116) n.r.
n.r. -
[116]
Detection of hepatitis B
surface antibodies in w
hole blood samples
LG: hepatitis B
Ag [protein]
AN
: hepatitis B A
b [crude samples]
In-house assembled
(200) n.r.
n.r. -
[117]
SAW
-MS, sensor chips w
ith bound complex
were digested and further used for M
ALD
I/MS
peptide mass fingerprinting
LG: throm
bin (via RN
A-aptam
ers) [protein] A
N: antithrom
bin III [protein]
S-Sense K5
(n.r.) n.r.
n.r. -
[118]
(n.r.): not reported
Keyw
ords Sum
mary
Biosensor
(frequency in MH
z) K
D (nM)
SD on K
D (nM
) Purpose of K
D determ
ination R
ef.
Medicine/
Diagnostics
Developm
ent of SAW
method to detect
different pathogens LG
: specific Ab [protein]
AN
: IgG, E. coli, M
13 bacteriophage [protein, bacteria, virus]
In-house assembled
(118) n.r.
n.r. -
[119]
Specificity and affinity analysis between
humanin peptide (24aa) and β-am
yloid (Aβ)
peptides LG
: Aβ [peptide]
AN
: biotinylated humanin [peptide]
S-Sens K5
(n.r.)
0.61 x 103
(Aβ /pep4)
0.53 x 103
(Aβ /pep6)
0.26 x 103
(n=1) n.r.
(n=1)
• K
D determined to
compare affinity of
different analytes •
Elucidation (target) m
olecular pathway
• Q
uantitative, but no further (statistical) analysis
[120]
Binding betw
een tyrosine-nitrated peptides of prostacyclin synthase and anti-3-nitrotyrosine
antibody LG
: peptide 5 [peptide]; AN
: 3-NT-A
b [protein]
S-Sens K5
(n.r.) 63
20 (n=1)
• K
D used for characterisation of affinity
• Q
uantitative, but no further (statistical) analysis
[121]
Online SA
W-ESI-M
S for simultaneous
detection, ID and quantification of protein-
ligand interactions LG
: Aβ (1-16) [m
AB
], Antiα-Syn (C
-20)-R
[polyclonal Ab], Substance P [peptides]
AN
: nitrotyrosine peptide-1 [peptides], hα-Synuclein [protein], C
almodulin [protein]
S-Sens K5
(n.r.)
106.9 (m
Ab/ Peptide 1)
43.6 (Substance P/ C
almodulin)
164.7 (pA
b/ hα-Synuclein)
14.1 (n=1) 15.8 (n=1) 17.5 (n=1)
• K
D determination for
binding affinity study •
Quantitative, but no
further (statistical) analysis
[122]
Interaction analysis between integrin V
LA-4
and heparin LG
: VLA
-4 [mem
brane vesicles] A
N: heparin [glycosam
inoglycan]
S-Sens K5
(n.r.)
10.1 (V
LA-4/V
CA
M-1)
4.61 x 103
(VLA
-4/Tinzaparin)
9 2.68 x 10
3
(n≥3)
• K
D used to confirm
affinity and to com
pare with other
KD
• Target screening
• Q
ualitative
[123]
(n.r.): not reported
K
eywords
Summ
ary B
iosensor (frequency in M
Hz)
KD (nM
) SD
on KD
(nM)
Purpose of KD
determination
Ref.
Medicine/
Diagnostics
Aptam
ers and HIV
proteins + protein identification via M
ALD
I-ToF MS.
Protein was digested on chip, peptides w
ere analysed via peptide m
apping. LG
: biotyinylated aptamers [peptides]
AN
: V3 loop of H
IV-1 envelope gp150 and
HIV
-1 reverse transcriptase [proteins]
S-Sens K5
(n.r.)
406 (S66A
-C6/H
IV-1 V
3) 791
(S68B-C
5/HIV
-1 V3)
555 (S69 A
-C15/ H
IV R
T-984-2)
n.r.
• K
D were com
pared w
ith KD from
titration w
ith nitrocellulose filter
• K
D determined for
kinetics studies. •
Target characterization
• Q
uantitative, but no further (statistical) analysis
[124]
Food analysis
Measurem
ent of protein concentration in com
plex matrices (e.g. m
eat juice) LG
: Ab [protein]; A
N: acute phase protein
(APP) [protein]
Sam5
(n.r.) 0.96
n.r.
• K
inectic evaluation •
Quantitative, no
further (statistical) analysis
[22]
Detection of okadaic acid w
ith Love wave
biosensor LG
: Ab [protein] AN
: okadaic acid [fatty acid]
In-house assembled
(117) n.r.
n.r. -
[25]
Biom
aterials B
inding of peptides to inorganic materials
LG: m
ineral TiO2 ; A
N: aptam
er peptides S-Sens K
5 (n.r.)
81 n.r.
• K
D used to study kinetics
• Q
uantitative, but no further (statistical) analysis
[125]
Polymer
science
Study of the properties of a responsive brush later
LG: 4,5-dim
ethoxy-2-nitrobenzyl methacrylate
[polymer] A
N: U
V and pH
change
In-house assembled
(150) n.r.
n.r. -
[126]
Cell biology
Interaction analysis of cell mem
brane receptors to im
mobilized ligands
LG: protein G
/anti-HLA
[protein] A
N: LG
2 cell suspension [cells]
In-house assembled
(110) n.r.
n.r. -
[127]
(n.r.): not reported
K
eywords
Summ
ary B
iosensor (frequency in M
Hz)
KD (nM
) SD
on KD (nM
) Purpose of K
D determ
ination R
ef.
Rheology
The influence of molecular w
eight on a LW
acoustic sensor; LW-SA
W as a viscom
eter LG
: Polydimethylsiloxane (PD
MS)-chip
AN
: PEG [polym
er]
In-house assembled
(117) n.r.
n.r. -
[128]
Microbiology
Effector protein NIeF of E. coli N
IeF binds to caspase-4, -8 and -9. LG
: Capase-9 [protein]
AN
: NIeF [protein]
Sam5 B
lue (n.r.)
38.7
13.18
• K
D used to proved affinity
• Elucidation (target) m
olecular pathw
ay •
Qualitative
[24]
Developm
ent of a SAW
method for the
detection of bacteriophages LG
: mAB anti-M
13 [protein] AN
: M-13
bacteriophage [virus]
In-house assembled
(163) n.r.
n.r. -
[129]
Binding of antim
icrobial peptides to model
mem
branes of E. coli and P. mirabilis
LG: lipid bilayer [lipids]; A
N: [peptides]
S-Sens K5
(n.r.) n.r.
n.r. -
[130]
Detection of E.coli w
ith optimized surface
LG: E. coli Ab [protein] A
N: E. coli
[bacteria]
In-house assembled
(118) n.r.
n.r. -
[131]
Analysis of m
embrane activities of
antimicrobial peptides
LG: lipid bilayer (D
OC
P) [lipids] AN
: galliderm
in and vancoycin [peptides]
S-Sens K5
(n.r.) n.r.
n.r. -
[132]
Binding of peptides to bilayers
LG: lipid bilayer [lipids] A
N: LL32
[peptides]
S-Sens K5
(n.r.) n.r.
n.r. -
[133]
Environm
ental science
Detection of heavy m
etals in liquid medium
w
ith love wave biosensor
LG: E. coli [bacteria]
AN
: Cadm
ium, M
ercury [metals]
In house assembled
(118) n.r.
n.r. -
[134]
(n.r.): not reported
K
eywords
Summ
ary B
iosensor (frequency in M
Hz)
KD (nM
) SD
on KD (nM
) Purpose of K
D determ
ination R
ef.
Genom
ics
DN
A hybridization analysis of point m
utation cancer related gene fragm
ents LG
: biotynilated DN
A fragm
ents [DN
A]
AN
: DN
A [D
NA
]
S-Sens K5
(148)
144 (w
t BR
CA
1/ wt
BR
CA
1) 123
(mut B
RC
A1/ m
ut B
RC
A1)
30 (w
t ptch/ wt ptch)
108 (m
ut ptch/ mut ptch)
52 (w
t p53/ wt p53)
103 (w
t p53/ mut p53)
139 (m
ut p53/ mut p53)
3.856 x 103
(mut p53/ w
t p53)
n.r.
• K
D is used for detection of m
utations (hit/no-hit)
• Q
ualitative
[23]
Evaluation of geometrical characteristics
of DN
A m
olecules resulting from the
formation of triple-helical D
NA
s LG
: biotinylated DN
A triplexes A
N: D
NA
In-house assembled
(155) n.r.
n.r. -
[135]
(n.r.): not reported
