Post on 26-Mar-2018
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutical Analysis
Drug Quality and Registration
Academic Year 2012-2013
CHARACTERIZING BINDING AFFINITY OF SOMATROPIN AND DERIVED STRUCTURES
TO HGHAB BY SURFACE ACOUSTIC WAVES AND SIZE EXCLUSION
CHROMATOGRAPHY
Apr. Evelyn Buyst
Master of Industrial Pharmacy
Promotor
Prof. dr. Apr. Bart De Spiegeleer
Mentor
Nathalie Bracke
Board of commissioners
Prof. Erwin Adams
Prof. Roger Kemel
Prof. Ann Van Schepdael
Prof. Sandra Apers
Prof. Ann Van Eeckhaut
Prof. Yvette Michotte
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutical Analysis
Drug Quality and Registration
Academic Year 2012-2013
CHARACTERIZING BINDING AFFINITY OF SOMATROPIN AND DERIVED STRUCTURES
TO HGHAB BY SURFACE ACOUSTIC WAVES AND SIZE EXCLUSION
CHROMATOGRAPHY
Apr. Evelyn Buyst
Master of Industrial Pharmacy
Promotor
Prof. dr. Apr. Bart De Spiegeleer
Mentor
Nathalie Bracke
Board of commissioners
Prof. Erwin Adams
Prof. Roger Kemel
Prof. Ann Van Schepdael
Prof. Sandra Apers
Prof. Ann Van Eeckhaut
Prof. Yvette Michotte
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."
April 22, 2013
Appreciations
First of all,
I would like to enounce my appreciations towards Prof. dr. Apr. Bart De Spiegeleer.
He gave me the opportunity to work in the DruQuaR laboratory. He arranged an internship at
the very last moment, offered me a very interesting and defiant topic to work on, and he was
always available for questions and his professional and expansive view on many things.
Secondly, I want to thank tremendously my mentor, Nathalie Bracke,
who was always and at every time available for questions and good advice.
It was really a pleasure to work with her.
Another word of thanks is for all other colleagues in the laboratory for their daily help and
the positive atmosphere they created to work in.
Thanks to all other students for the good company
and pleasant leisure time during this last year.
Last but not least,
I thank my parents and my family for all the opportunities
they gave me, their never ending support and encouraging words.
TABLE OF CONTENTS
1. INTRODUCTION ....................................................................................................... 1
1.1. BIOLOGICAL FUNCTION OF HUMAN GROWTH HORMONE AND ITS ROL
IN CANCER .............................................................................................................. 1
1.1.1. Neuro-endocrine tumors ....................................................................... 1
1.1.2. Growth hormone ................................................................................... 2
1.2. PROTEIN AND PEPTIDE THERAPEUTICS .................................................................. 3
1.2.1. Protein scaffolds .................................................................................... 3
1.2.2. Peptide therapeutics .............................................................................. 4
1.3. BIOSENSORS IN DRUG DEVELOPMENT .................................................................. 5
1.3.1. What are biosensors? ............................................................................. 5
1.3.2. Surface acoustic waves biosensors ......................................................... 6
1.3.3. Sensorgram of surface acoustic wave biosensors .................................... 7
1.3.4. Other tools for the characterization of binding events ........................... 8
1.3.4.1. Surface plasmon resonance............................................................. 8
1.3.4.2. Quarz crystal microbalance ............................................................ 8
1.3.4.3. Isothermal titration calorimetry ..................................................... 9
1.3.5. Comparison of biosensors and other techniques .................................... 9
2. OBJECTIVES ............................................................................................................ 11
3. MATERIALS AND METHODS .................................................................................... 12
3.1. SENSOR CHIP .......................................................................................................... 12
3.1.1. Cleaning sensor chips ............................................................................. 12
3.1.1.1. Pre-cleaning .................................................................................... 12
3.1.1.2. Chemical etching with AMP ............................................................ 12
3.1.2. Coating sensor chip with dextran ........................................................... 12
3.2. LIGAND IMMOBILIZATION ...................................................................................... 13
3.3. SAM5 BINDING EXPERIMENTS ............................................................................... 14
3.3.1. Screening for a regeneration condition .................................................. 14
3.3.2. Analytes (modified) somatropin ............................................................ 14
3.3.2.1. Binding experiment between hGHAb and (modified) somatropin with
regeneration ................................................................................... 14
3.3.3. Somatropin derived peptides ................................................................. 15
3.3.3.1. QC analysis of somatropin derived peptides ................................... 15
3.3.3.2. Binding experiments with somatropin derived peptides ............... 15
3.3.4. Data processing of SAW binding experiments for (modified) somatropin and
peptides ................................................................................................ 16
3.4. SIZE EXCLUSION CHROMATOGRAPHY .................................................................... 18
4. RESULTS AND DISCISSION ....................................................................................... 19
4.1. LIGAND IMMOBILIZATION ...................................................................................... 19
4.2. SOMATROPIN ......................................................................................................... 21
4.2.1. Screening for regeneration conditions ................................................... 21
4.2.2. SAM5 binding experiments .................................................................... 22
4.2.2.1. Binding experiment between hGHAb and somatropin .................. 22
4.2.3. SEC experiments .................................................................................... 24
4.2.3.1. Calibration curve of the SEC column .............................................. 24
4.2.3.2. SEC analysis of hGHAb .................................................................... 25
4.2.3.3. SEC analysis of somatropin ............................................................. 25
4.2.3.4. SEC analysis of mixtures of hGHAb and somatropin ...................... 27
4.3. MODIFIED SOMATROPIN ........................................................................................ 30
4.3.1. 1:1 NOTA-somatropin ............................................................................ 30
4.3.1.1. SAM5 binding experiment with regeneration ................................ 30
4.3.1.2. SEC experiments ............................................................................. 31
4.3.1.2.1. SEC analysis of 1:1 NOTA-somatropin ............................ 31
4.3.1.2.2. SEC analysis of mixtures of hGHAb and
1:1 NOTA-somatropin ..................................................... 32
4.3.2. 1:3 NOTA-somatropin ............................................................................ 33
4.3.2.1. SAM5 binding experiment with regeneration ................................ 33
4.3.2.2. SEC experiments ............................................................................. 34
4.3.2.2.1. SEC analysis of 1:3 NOTA-somatropin ............................ 34
4.3.2.2.2. SEC analysis of mixtures of hGHAb and
1:3 NOTA-somatropin ..................................................... 35
4.3.3. 1:10 NOTA-somatropin .......................................................................... 36
4.3.3.1. SAM5 binding experiments ............................................................. 36
4.3.3.1.1. Binding experiment with regeneration ........................... 36
4.3.3.1.2. Duplication of binding experiment with regeneration ... 37
4.3.3.2. SEC experiments .............................................................................. 38
4.3.3.2.1. SEC analysis of 1:10 NOTA-somatropin .......................... 38
4.3.3.2.2. SEC analysis of mixtures of hGHAb and
1:10 NOTA-somatropin ................................................... 39
4.3.4. Comparison of the modified somatropin structures ............................... 41
4.4. SOMATROPIN DERIVED PEPTIDES .......................................................................... 45
4.4.1. Choise of peptides ................................................................................. 45
4.4.2. QC analysis somatropin derived peptides .............................................. 45
4.4.3. SAM5 binding experiments .................................................................... 46
4.4.3.1. Feasability calculations ................................................................... 46
4.4.3.2. Binding experiments ....................................................................... 47
4.4.4. SEC experiments .................................................................................... 50
4.4.4.1. SEC analysis of P0320 ...................................................................... 50
4.4.4.2. SEC analysis of mixtures of hGHAb and P0320 ............................... 52
5. CONCLUSION AND PERSPECTIVES ............................................................................ 53
6. REFERENCES ........................................................................................................... 54
SUMMARY
The global aim of the project was to develop a Surface Acoustic Wave (SAW) biosensor
technology into a functional quality characterization tool. In this work, we have used
somatropin or recombinant human growth hormone (GH) as analyte. The more specific
project goals are (i) the development of a method for selective immobilization of human GH
antibody (hGHAb), (ii) the development of robust operational conditions and (iii) the
quantitative binding characterization (affinity, on- and off-rates) of 1,4,7-triazacyclononane-
1,4,7-triacetic acid (NOTA) modified somatropin (equimolar (1:1) or 3 to 10 times molar
excess of NOTA (1:3 and 1:10) added to somatropin) and multiple somatropin derived
peptides. This quantitative binding was also investigated by SEC.
In the SAW binding experiments, the hGHAb ligand was immobilized via the amine coupling
chemistry. When the ligand has multiple copies of the functional group (-NH2) that mediates
immobilization, proteins are coupled heterogeneously and sometimes at multiple sites. This
random immobilization therefore influences the accessibility and/or activity of the ligand,
and hence, the binding signal upon analyte binding. One of the most important finding was
the overall variability of the binding results (within-measurement variability as well as
between-measurement variability), which indicate a lack of reproducibility. Therefore
attention must be paid to the quality of the operations, of materials and of the chip. The
binding affinity of the multiple modified somatropin forms are at least similar than
unmodified somatropin. The experiments with somatropin derived peptides have to be
interpreted even more carefully, because of the presence of impurities in almost all peptide
samples, which can have a major influence on the affinity. Our pilot data indicate a possible
interaction between two of the peptides and the hGHAb. The observed binding in the SAW
experiments was confirmed with size exclusion chromatography (SEC). Binding was observed
with somatropin and 1:1 NOTA-somatropine, but not for the 1:3 and 1:10 NOTA-derivates.
As a final conclusion, we can state that the SAW biosensor is a very promising instrument to
be involved in different stages of the drug discovery process, but further improvements,
especially in robustness, are absolutely necessary.
SAMENVATTING
Het algemene doel van dit project was een Surface Acoustic Wave (SAW) biosensor
technologie te ontwikkelen als een functioneel instrument voor kwalitatieve karakterisatie.
In dit werk werd gebruikt gemaakt van somatropine of recombinant groeihormoon (GH) als
analiet. De meer specifieke doelen zijn (i) de ontwikkeling van een methode voor selectieve
immobilisatie van humaan GH antilichaam (hGHAb), (ii) de ontwikkeling van robuuste
operationele condities en (iii) de kwantitatieve bindingskarakterisatie (affiniteit, on- en off-
rates) van 1,4,7-triazacyclonaan-1,4,7 triazijnzuur (NOTA) gemodificeerd somatropine
(equimolair (1:1) of 3 tot 10 keer molaire overmaat van NOTA (1:3 en 1:10) toegevoegd aan
somatropine) en meerdere somatropine afgeleide peptiden. Deze kwantitatieve binding
werd ook onderzocht door gebruik te maken van size exclusion chromatografie (SEC).
In de SAW bindingsexperimenten werd de hGHAb ligand geïmmobiliseerd via
aminekoppeling. Wanneer de ligand meerdere functionele groepen (-NH2) heeft die
beschikbaar zijn voor immobilisatie, werden proteïnen heterogeen gekoppeld en soms via
meerdere groepen gekoppeld. Deze random immobilisatie beïnvloedt de beschikbaarheid
en/of de activiteit van de ligand, en bijgevolg ook het bindingssignaal. Een van de
belangrijkste bevindingen is de algemene variabiliteit van de bindingsresultaten (zowel
variabiliteit tussen metingen als binnen metingen), wat wijst op een gebrek aan
reproduceerbaarheid. Daarom moet er aandacht worden besteed aan de kwaliteit van het
experiment, de gebruikte materialen en de chip. De bindingsaffiniteiten van de meerdere
vormen gemodificeerd somatropine zijn gelijkend aan het niet-gemodificeerde somatropine.
De experimenten met de somatropine afgeleide peptiden moeten echter worden
geïnterpreteerd met enige voorzichtigheid, aangezien de aanwezigheid van onzuiverheden
in bijna alle peptide stalen een invloed kunnen hebben op de affiniteit. De data van piloot
experimenten geven een mogelijke interactie van twee van de peptiden met hGHAb aan. De
waargenomen binding in de SAW experimenten werd bevestigd met SEC. Binding werd
waargenomen met somatropine en 1:1 NOTA-somatropine, maar niet met de 1:3 en 1:10
NOTA afgeleide structuren.
Finaal kunnen we besluiten dat de SAW biosensor een veelbelovende techniek is die kan
gebruikt worden in meerdere stadia bij het ontwikkelen van geneesmiddelen, maar verdere
vooruitgang, inzake robuustheid van de techniek, is absoluut nodig.
LIST OF ABBREVIATIONS
5-HIAA 5-hydroxyindoleacetic acid
ACN Acetonitrile
AMP Ammonia-peroxide-mixture
CE Capillary electrophoresis
Cgs Chromogranins
CT Computed tomography
DAD Diode array detector
DMSO Dimethylsulfoxide
DruQuaR Drug Quality and Registration
DSA Digital substraction angiography
DTE Dithioerythritol
EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
ELISA Enzyme-linked immunosorbent assay
EMA European Medicines Agency
FDA Food and Drug Administration
GH Growth hormone
GHBp Growth hormone binding protein
GHIH Growth hormone-inhibiting hormone
GHR Growth hormone receptor
GHRH Growth hormone-releasing hormone
GLP Good laboratory practice
GLP-1 Glucagon-like-peptide-1
GMP Good manufacturing practice
HBS HEPES buffered saline
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hGHAb Human growth hormone antibody
hGHR Human growth hormone receptor
HILIC Hydrophilic interaction liquid chromatography
HPLC High performance liquid chromatography
IDT Interdigital transducer
ITC Isothermal titration calorimetry
IUPAC International Union of Pure and Applied Chemistry
JAK2 Janus kinase 2
KD Binding constant
koff Dissociation constant
kon Association constant
MRI Magnetic resonance imaging
NET Neuro-endocrine tumor
NHS N-Hydroxysuccinimide
NOTA 1, 4, 7-triazacyclononane-1,4,7-triacetic acid
PDA Photodiode array
PDEA 2-(2-pyridinyldithio)ethaneamine
PEG Polyethylene glycol
PET Positron emission tomography
QC Quality control
QCM Quarz cristal microbalance
RP Reversed phase
RU Resonance Unit
SAM Self assembled monolayer
SAW Surface acoustic waves
SDS Sodium dodecyl sulfate
SEC Size exclusion chromatography
SPECT Sigle-photon emission computed tomography
SPR Surface plasmon resonance
SSRS Somatostatin receptor scinctigraphy
TIR Total internal reflection
TSM Thickness shear mode
UPLC Ultra performance liquid chromatography
USA United States of America
1
1. INTRODUCTION
1.1. BIOLOGICAL FUNCTION OF HUMAN GROWTH HORMONE AND ITS ROLE IN CANCER
Cancer is a major health problem worldwide. It is a genetic disease, characterized by an
uncontrolled growth of cells, which sometimes is induced by bacteria and viruses or can be
heritable. However, it is caused most of the times by exposure to exogenous factors such as
carcinogens, radiation or hormones that initiate mutations of the DNA. Particularly,
mutations of oncogenes (including growth factors and their receptors, signal transducer
molecules and transcription factors), tumor suppressor genes and genes responsible for DNA
repair can lead to disturbed cell proliferation [1-2].
In 2008 approximately 12.7 million cancer cases and 7.6 million people died of cancer
worldwide [4]. Statistics in Belgium reported 59 996 new diagnoses of cancer in 2008,
whereof 54% were diagnosed in men (622 per 100 000 individuals) and 46% in females (505
per 100 000 individuals). Leading cancer sites are breast cancer (16%), prostate cancer
(15%), colorectal cancer (14%) and lung cancer (12%). They cover more than 56% of all newly
diagnosed tumors in Belgium [4].
1.1.1. Neuro-endocrine tumors
Neuro-endocrine tumors (NETs) are a rare but life-threatening type of tumors with an
incidence of 0.0053%. They arise from cells in the neuroendocrine system, composed of both
nervous and endocrine cells. These tumors are able to develop at any epithelial site in the
human body, but the main primary sites are the gastrointestinal tractus, with most occurring
places: ileum, appendix, rectum and the bronchopulmonary system [5-6]. The NETs can be
divided into two groups, the poorly differentiated and well differentiated tumors. The poorly
differentiated tumors are in general relative indolent and are characterized by small
resemblance to neuroendocrine cells, a more sheetlike or diffuse architecture, irregular
nuclei and less cytoplasmic granularity. The well-differentiated tumors have an aggressive
nature and show more or less uniform cells. They are responsible for the production of
abundant neurosecretory granules containing overexpressed neuroendocrine markers.
General tumor markers for both types are for example chromogranins (Cgs) and pancreatic
polypeptides. Specific markers produced by well-defined tumors are for example
2
neuropeptide K, supstance P and 5-hydroxyindoleacetic acid (5-HIAA), which is a metabolite
of serotonin. Tumormarkers can also be specific to the primary site as for example endocrine
pancreatic tumors produce for example gastrin, insulin, vasoactive intestinal polypeptide
and glucagon. Also, an expression of somatostatin receptors was observed in multiple NETs
[7-9].
Because clinical symptoms are lacking and the disease is characterized with slow growth and
progression, diagnosis comes most often too late. Diagnosis is based on detection of
biochemical markers, especially Cgs, 5-HIAA and the more specific biomarkers in blood and
urine. Visualisation of the tumors is performed by using computed tomography (CT),
magnetic resonance imaging (MRI), somatostatin receptor scintigraphy (SSRS) and digital
substraction angiography (DSA) [9-11]. The choice of treatment depends on the symptoms and
the stage of the disease. Surgical therapy is only an option for a small group, when
diagnosed in an early stage. Symptomatic treatment, by using somatostatin-analogues
(growth hormone inhibiting analogues) can provide a relief of clinical symptoms. Also
targeted radionuclide therapy becomes more and more important. Still, the available
treatments are limited and the need for new therapies is high [9-14].
1.1.2. Growth hormone
Growth hormone (GH) or somatropin is a heterogeneous peptide hormone consisting of
multiple isoforms. The gene cluster is located on chromosome 17q and includes 2 genes GH1
and GH2. The main product of the GH1 gene is GH expressed by the pituitary. It has a
sequence of 191 amino acids an is a 22 129 Da single chain protein containing two disulfide
bridges. Isoforms arise by differences in mRNA splicing, post-translational modifications and
metabolism [15].
GH is secreted by the pituitary and is controlled by the hypothalamus which produces the
stimulating growth hormone-releasing hormone (GHRH) and the growth hormone-inhibiting
hormone (GHIH) or somatostatin. The release of both hormones is controlled by a feedback
system [17]. One GH molecule binds two extracellular growth hormone receptor molecules
sequential (Figure 1) what results in the activation of Janus kinase 2 (JAK2), a GH receptor
associated tyrosine kinase, and influences the transcription of genes for a variety of proteins,
3
especially concerning cell metabolism, differentiation and proliferation. GH has major
importance in postnatal growth and plays a key role in metabolism, reproductive,
gastrointestinal, cardiovascular and renal systems [18].
Figure 1: Growth hormone receptor complex (PDB: 3HHR [16]
).
Binding site I colors blue and binding site II green.
Up till now, GH has been used several decades as a first-line therapeutic, mainly within
treatment of children with short stature, short stature-related diseases or growth hormone
deficiency. Recently, it is also administered to adults with growth hormone deficiency and
short bowel syndrome. Short-term use of this therapeutic is suggested to be safe; however,
immune responses can occur and a periodic overuse of GH can cause acromegaly. Long-term
effects and/or administering higher concentrations of GH, are correlated with significantly
increased (colorectal) cancer risks and Hodgkin’s disease [19-20]. Within multiple cancer cell
lines, e.g. prostate cancer and breast cancer, an overexpression of GH and its receptor was
observed [21-24]. GH can act as a lymphangiogenic factor, which is associated with lymphatic
vessel activation in pathological conditions, as well as in growth and metastasis [25].
Up till now, experimental evidence for the GH/GHR role in NETs is still lacking. However,
treatment of NETs with somatostatin (GHIH) analogues proved to be successful and inhibited
the function of NETs, what might indicate a potential role of the GH system in NETs [9-14,26].
1.2. PROTEIN AND PEPTIDE THERAPEUTICS
1.2.1. Protein scaffolds
R&D initiatives in targeting protein-protein interfaces are a relative recent venture in the
pharmaceutical sector. Since the commercialization of insulin in 1923 (FDA approved in
4
1939) as a protein therapeutic for the treatment of diabetis, protein therapeutics are
introduced in many categories of medically important applications [27].
Many advantages can be seen compared to small-molecule drugs, for example because of
the high affinity and highly specific interaction capacity, i.e. less adverse reactions are
observed. Because the human body produces many of the proteins that are therapeutically
used, good toleration and fewer occurrence of immune responses are observed. Also, the
clinical development and the regulatory approval by official agencies such as FDA is generally
faster than within conventional drugs. For treatment and diagnose of cancer, mostly
antibody derived structures are available e.g. rituximab and cetuximab. In 2008, more than
300 protein therapeutics were on the market and used for many applications and about 60
peptide drugs are currently available. In the near future, many data from clinical studies
using protein scaffolds are expected [27-30].
1.2.2. Peptide therapeutics
Peptides can be defined as polypeptide chains containing less than 50 amino acids and
having a molecular weight lower than 5 000 Da what can result in a highly developed
secondary structure, without tertiary structure. They have multiple advantages over proteins
including a higher affinity/specificity for targeting, and because of their limited seize, lower
toxicity profiles and a better tissue penetration can be observed. For some cases, a
disadvantage can be their short half-life because of rapid renal clearance and protease
degradation. Structural changes, including unnatural aminoacids and modifications, can be
introduced within peptides to improve the stability and the therapeutic potential.
Modifications with polyethylene glycol (PEG), which functions as a carrier for peptides, can
be used to improve the solubility in aqueous environments and is characterized by a low
immunogenicity. Research on peptide therapeutics has increased the latest years because of
their advantages compared to small molecules and because the technical production and
quality improvements. In 2010, about 60 peptide therapeutics were approved by the FDA as
for example Byetta, containing exenatide, a 39 amino acid peptide, which is an analogue of
the glucagon-like peptide-1 (GLP-1) and is used for the treatment of diabetis [31-33].
5
1.3. BIOSENSORS IN DRUG DEVELOPMENT
1.3.1. What are biosensors?
According the IUPAC, a biosensor is: “a self-contained integrated device which is capable of
providing specific quantitative or semi-quantitative analytical information using a biological
recognition element (biochemical receptor) which is in direct spatial contact with a
transducer element. A biosensor should be clearly distinguished from a bioanalytical system,
which requires additional processing steps such as reagent addition. Furthermore, a
biosensor should be distinguished from a bioprobe which is either disposable after one
measurement, i.e. single use, or unable to continuously monitor the analyte concentration”
[34]. An immunosensor is a specific form of a biosensor, including a recognition element
composed of antibodies. Most of these biosensors are based on the ELISA principle, whereby
the substrate is converted by enzymes into a detectable signal, for example a visible light
sensitive products [35-36]. Electrochemical immunosensors on the other hand have the
advantage they can be automated and have great potential for miniaturization. Because of
the high affinity and specificity for its antigens and the high diversity potential, this is a very
powerful tool [36].
One of the major aims of biosensors in R&D is the characterization of biomolecular
interactions. Specific, detailed, qualitative and quantitative information can be gathered on
the binding affinity and kinetics of an interaction. Also cell-based assays can be performed
with these instruments. A major advantage is that characterization can be performed in real-
time in a near-native state. The analysis has a high reproducibility, a high sensitivity and only
a minimum of sample is used. This explains why biosensors are effective workhorses used in
many stages of drug discovery research, like for example target identification, ligand fishing,
screening drugs that interfere with cell adhesion, hit selection and optimization or early
ADME and/or toxicological screens [37-38].
In addition, biosensors are promising techniques in quality control in GLP/GMP
environments. FDA and EMA directives require a validated binding assay as part of the
product-release portfolio for all therapeutic biological products. Traditional biological assays
for drug response, which use in vitro cell cultures or in vivo animal models, are expensive
6
and not always reliable. Provided that there are sufficient physicochemical data for drug
response (receptor binding does not always correlate with drug potency), binding assays
that use purified receptors or membrane fragments can form an alternative [74-75].
Biosensors have a lot of possible applications in many other fields like for example screening
for (veterinary) drug and/or toxic residues in the food industry [39-40], environmental
detection of pollutants [41], screening for specific targets in body fluids in forensic cases [42],
military screening for biological of chemical warfare agents [43] and describing disease
parameters by for example continuous glucose monitoring [44]. However, these applications
are still to be further developed.
1.3.2. Surface acoustic waves biosensors
Surface Acoustic Wave (SAW) biosensors are devices based on physical properties to detect
and quantify label-free interactions in real time. The popularity of this method has increased
enormously the latest years: between 1996 and 2006, publications using this technique
increased by a factor of 1.5-78 [45-51]. A setting is shown in Figure 2 [51-52].
Figure 2: Principle of a SAW biosensor. (a) A SAW biosensor is composed of a piezoelectric crystal or substrate, a sensitive layer and a guiding layer. A surface acoustic wave is generated by an input IDT, propagates in the z-direction and is detected by the output IDT. (b) Placing of a biosensor, whereby a
sealed flow cell is required [52].
A SAW biosensor is composed of a piezoelectric crystal, two interdigital transducers (IDTs), a
guiding layer and a sensitive layer containing a ligand, which can be modified in function of
7
the investigated analyte. An electric signal is converted by the input IDT into a polarized
transversal wave, which propagates through the substrate and is afterwards converted again
by the output IDT into an electrical signal, which can be detected. The propagation of the
wave is performed within the guiding layer and the wave energy is retained near the surface.
Responsible for this is the lower acoustic wave velocity in the guiding layer, what results in
being guided in only this layer and a minimization of loss into the bulk of the substrate or
into the liquid/gas that passes above the sensor surface. Thereby, the thickness of the
substrate doesn’t have influence on the detection capacities of the SAW sensors [51-54].
Investigation of the binding properties of an analyte to its ligand requires a sensor chip of
good quality. On this sensor chip, the ligand is immobilized and the passing analyte is able to
associate and dissociate with the immobilized ligand. The quality of the sensor chip and
therefore also its sensitivity, is related to the ligand immobilization, which has to be as
homogeneous and representative as possible. A gold sensor chip is mostly used because of
its potential to easily form strong bonds by multiple methods. In addition, gold is an inert
material and has the advantage of being resistant to corrosion due to aqueous buffer
solutions [53, 55-56].
Changes at the gold surface influence the oscillation behaviour of the surface acoustic
waves. A divergent phase is related to mass changes (i.e. acting as microbalance), while a
changed amplitude is related to viscoelastic and conformational changes [52-57]. Other
parameters such as electrical conductivity, liquid viscosity, liquid density pressure and
temperature can have influence on the waves as well [58].
1.3.3. Sensorgram of surface acoustic wave biosensors
A typical sensorgram of a binding process, where the analyte is interacting with an
immobilized ligand, is represented in Figure 3. While the analyte is injected, an exponential
curve can be noticed, which flattens when saturation of the surface is obtained. During the
post-injection phase, when only buffer has contact with the surface, the unbounded analyte
is washed away and bound analyte dissociates, which can be noticed by a descending curve.
When a regeneration step is performed, and the analyte is removed from the immobilized
ligand by specific conditions, the curve is brought back to the baseline [59-61].
8
Figure 3: SAW sensorgram [based on 60]
.
1.3.4. Other tools for the characterization of binding events
1.3.4.1. Surface plasmon resonance
The most frequent used biosensor technique of the latest years is the surface plasmon
resonance biosensor (SPR). This optical biosensor performs a non-destructive analysis that
uses plane-polarized light to investigate the interaction of molecules on the sensor chip. The
chip is constructed out of an inert metal coating and glass, where total internal reflection
(TIR) of polarized light can be observed. The surface plasmons are sensitive to fluctuations of
electron density at the interface of two materials. This means that when analyte and ligand
interact at the interface, the angle of reflected light is modified. The amount of bound
analyte, affinity to its ligand and interaction kinetics can be observed by optical signals [46, 49-
51, 62].
1.3.4.2. Quartz crystal microbalance
The quartz crystal microbalance (QCM) sensor, is based on the properties of piezoelectric
materials. These sensors work with acoustic waves which are generated in the whole
substrate, which are afterwards converted back into an electric signal and is applied for
detection [63-67]. The binding of molecules to the surface results in modified waves passing
the substrate. The mass and viscosity changes of this process are converted on the waves by
a proportional change in resonant frequency and a signal attenuation which can be detected
in real time [68].
9
1.3.4.3. Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) is a technique giving information about binding
constants, reaction stoichiometry and the thermodynamic profile (enthalpy and entropy) of
an interaction [46, 69-73]. The system is composed out of two identical cells with equal
temperature at any time. When an analyte is added to the sample cell, which contains a
ligand solution, an enthalpic change is induced by interaction and a thermodynamic effect,
being an exothermic or endothermic reaction, can be observed. Respectively less or more
heat per time unit has to be produced to retain an equal temperature of the sample cell
compared to the reference cell. Registration of this dispensed heat and plotting against the
time or concentration of analyte, can give the thermodynamic profile of an interaction [46, 69-
73].
1.3.5. Comparison of biosensors and other techniques
SPR is based on the optical properties of molecules for measurements, which makes
detection of more complex solutions often complicated and limited. SAW sensors are
microbalances and are unaffected by the complexity of the solution. For example, fragments
and small molecules are often dissolved in DMSO whose high refractory index typically
adversely affects optical detection methods such as SPR. In addition, SAW based biosensors
are able to registrate changes in viscoelastiscitiy. Both SAW and SPR make use of inert
materials for the sensor chip surface, mostly gold. SAW sensors however are able to use
alternative new materials for sensor surfaces as for example SiO2 and ZnO what possibly
might lead to chromatographic techniques on the chip in the future [45-58].
Both SAW and QCM sensor techniques use acoustic waves to investigate binding
characteristics. The major difference between the two are the used waves. Within SAW
sensors, the waves propagate within the guiding layer versus the whole substrate by QCM
what results in a reduction of energy and sensitivity. Therefor, SAW biosensors are more
suitable to investigate molecular interactions of for example small peptides and proteins,
and QCM is more likely to be used to observe interactions with cells [52, 63-69]. However,
interaction studies on cells are also reported with the SAW biosensor.
10
In ITC, the molecular interactions are studied in their native state, meaning that no
modification like surface immobilization is necessary. However, both cells (reference and
sample cell) have to contain the exact same buffer solution to prevent disturbances in the
results. A high concentration (> µM) of both ligand and receptor are necessary to perform
the analysis, what decreases its potential in industrial applications [69-73]. Because its
thermodynamic information output, it can be seen as a complementary technique to SAW
and SPR.
11
2. OBJECTIVES
The global aim of the project is to develop a SAW biosensor technology into a functional
quality characterization tool. In this work, we have used somatropin or recombinant human
growth hormone (GH) as analyte, as well as 1,4,7-triazacyclononane-1,4,7-triacetic acid
(NOTA) modified somatropin (equimolar (1:1) or 3 to 10 times molar excess of NOTA (1:3
and 1:10) added to somatropin) and multiple somatropin derived peptides. This NOTA group
can allow the incorporation of radiometals for SPECT/PET-diagnostic (67Ga, 68Ga, 111In) or
therapeutic (90Y) purposes for further investigations in the future.
The more specific project goals are
(i) The development of a method for selective immobilization of antibody (hGHAb),
thereby preserve the binding capacity and binding characteristics of the antibody
for the analyte somatropin.
(ii) The development of robust operational conditions, to obtain reproducible
binding studies and minimal variation.
(iii) The quantitative binding characterization (affinity, on- and off-rates) of NOTA-
modified somatropin and multiple somatropin derived peptides. The binding
characteristics will be a benchmark for the functional quality control since the
addition of NOTA under different synthesis procedures can affect the
functionality of somatoprin.
(iv) Confirmation binding experiments with size exclustion chromatography were
performed.
12
3. MATERIALS AND METHODS
3.1. SENSOR CHIP
3.1.1. Cleaning sensor chips
3.1.1.1. Pre-cleaning
The pre-cleaning process of the sensor chip is composed out of three steps. To remove salts
and other polar compounds like carbohydrates, the chip was sonicated during three minutes
in water. The chip was placed in 100% acetone (Fisher Scientific) and exposed to ultrasonic
vibrations to remove nonpolar, lipophilic compounds like lipids, membranes, cell contents
and many proteins. The chip had to be placed immediately in 100% isopropanol (Fluka) to
prevent stains on the gold surface due to drying. Washing and sonication in isopropanol also
removed nonpolar compounds and some sugars. Advantageous is the quick drying property
of isopropanol, especially when N2 or O2 is used.
3.1.1.2. Chemical etching with AMP
Chemical etching is necessary when the chip was already modified. The chip was boiled in a
freshly prepared solution (70°C) of 5:1:1 water, ammonia (32%) (PRS Panreac) and hydrogen
peroxide (30%) (Merck). After three minutes, the chip was dried in a steam of N2 or O2.
3.1.2. Coating sensor chip with dextran
First, an alkanethiol sensor chip was prepared by covering the golden sensor chip (SAW
instruments) in a 2 mM 11-mercapto-1-undecanol (Sigma Aldrich) in 100% ethanol solution
(Sigma Aldrich) overnight in dark at room temperature. During this process, the thiol groups
were coupled to the golden surface and free –OH groups were created on the surface. After
the chip was sonicated three times in ethanol, the free terminating hydroxyl groups of
alkanethiol were activated. For this purpose, the chip was incubated during 4 hours in 0.6 M
epichlorohydrin (Sigma Aldrich) in a 1:1 mixture of diglyme (Sigma Aldrich) and 0.4 M NaOH
(Sigma Aldrich). The extremely reactive epoxides, formed by this reaction, will be able to
react easily with the dextran molecules by covalent bindings. Next, the sensor chip was
washed and sonicated three times in water, two times in ethanol and again three times in
water and dried in streaming N2 or O2. The dextran hydrogel sensor chip was prepared by
13
dripping a dextran (Sigma Aldrich) solution of 0.30 mg/ml in 0.1 M NaOH on the sensor chip
surface and incubation during night by room temperature. The unbounded dextran was
removed the next day by vigorously washing in 50°C water for multiple times and alternated
by sonication. Free carboxyl groups, whereon immobilization of the ligand can take place,
were created by incubating the chip in 1 M bromoacetic acid (Sigma Aldrich) in 2 M NaOH
overnight at room temperature. After this carboxylation, the chip was washed again
vigorously with 50°C water and exposed to ultrasonic vibrations to speed up dissolution. The
chip was dried in a steam of N2 or O2 and stored in a refrigerator at 4°C to prevent bacterial
or fungal growth.
3.2. LIGAND IMMOBILIZATION
A dextran hydrogel sensor chip was placed in the SAM5 instrument (SAW Instruments). The
binding of biomolecules is promoted by the hydrophilic environment, created by the dextran
layer. Positive loaded compounds also have the advantage in binding because of the
electrostatic attraction due to the negative charge of dextran [51-53]. The complete ligand
immobilization procedure was completed within one sequence. A flow rate of 13 µl/min was
maintained during the immobilization procedure with running buffer (HBS containing 20 mM
HEPES (Fluka), 150 mM NaCl (Sigma Aldrich), pH 7.4). The carboxyl groups were activated by
forming reactive succinimide esters by injecting 130 µl 1:1 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC) (Sigma Aldrich) and N-hydroxysuccinimide (NHS)
(Sigma). Next, six injections of 65 µl 50 µg/ml hGHAb (Thermo) in a HBS and acetic acid
(Sigma Aldrich) pH 4.5 solution were injected, each with an equilibration time of ten
minutes. The immobilization procedure was terminated by inactivation of the reactive
succinimide ester groups by injecting 100 µl ethanol amine (Sigma Aldrich), followed by 30
minutes equilibration.
A SAM5 feasability test was performed, whereby the amount of analyte that can be detected
(in pg/mm²) has to be determined, with the assumption that all binding sites of the ligand
are available and active. The calculated amount of bound analyte has to exceed 0.5 pg/mm²
to obtain a detectable signal. The theoretical formula is represented below, where X is the
theoretical amount of bound analyte (in pg/mm²), P is the phase shift (in °) related to the
14
amount of surface bound ligand, Mw2 is the molecular weight of the analyte (in pg/pmol),
Mw1 is the molecular weight of the ligand (in pg/pmol) and S, the conversion factor, is a
sensor sensitivity constant (515 °cm²/µg).
3.3. SAM5 BINDING EXPERIMENTS
3.3.1. Screening for a regeneration condition
A regeneration experiment was performed with 0.1% sodiumdodecylsulphate (SDS). HBS (20
mM HEPES, 150 mM NaCl, pH 7.4) was used as running buffer with a flow rate of 30 µl/min.
180 µl of a 500 nM 1:10 NOTA-somatropin solution was injected first to generate binding to
the immobilized ligand. After 5 minutes while running only buffer flowed over the chip, 30 µl
of 0.1% SDS was injected. This reloading and regeneration step was repeated once more
after 10 minutes while running buffer flowing over the sensor chip to ensure all unbounded
particles were removed.
3.3.2. Analytes (modified) somatropin
3.3.2.1. Binding experiments between hGHAb and (modified) somatropin with regeneration
A dilution series was made from the 10 µM somatropin (Zomacton, Ferring) stock solution
and from the 8 µM stocks of modified somatropin, including 1:1 NOTA-somatropin, 1:3
NOTA-somatropin and 1:10 NOTA-somatropin. HBS (20 mM HEPES, 150 mM NaCl, pH 7.4)
was used as running buffer with a flow rate of 30 µl/min. After a wait of 10 minutes, 180 µl
of the 75 nM somatropin sample was injected, followed by the increasing concentrations of
100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 500 nM, 500 nM (duplication), 600 nM, 750 nM,
900 nM, 1 000 nM and 500 nM. After every injection, only buffer was running 20 minutes for
providing sufficiently dissociation and equilibration of the analyte. 0.1% SDS (Sigma Aldrich)
was injected for one minute to regenerate the coated chip whereon the growth hormone
antibody was bound. An equilibration period of 10 minutes was maintained here as well.
This procedure was finished by an extra injection with 5% glycerol (Merck). The experiment
with 1:10 NOTA-somatropin was performed 2 times.
15
3.3.3. Somatropin derived peptides
3.3.3.1. QC analysis of somatropin derived peptides
Mobile phases A and B consisted of 0.1% (m/V) FA (Fisher scientific) in respectively water or
ACN (Fisher scientific). All somatropin derived peptides P0326, P0320, P0318, P0368, P0355
and P0389 (GC Biochem) (Attachment 1) were solved to a concentration of 1 mg/ml. For the
UPLC-RP, the peptide solvent was 95:5 A:B. The UPLC-HILIC analysis used 5:95 A:B as peptide
solvent. All somatropin derived peptides were analysed by UPLC-RP, and P0355 and P0389
were analysed by UPLC-HILIC. The certificates of both columns (Aquity) were enclosed in
Attachments 2 and 3. The temperatures for the column and the sample were respectively
30°C and 5°C. The flow rate was 0.5 ml/min and 2 µl was injected. Detection was performed
using a PDA 190-400 nm, with quantification at 210 nm. The gradient program of both the
UPLC-RP and UPLC-HILIC experiments was represented in Table 1 and Table 2.
Table 1: Gradient program (RP).
Time (min) Flow rate (ml/min)
Solvent composition
MP A (%) MP B (%)
Initial
0.5
95 5
20.00 20 80
22.00 20 80
23.00 95 5
30.00 95 5
Table 2: Gradient program (HILIC).
Time (min) Flow rate (ml/min)
Solvent composition
MP A (%) MP B (%)
Initial
0.5
10 90
20.00 90 10
22.00 90 10
23.00 10 90
30.00 10 90
3.3.3.2. Binding experiments with somatropin derived peptides
This experiment was performed with all somatropin derived peptides, namely P0318, P0320,
P0326, P0355, P0368 and P0389. The HBS running buffer had a flow rate of 30 µl/min. After
10 minutes only buffer running, 180 µl of a blank was injected, followed by a 180 µl injection
of the 100 nM peptide sample after 10 minutes equilibration period. Next injections
included the ascending concentrations of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700
16
nM, 800 nM, 800 nM (duplication), 900 nM, 1 000 nM, 2 000 nM, 3 000 nM, 4 000 nM, 6 000
nM, 8 000 nM, 10 000 nM and 800 nM. After every injection, buffer was running 10 minutes
for sufficiently dissociation and equilibration of the analyte was provided. No regeneration
procedure was performed between the multiple injections.
3.3.4. Data processing of SAW binding experiments for (modified) somatropin and
peptides
For the protein binding study a one-to-one kinetic model was used, assuming that the two
antigen binding sites of the hGHAb ligand are individual, independent binders for the mobile
analyte somatropin [76-77].
[
[ [ [
kon is the association rate constant (units M-1s-1) and koff the dissociation rate constant (units
s-1). In the biosensor, 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 total analyte concentration C. The reaction between immobilized
ligand and analyte in solution can therefore 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:
( )
( ) ⌊ ⌋
kon
koff
17
Where,
The blanc subtracted association curves (0-300 s) of the sensorgrams were analysed using
the integrated rate equation. For each concentration of (modified) somatropin, an apparent
rate constant was determined (kobs) as well as a phase equilibrium Peq, i.e. the analyte phase
response where an equilibrium between association and dissociation or steady state
condition was reached. These apparent rate constants were plotted against the analyte
concentration C (nM). The intercept is the dissociation rate constant at equilibrium (koff). The
slope is the association constant (kon). The binding constant (KD expressed in nM) is
determined by:
The peptide interaction studies, characterized with extreme fast association, were unable to
be analyzed with a one-to-one binding model. Therefore, a steady state model was applied
where P equals to the blanc substracted phase signal at 200 s, C is equal to the used peptide
concentration and Pmax is the maximal phase signal (Figure 4).
( )
Figure 4: Steady state model.
18
3.4. SIZE EXCLUSION CHROMATOGRAPHY
Gel filtration HPLC was performed on a Alliance 2695 separations module and 2996
photodiode array detector (Waters) with a BioSep-SEC-S 2000 (300 x 7.8 mm) column
(Phenomenex) (Attachment 4) protected with a suitable guard column (Phenomenex) in HBS
(20 mM HEPES, pH 7.5, 150 mM NaCl). The (modified) somatropin concentrations applied to
the column contained 45.1 pM in HBS. The hGHAb concentration was 4.43 pM in HBS. The
mixture of hGHAb and (modified) somatropin contained 4.34 pM and 45.1 pM in HBS,
respectively. The P0320 peptide concentration was 8.81 pM, the hGHAb concentration was
0.67 pM, all in HBS. The peptide and hGHAb concentrations were maintained in the
mixtures. For the SEC/HPLC analysis an injection volume of 10 µl was applied, with a flow
rate of 10 µl/min. The column and sample temperature were maintained at respectively
20°C and 5°C during analysis, and detection was performed at 280 nm.
19
4. RESULTS AND DISCUSSION
4.1. LIGAND IMMOBILIZATION
The hGHAb was immobilized via the amine coupling chemistry (Figure 5). Amine coupling is a
direct covalent immobilisation procedure which can be used for any protein: amines present
on the ligand (e.g. lysine residues) are covalently bond to activated carboxyl-groups on the
dextran surface [86]. This procedure however, has two drawbacks. Firstly, because proteins
usually have multiple copies of the functional group (-NH2) that mediates immobilization,
proteins are coupled heterogeneously or random and sometimes at multiple sites. Secondly,
direct coupling often decreases or completely abrogates binding to analyte.
Figure 6 shows the sensorgram of the immobilization procedure. An injection of NHS/EDC
was performed to activate the carboxyl groups on the alkanethiol sensor chip to reactive
succinimide esters (Figure 5). Successive injections of hGHAb revealed an increased phase
shift. Multiple injections were performed to maximize the amount of immobilized ligand.
Not all active NHS esters react with a ligand and it is therefore crucial to eliminate unreacted
esters by deactivation, else they could attack amino groups in buffer or analyte during the
interaction experiment. The deactivation step with a high concentration of ethanolamine
changes the active ester into an inactive hydroxyethyl amide.
Figure 5: Immobilization procedure of ligands
[56]. Activation of carboxyl terminating groups in
dextran layers on the golden sensor chip by EDS/NHS, followed by amine coupling of ligands.
Table 3 shows the immobilization efficiency of every hGHAb injection, calculated as the
percentage phase shift of each hGHAb injection compared to total surface bound ligand per
channel. The injection efficiency decreased over the different injections (e.g. 38% for
injection 1 to 7% for injection 6 in channel 1). This could indicate that:
(i) the surface becomes saturated with hGHAb. Less reactive succinimide esters
are (sterically) available for binding of primairy amines on the ligand. If we
assume that the immobilization is a covalent irreversible reaction and the
20
ligand solution (L) is constantly replenished, then the immobilization is
dependent on the amount of (sterically) available reactive groups on the
surface (S).
(ii) the reactivity of the succinimide esters decreases overtime.
Table 3: hGHAb immobilization efficiency for each injection (% bound).
Final phase
shift Injection 1 (% bound)
Injection 2 (% bound)
Injection 3 (% bound)
Injection 4 (% bound)
Injection 5 (% bound)
Injection 6 (% bound)
Channel 1 5.41° 38 21 15 11 9 7
Channel 2 3.43° 36 21 15 12 9 7
Channel 3 2.22° 33 21 16 12 10 8
Channel 4 2.09° 33 21 16 13 10 7
Channel 5 3.07° 35 21 15 12 9 7
Mean 3.24° 34.9 21.2 15.4 11.9 9.3 7.2
St. Dev. 1.34° 2.0 0.1 0.5 0.6 0.5 0.4
A decrease in surface bond ligand was observed over the different channels. Channel 1 had
the highest final phase shift of 5.41°, followed by 3.43°, 3.07°, 2.22° and 2.09° for channel 2,
channel 5, channel 3 and channel 4, respectively. The difference in surface bound ligand over
the channels can be caused by a heterogeneous carboxymethylated dextran hydrogel.
Figure 6: Sensorgram of the hGHAb immobilization process. EDC/NHS injection, followed by six injections of hGHAb and an ethanolamine injection.
Using the feasibility calculations, a binding stoichiometry of 2, a Mw of 150 000 Da for
hGHAb and 22 125 Da for somatropin, we calculated the quantity of surface bound hGHAb
and the amount of somatropin (analyte) that can bind to the surface, assuming all binding
sites are active (Table 4). The theoretically calculated ad hoc signal value from analyte
0
2
4
6
8
10
12
0 2000 4000 6000 8000 10000
Ph
ase
sh
ift(
°)
Time (s)
Channel 1
Channel 2
Channel 3
Channel 4
Channel 5
21
somatropin exceeds 0.5 pg/mm2 (area/concentration), which is necessary to obtain a
detectable signal.
Table 4: Quantitative overview of ligand immobilization and ad hoc analyte binding signal.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
pg hGHAb per mm2
105.11 66.64 43.13 40.52 59.64
pg somatropin per mm2
31.01 19.66 12.72 11.95 17.59
4.2. SOMATROPIN
4.2.1. Screening for regeneration conditions
The hGHAb bound sensor chip was exposed to an injection of 500 nM 1:10 NOTA-
somatropin, which resulted in a positive phase shift. A regeneration step with 0.1% SDS
should result in a decreasing phase. Repetition of this loading and regeneration experiment
gave similar trends in the results (Figure 7 and Table 5).
Figure 7: Sensorgram screening for regeneration condition with 0.1% SDS.
(A) association, (D) dissociation and (R) regeneration.
Table 5: Phase overview at multiple steps of regeneration procedure.
Phase at t1 (°) Phase at t2 (°) Phase at t3 (°) Phase at t4 (°) Phase at t5 (°)
Channel 1 -0.0123 0.0754 -0.1065 0.0288 -0.1076
Channel 2 -0.0133 0.0512 -0.0911 0.0152 -0.0993
Channel 3 0.0116 0.0670 -0.0424 0.0414 -0.0624
Channel 4 -0.0019 0.0419 -0.0577 0.0162 -0.0762
Channel 5 0.0177 0.0873 -0.1103 0.0239 -0.1371
Ideal regeneration conditions are able to generate a signal which is brought back to the
baseline. By testing 0.1% SDS, a negative phase shift was observed i.e. below baseline. This
could indicate that:
the anionic detergent is too strong and can affect the binding capacity of the hGHAb.
-0.4
-0.2
0
0.2
0.4
0.6
0.8
3000 3500 4000 4500 5000 5500 6000Ph
ase
shif
t (°
)
Time (seconds)
Channel 1
Channel 2
Channel 3
Channel 4
Channel 5t1
t5
t4 t3
t2
A A D D R R
22
non-covalently bound hGHAb (Figure 8) are washed away by this regeneration
procedure.
A. B. C. D.
Figure 8: Schematic overview of random binding between hGHAb and the dextran layer. (A) All antigen binding sites are still available for analyte binding, (B) no analyte can bind on the
antigen binding sites, (C) limited antigen binding sites are available to analyte molecules and
(D) non-covalently bound antibodies with (limited) available antigen binding sites.
4.2.2. SAM5 binding experiments
4.2.2.1. Binding experiment between hGHAb and somatropin
Figure 9 shows the binding of somatropin to the immobilised hGHAb in channel 1. The
association (0-300s) was fitted for all injections and for all channels (Attachment 5). All fitted
curves had an R2 of at least 0.90.
Figure 9: Sensorgram of somatropin binding to the hGHAb in channel 1.
The non-linear regression resulted in a pseudo-first order kinetic constant (kobs) for each
injection. The kon and koff for each channel and the resulting KD are shown in Table 6.
Affinities going from 1 276 nM to 1 900 nM were calculated indicating a high variation
between the channels. Also a large standard error was calculated.
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
-200 0 200 400 600 800 1000
Ph
ase
shif
t (°
)
Time (s)
75 nM
100 nM
150 nM
250 nM
300 nM
500 nM
500 nM
550 nM
600 nM
750 nM
900 nM
1000 nM
Blanc
23
The maximal phase signal at 1 000 nM is presented in Table 7. This was obtained by
subtraction of the phase signal at -80 s and 300 s. Assuming that the injection of 1 000 nM
somatropin is capable of saturating all hGHAb binding sites, the percentage of active hGHAb
was calculated by using the amount of analyte somatropin that was theoretically able to
bind in every channel (Table 4). The calculated percentage of active hGHAb is low and can be
due to the random immobilization procedure (amine coupling) (Figure 8), which can make
the somatropin binding sites on hGHAb sterically unavailable and/or influence its
functionality.
Table 6: Schematic overview of the dissociation constant (KD) and kinetic parameters (kon and koff).
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
kon (M-1s-1)
± SE 8.32E-06
± 3.98E-06 8.22E-06
± 4.19E-06 8.22E-06
± 5.34E-06 7.16E-06
± 4.74E-06 7.31E-06
± 2.19E-06
koff (s-1)
± SE 0.011
± 0.002 0.012
± 0.002 0.013
± 0.003 0.014
± 0.003 0.010
± 0.001
KD (nM) ± SE
1 276 ± 589
1 424 ± 706
1 579 ± 1 084
1 900 ± 1 308
1 519 ± 484
Table 7: Binding capacity of 1 000 nM NOTA-somatropin to surface immobilized hGHAb.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
Phase signal analyte binding at 1 000 nM (°)
0.332 0.255 0.195 0.177 0.323
Amount pg/mm2
6.447 4.954 3.792 3.428 6.273
% active hGHAb 21 25 30 29 36
The observed phase shift seems low but can be related to the used immobilization chemistry
which is an ad random procedure, whereby antigen recognition sites of the antibody can
possibly be non-functional. When direct capturing approaches are used, as for example the
streptavidine-biotine capture or antibody based capture (Fc region), the ligand is attached to
the surface in a specific orientation (determined by the location of the binding site for the
capturing molecule) so that attachment to the surface does not introduce heterogeneity in
the ligand population [78-79].
24
4.2.3. SEC experiments
4.2.3.1. Calibration curve of the SEC column
Figure 10 represents the calibration curve of the BioSep-SEC-S 2000 column, which has a
supplier-specified separation range from 244 to 670 000 Da (Attachment 4). This curve was
based on the logarithm of the molecular weight against the retention time of both own
experimental and supplier-originated data (Table 8 and Attachment 4). Multiple models and
corresponding calibration curves are described in literature, with higher order equations
being more precisely [80]. However, a linear calibration curve was chosen above a more
complex polynominal equation because of the limited available calibration points and to
minimize deviations of the model. Still, an observed error of 3-23% between the theoretical
weight and the weight was determined. The premised model had a sufficient sensitivity to
detect small differences in molecular weight.
Table 8: Calibration curve of the SEC column.
Compound Molecular
weight (Da) Retention time
(minutes) Origin
Modelled molecular weight (Da)
Deviation (%)
IgG 150 000 6.46 Supplier info 124 433 17.0
Ovalbumin 44 000 7.65 Supplier info 45 501 3.4
Somatropin 22 125 8.41 Experimental 23 932 8.2
Myoglobin 17 000 8.57 Supplier info 20 904 23.0
Obestatin (mouse/rat)
2 517 11.23 Experimental 2 206 12.4
Contributing to the deviation of the model can be the use of different specific equipment,
wich have their own parameters such as dead volume, tubings, e.g., other values could also
be obtained by using other buffer solutions, for example the supplier used a phosphate
buffer compared to an HBS buffer within all described SEC experiments.
Figure 10: Calibration curve of the SEC column.
y = -0.3672x + 7.4668 R² = 0.9885
0
1
2
3
4
5
6
5 7 9 11 13
log
Mw
(D
a)
Time (minutes)
25
4.2.3.2. SEC analysis of hGHAb
Figure 11 represents the chromatogram of an hGHAb injection (4.34 pM) obtained by SEC.
The characteristics of the multiple injections are presented in Table 9, where an average
retention time of 6.46 minutes was calculated. Using the calibration curve (Figure 10), this
elution time corresponded to a compound with a calculated molecular weight of 124 362 Da.
The theoretical Mw of the hGHAb was not given by the supplier, however, most antibodies
have an Mw of approximately 150 000 Da. Comparing both molecular weights by the
selected model indicates a deviation of 17%.
Figure 11: SEC chromatogram of hGHAb.
Table 9: SEC results of hGHAb.
RT Area
1 6.49 152 258
2 6.44 150 916
3 6.45 148 268
4 6.45 153 000
5 6.47 156 142
Average 6.46 152 117
SD 0.02 2 883
4.2.3.3. SEC analysis of somatropin
Figure 12 represents the injection of a 45.1 pM somatropin sample derived from a Zomacton
dilution within HBS. Multiple peaks were visible: the major peak at RT 8.41 min (Table 10)
corresponds with somatropin and has a calculated molecular weight of 23 914 Da according
to the previous calibration model. The smallest peak, eluting before somatropin at RT 7.73
min corresponds to a compound with a molecular weight of 42 496 Da according to the
model and has an AUC which is 3.5% of the main peak and lower than the limit of 4%
allowed impurities by the Ph. Eur. There is also a second peak at RT 10.39 min,
26
corresponding to a molecular weight of 4 483 Da and which is 4.5 % of the main peak. Out of
the DAD spectra seemed this peak was not originating from peptides and is considered as a
contaminant.
Figure 12: SEC chromatogram of somatropin (Zomacton).
Table 10: SEC results of somatropin (Zomacton).
RT Area Height
Extra peak
1 7.733 19 216 707
2 7.729 13 395 500
Average 7.73 16 306 604
SD 0.0028 4 116 146
Somatropin (Zomacton)
1 8.413 479 800 28 271
2 8.411 456 352 27 676
Average 8.41 468 076 27 974
SD 0.0014 16 580 421
Extra peak
1 10.392 20 842 1 888
2 10.396 21 634 1 633
Average 10.39 21 238 1 761
SD 0.0028 560 180
Table 11 represents the injection of a 45.1 pM somatropin (lyophilized) sample where the
component had undergone the same lyophilisation procedures as the NOTA-modified
somatropin samples (discussed later). The lyophilized somatropin sample had a similar
chromatogram (not represented) and RT of 8.41 minutes as the Zomacton sample described
above and also an extra peak wich corresponds to 3.4% of the main peak and is lower than
the maximal limit for impurities of the Ph. Eur.
27
Table 11: SEC results of somatropin (lyophilized).
RT Area Height
Extra peak
1 7.658 14 658 521
2 7.644 9 598 539
Average 7.65 12 128 530
SD 0.01 3 578 13
Somatropin (lyophilized)
1 8.407 361816 21492
2 8.412 361906 21323
Average 8.41 361861 21408
SD 0.0035 63.64 119.50
4.2.3.4. SEC analysis of mixtures of hGHAb and somatropin
Figure 13 represents the chromatogram of a sample containing hGHAb (4.34 pM) and a 10
fold molar excess of somatropin (45.1 pM) in HBS. Within the chromatogram, an extra peak
was visible, characterized by an average retention time of 5.76 minutes (Table 12), which
corresponds to a calculated molecular weight of 224 765 Da. The hGHAb (RT 6.32 min) and
somatropin (RT 8.41 min) corresponded to a calculated molecular weight of respectively 139
990 Da and 23 914 Da. The SEC system is sufficiently sensitive to separate both compounds.
The importance of SEC in studying binding characteristics between molecules has become
more important, also because it can replace the deficiancies of the CE technique which is
often used as well [81].
Figure 13: SEC chromatogram of hGHAb and somatropin (Zomacton).
Table 12: SEC results of mixture hGHAb and somatropin (Zomacton).
RT Area Height
Extra peak
1 5.745 9 068 876
2 5.765 11 442 1 105
Average 5.76 10 255 991
SD 0.014 1 679 162
28
Table 12 continued: SEC results of mixture hGHAb and somatropin (Zomacton).
hGHAb
1 6.312 139 901 4 748
2 6.324 141 513 4 726
Average 6.32 140 707 4 737
SD 0.0085 1 140 16
Extra peak
1 7.653 17 952 685
2 7.600 17 573 703
Average 7.63 17 763 694
SD 0.038 268 13
Somatropin (Zomacton)
1 8.407 468 355 28 152
2 8.414 469 857 28 248
Average 8.41 469 106 28 200
SD 0.0050 1 062 68
Assuming additive UV properties (at 280 nm), the compounds ‘alone’ and the ‘mixture’ are
reported to give identical peak areas. This is indeed confirmed by Table 13, whereby the loss
in hGHAb peak area is compensated by the extra peak area at 5.74 minutes. Moreover the
hGHAb peak is shifted from 6.41 minutes (alone) to 6.32 minutes (mixture), while the peak
has widened, what indicates no certainity this peak is pure.
Table 13: Area-balance (at 280 nm) of somatropin (Zomacton).
Alone Mixture
hGHAb 152 117 140 707
Somatropin (Zomacton) 468 076 469 106
Extra complex - 10 255
Total area 620 193 620 068
Figure 14 represents an injection of 4.34 pM hGHAb and a 10 fold excess of somatropin
(lyophilized) in HBS. The results of all runs were gathered in Table 16. An additional peak was
observed with an average retention time of 5.78 minutes, corresponding to a calculated
molecular weight of 220 996 Da. The antibody and somatropin peaks had average retention
times of 6.33 and 8.41 minutes which corresponded to a molecular weight of respectively
138 811 Da and 23 914 Da. The variation to the theoretical weight could be explained to an
error of the model (Table 8). Also the already discussed impurity peak was visible here at
7.64 minutes.
29
Figure 14: SEC chromatogram of hGHAb and somatropin (lyophilized).
Table 16: SEC results of hGHAb and somatropin (lyophilized).
RT Area Height
Extra peak
1 5.781 3 542 518
2 5.783 3 971 577
Average 5.78 3 757 548
SD 0.0014 303 42
hGHAb
1 6.330 176 136 5 513
2 6.329 164 870 5 328
Average 6.33 170 503 5 421
SD 0.00071 7 966 131
Extra peak
1 7.681 20 240 723
2 7.602 14 877 616
Average 7.64 17 559 670
SD 0.06 3 792 76
Somatropin (lyophilized)
1 8.412 359 054 21 112
2 8.407 354 596 21 056
Average 8.41 356 825 21 084
SD 0.0035 3152 40
Assuming additive UV properties (at 280 nm), the solutions ‘alone’ and the ‘mixture’ are
reported to give practically identical peak areas, where small differences probably are due to
differences within pipetting or integration. This is indeed confirmed Table 17, whereby the
loss in hGHAb peak area was compensated by the extra peak area at 5.78 minutes. An other
important remark is the shift of the hGHAb peak from 6.41 to 6.33 minutes and the widening
of the peak what assumes the hGHAb peak is not pure.
30
Table 17: Area-balance (at 280 nm) of somatropin (lyophilized).
Alone Mixture
hGHAb 152 117 170 503
Somatropin (lyophilized) 361 861 356 825
Extra complex - 3 757
Total area 513 978 531 085
However, by considering the results of somatropin (Zomacton and lyophilized), smaller
values were observed for the extra peak of the lyophilized sample. This could be an
indication that the lyophilized somatropin is less reactive then the Zomacton sample, which
could be due to the lyophilisation process or to a decrease in stability of the compound.
4.3. MODIFIED SOMATROPIN
4.3.1. 1:1 NOTA-somatropin
4.3.1.1. SAM5 binding experiment with regeneration
Figure 15 shows the binding of 1:1 NOTA-somatropin to the immobilized hGHAb. The
binding curve showed spiking at the beginning (0 s) and at the end (300 s) of an injection.
Injections were performed with injection parameter ‘burst on’ to quickly change from the
buffer solution to the analyte at the beginning and end of an injection. If the burst is turned
off, the interface between analyte and buffer solution will continuously flow with the regular
flow rate over the sensor chip. This will cause a delay of signal on the last sensor. With the
burst turned on, the flow rate (and the pressure) is increased shortly when the interface
reaches the sensor chip. The spiking has therefore a physical and chemical origin. However,
this spiking was not always observed in experiments with identical injection parameters.
Figure 15: Sensorgram of 1:1 NOTA-somatropin binding to hGHAb in channel 1.
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
-200 0 200 400 600 800 1000
Ph
ase
sh
ift
(°)
Time (s)
100 nM
150 nM
200 nM
250 nM
300 nM
500 nM
500 nM
550 nM
600 nM
750 nM
900 nM
1000 nM
500 nM
Blanc
31
The one-to-one binding model was used for the calculation of the kon and koff for each
channel, and the resulting KD (Table 18 and Attachment 6). The maximal phase signal at
1 000 nM somatropin is presented in Table 19. As with somatropin (section 4.2.2.1), the
calculated percentage of active ligand concentration is relative low: 18% to 35% of all
surface bound ligand is active and available for analyte interaction.
Table 18: Schematic overview of the dissociation constant and kinetic parameters.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
kon (M-1s-1)
± SE 3.80E-05
± 3.96E-06 2.51E-05
± 2.24E-06 2.13E-05
± 2.01E-06 1.64E-05
± 1.99E-06 1.57E-05 1.52E-06
koff (s-1)
± SE 0.0099
± 0.0022 0.0135
± 0.0012 0.0133
± 0.0011 0.0013
± 0.0011 0.0153
± 0.0008
KD (nM) ± SE
260 ± 138
538 ± 68
623 ± 78
796 ± 116
979 ± 108
Table 19: Binding capacity of 1 000 nM 1:1 NOTA-somatropin to surface immobilized hGHAb.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
Phase signal analyte binding at 1000 nM (°)
0.285 0.231 0.173 0.515 0.315
Amount pg/mm2
5.540 4.491 3.359 2.942 6.113
% hGHAb active 18 23 26 25 35
4.3.1.2. SEC experiments
4.3.1.2.1. SEC analysis of 1:1 NOTA-somatropin
Figure 16 represents the injection of 1:1 NOTA-somatropin with a concentration of
approximately 45.1 pM. The results of the two runs were collected in Table 20. This
modification had an average retention time of 8.33 minutes, what corresponds to an
calculated molecular weight of 25 587 Da. The higher molecular weight, compared to
somatropin (23 914 Da) can be related to the coupling of NOTA-chelator groups to free
amines of somatropin (Mw NOTA is 450 Da).
32
Figure 16: SEC chromatogram of hGHAb and 1:1 NOTA-somatropin.
Table 20: SEC results of 1:1 NOTA-somatropin.
RT Area Height
1 8.325 62 650 2912
2 8.338 64 997 2987
Average 8.33 63824 2950
SD 0.0092 1660 53
4.3.1.2.2. SEC analysis of mixtures of hGHAb and 1:1 NOTA-somatropin
Figure 17 represents the chromatogram of an injected sample containing both hGHAb and a
tenfold molar excess of 1:1 NOTA-somatropin (Table 21). An extra peak was observed with
an average retention time of 5.80 minutes, which corresponds to a molecular weight of 217
290 Da according to the calibration model.
Figure 17: SEC chromatogram of hGHAb and 1:1 NOTA-somatropin.
Table 21: SEC results of hGHAb and 1:1 NOTA-somatropin.
RT Area Height
Extra peak
1 5.792 12 751 1 390
2 5.815 11 128 1 255
Average 5.80 11 940 1 323
SD 0.016 1 148 95
33
Table 21 continued: SEC results of hGHAb and 1:1 NOTA-somatropin.
hGHAb
1 6.353 143 790 4 884
2 6.390 152 690 4 458
Average 6.37 148 240 4 671
SD 0.026 6 293 301
1:1 NOTA-somatropin
1 8.325 76 072 3 548
2 8.366 79 795 3 517
Average 8.35 77 934 3 533
SD 0.029 2 633 22
Assuming additive UV properties (at 280 nm), the solutions ‘alone’ and the ‘mixture’ are
reported to give a small difference in peak areas, which probably were due to variation in 1:1
NOTA-somatropin concentration between the samples (Table 22). The similar trend in peak
shift and peak widening was also observed here, indicating the peak might not be pure en
may be partially deriving from other compounds than hGHAb.
Table 22: Area-balance (at 280 nm) of 1:1 NOTA-somatropin.
Alone Mixture
hGHAb 152 117 148 240
1:1 NOTA-somatropin 63 824 77 934
Extra complex - 11 940
Total area 215 941 238 114
4.3.2. 1:3 NOTA-somatropin
4.3.2.1. SAM5 binding experiment with regeneration
Figure 18 shows the binding of 1:3 NOTA-somatropin to the immobilised hGHAb. The
association was fitted using the one-to-one binding model (Attachment 7). The results are
shown in Table 23. For channel 5, the kon and koff were not significantly different from 0. The
maximal phase signal at 1 000 nM is presented in Table 24. The calculated percentage of
active hGHAb ligand is lower than in previous experiments, what may point to
(i) a loss in ligand activity by the successive interaction and regeneration cycles,
and/or,
(ii) the influence of the NOTA-group on binding.
34
Figure 18: Sensorgram of 1:3 NOTA-somatropin binding to the hGHAb in channel 1.
Table 23: Schematic overview of the dissociation constant and kinetic parameters.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
kon (M-1s-1)
± SE 1.15E-05
± 3.89E-06 1.33E-05
± 2.24E-06 1.64E-05
± 2.01E-06 1.31E-05
± 1.99E-06 1.41E-05
± 1.57E-05
koff (s-1)
± SE 0.00637 ± 0.0021
0.0074 ± 0.0012
0.0031 ± 0.0012
0.0060 ± 0.0012
0.0073 ± 0.0153
KD (nM) ± SE
551 ± 90
560 ± 96
188 ± 60
460 ± 67
517 ± 35
Table 24: Binding capacity of 1 000 nM 1:3 NOTAsomatropin to surface immobilized hGHAb.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
Phase signal analyte binding at 1000 nM (°)
0.225 0.180 0.131 0.119 0.258
Amount pg/mm2
4.369 3.491 2.550 2.318 5.018
% hGHAb active 14 18 20 19 29
4.3.2.2. SEC experiments
4.3.2.2.1. SEC analysis of 1:3 NOTA-somatropin
Figure 19 represents the injection of 1:3 NOTA-somatropin with a concentration of
approximately 45.1 pM. The results of the two runs are collected in Table 25. This
modification had an average retention time of 8.26 minutes, what corresponds to a
molecular weight of 27 147 Da. The higher molecular weight, compared to somatropin, can
be related to the coupling of NOTA-chelator groups to free amines of somatropin (Mw NOTA
is 450 Da). Again, a small impurity peak was observed, here at an average retention time of
7.54 minutes, corresponding to a molecular weight of 49 901 Da and remained 3.8% of the
main peak what was beneath the acceptable limit of the Ph. Eur. The mean peak
-0.05
0.05
0.15
0.25
0.35
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Ph
ase
sh
ift
(°)
Time (s)
50 nM
75 nM
100 nM
150 nM
250 nM
300 nM
500 nM
500 nM
550 nM
600 nM
750 nM
900 nM
1000 nM
Blanc
35
corresponding to 1:3 NOTA-somatropin showed a higher peak area and high compared to
the 1:1 NOTA-somatropin. This will be discussed in the 1:10 NOTA-somatropin section.
Figure 19: SEC chromatogram of 1:3 NOTA-somatropin.
Table 25: SEC results of 1:3 NOTA-somatropin.
RT Area Height
Extra peak
1 7.507 15 541 695
2 7.582 17 897 724
Average 7.54 16 719 710
SD 0.05 1 666 21
1:3 NOTA-somatropin
1 8.258 436 422 20 562
2 8.271 452 057 20 265
Average 8.26 444 240 20 414
SD 0.0092 11 056 210
4.3.2.2.2. SEC analysis of mixtures of hGHAb and 1:3 NOTA-somatropin
Figure 20 represented the chromatogram of an injected sample containing both hGHAb and
a tenfold excess of 1:3 NOTA-somatropin. All results were assembled in Table 26. No extra
peak was here detected at 280 nm, which can be due to an insufficient detection limit.
Figure 20: SEC chromatogram of hGHAb and 1:3 NOTA-somatropin.
36
Table 26: SEC results of hGHAb and 1:3 NOTA-somatropin.
RT Area Height
hGHAb
1 6.328 99 121 3 002
2 6.305 94 264 2 684
Average 6.32 96 693 2 843
SD 0.016 3 434 225
Extra peak
1 7.467 23 003 879
2 7.477 34 045 866
Average 7.47 28 524 873
SD 0.007 7 808 9
1:3 NOTA-somatropin
1 8.252 451 110 20 775
2 8.275 470 131 20 613
Average 8.26 460 621 20 694
SD 0.016 13 450 115
Assuming additive UV properties (at 280 nm), the solutions ‘alone’ and the ‘mixture’ are
reported to give difference in peak areas (Table 27), which probably were due to variation in
hGHAb and 1:3 NOTA-somatropin concentration between the samples. An extra peak
corresponding to the complex of hGHAb and 1:3 NOTA-somatropin was not observable. This
lacking peak is due to the lacking sensitivity of the system to detect this complex. Also the
antibody peak showed a shift as previously described what could include a part of the
complex. Deconvolution of the peaks could give further information about this.
Table 27: Area-balance (at 280 nm) of 1:3 NOTA-somatropin.
Alone Mixture
hGHAb 152 117 96 693
1:3 NOTA-somatropin 444 240 460 621
Extra complex - -
Total area 596 357 557 314
4.3.3. 1:10 NOTA-somatropin
4.3.3.1. SAM5 binding experiments
4.3.3.1.1. Binding experiment with regeneration
Figure 21 shows the binding of 1:10 NOTA-somatropin to the immobilised hGHAb. The
association (0-300s) was fitted for all injections and for all channels. The statistical analysis of
the non-linear regression is summarised in Attachment 8.
37
Figure 21: Sensorgram of 1:10 NOTA-somatropin binding to hGHAb in channel 1.
The on- and off-rates as well as the calculated affinities (KD) are shown in Table 28. We
observe a decrease in affinity from channel 1 to 5, including an increased standard error. The
maximal phase signal at 1 000 nM is presented in Table 29.
Table 28: Schematic overview of the dissociation constant (KD) and kinetic parameters (kon and koff).
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
kon (M-1s-1)
± SE 1.58E-05
± 4.45E-06 8.11E-06
± 2.79E-06 1.05E-05
± 4.03E-06 5.01E-06
± 1.69E-06 4.40E-06
± 1.49E-06
koff (s-1)
± SE 0.0114
± 0.0023 0.0147
± 0.0016 0.0112
± 0.0021 0.0129
± 0.0009 0.0103
± 0.0008
KD (nM) ± SE
724 ± 251
1808 ± 651
1062 ± 455
2571 ± 887
2336 ± 812
Table 29: Binding capacity of 1000 nM 1:10 NOTA-somatropin to surface immobilized hGHAb
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
Phase signal analyte binding at 1000 nM (°)
0.198 0.162 0.133 0.119 0.206
Amount pg/mm2 3.848 3.143 2.586 2.305 4.008
% hGHAb active 12 16 20 19 23
4.3.3.1.2. Duplication of binding experiment with regeneration
Figure 22 shows the binding of 1:10 NOTA-somatropin to the immobilised hGHAb. The
statistical analysis of the non-linear regression is summarised in Attachment 9. The binding
characteristics are summarized in Table 30. The calculated affinities deviated from the ones
calculated in previous experiment (Table 28) by a factor 2 to 4. Consequently, serious
considerations have to be made according the robustness of the system The calculated
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0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
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Ph
ase
sh
ift
(°)
Time (s)
100
150
200
250
300
500
500
600
750
1000
500
Blanc
38
active ligand binding sites (Table 31) are similar than in previous experiments with 1:3 and
1:10 NOTA-somatropin.
Figure 22: Sensorgram of 1:10 NOTA-somatropin binding to the hGHAb in channel 1.
Table 30: Schematic overview of the dissociation constant and kinetic parameters.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
kon (M-1s-1)
± SE 1.02E-05
± 1.51E-06 1.18E-05
± 1.23E-06 1.18E-05
± 1.03E-06 1.30E-05
± 9.81E-07 1.76E-05
± 2.81E-06
koff (s-1)
± SE 0.0032
± 0.0008 0.0042
± 0.0006 0.0038
± 0.0055 0.0040
± 0.0005 0.0097
± 0.0015
KD (nM) ± SE
316 ± 95
358 ± 67
322 ± 55
306 ± 47
554 ± 123
Table 31: Binding capacity of 1 000 nM 1:10 NOTA-somatropin to surface immobilized hGHAb.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
Phase signal analyte binding at 1000 nM (°)
0.268 0.208 0.148 0.127 0.287
Amount pg/mm2
5.210 4.048 2.875 2.460 5.566
% hGHAb active 17 21 23 21 32
4.3.3.2. SEC experiments
4.3.3.2.1. SEC analysis of 1:10 NOTA-somatropin
Figure 23 represents the injection of 1:10 NOTA-somatropin with a concentration of
approximately 45.1 pM. The results of the two runs were collected in Table 32. This
modification had an average retention time of 8.15 minutes, what corresponds to a
calculated molecular weight of 29 793 Da. The two extra peaks had an average retention
time of 7.41 minutes and 10.12 minutes, corresponding to the calculated weights of
respectively 55 699 Da and 5 633 Da and were identified as an impurity and a contamination.
-0.2
-0.1
0
0.1
0.2
0.3
-200 0 200 400 600 800 1000
Ph
ase
sh
ift
(°)
Time (S)
100 nM150 nM200 nM250 nM300 nM500 nM500 nM550 nM600 nM750 nM900 nM1000 nM500 nMBlanc
39
Figure 23: SEC chromatogram of 1:10 NOTA-somatropin.
Table 32: SEC results of 1:10 NOTA-somatropin.
RT Area Height
Extra peak
1 7.365 17 938 731
2 7.446 18 657 817
Average 7.41 18 298 774
Mean 0.06 508 61
1:10 NOTA-somatropin
1 8.143 558 746 25 007
2 8.152 566 399 24 170
Average 8.15 562 573 24 589
SD 0.0064 5 411 592
Extra peak
1 10.091 16 927 961
2 10.150 18 864 1 004
Average 10.12 17 896 983
Mean 0.04 1 370 30
4.3.3.2.2. SEC analysis of mixtures of hGHAb and 1:10 NOTA-somatropin
Figure 24 represented the chromatogram of an injected sample containing a mixture of
hGHAb and a tenfold excess of 1:10 NOTA-somatropin. All results were assembled within
Table 33 and in Table 34. No extra peak was here detected at 280 nm, which can be due to
low sensitivity and the peak of the complex can be included in the hGHAb peak because of
the peak shift and difference in shape. Deconvolution can give exclusion about this.
Figure 24: SEC chromatogram of hGHAb and 1:10 NOTA-somatropin.
40
Table 33: SEC results of hGHAb and 1:10 NOTA-somatropin.
RT Area Height
hGHAb
1 6.28 17 6575 4 946
Extra peak
1 7.47 33 298 1 039
1:10 NOTA-somatropin
1 8.15 572 275 23 618
Extra peak
1 10.16 32 273 1 054
Table 34: Area-balance (at 280 nm) of 1:10 NOTA-somatropin.
Alone Mixture
hGHAb 152 117 176 575
1:10 NOTA-somatropin 562 573 572 275
Extra complex - -
Total area 714 690 748 850
We can conclude that the binding experiments performed with SEC have to be interpreted
carefully because the observed error on the used model between the experimental and
theoretical data from 2 517 to 150 000 Da in this range varied between 3-23%. Also
differences in shape and compactness of a molecule can influence the retention
characteristics. A shorter retention time was observed by higher order NOTA-modifications.
Calculated molecular weights of 25 587 Da, 27 147 Da and 29 793 Da were determined by
respectively 1:1, 1:3 and 1:10 NOTA-somatropin, indicating the higher amounts of NOTA that
were used for modification, the more NOTA was bound on somatropin. This conclusion
could be made considering the separation of NOTA-groups (Mw NOTA is 450 Da) is possible
at the respectively retention times. Previous investigations indicated that within the 1:1
NOTA-somatropin batch about half of the molecules were modified with one NOTA group
and the other half was remained unmodified. Within the 1:3 NOTA-somatropin batch, 13%
had no modifications, 45% contained one NOTA-group, 35% were coupled to two groups and
7% were coupled to tree NOTA-groups. Within the 1:10 NOTA-somatropin batch, all
molecules were modified: 11% contained one NOTA-group and respectively 56% and 33%
were coupled to two and three NOTA-groups. The additional effect of the NOTA-groups on
280 nm, detection was reflected on the 1:3 and 1:10 NOTA-somatropin batches. The amount
of bound NOTA to somatropin was proportional to the difference in peak heights; the more
NOTA that was bound to somatropin, the higher the peak height. 1:1 NOTA-somatropin
showed proportionally a lower additional effect, which was probably due to production
41
variation. The 1:1 NOTA-somatropin modification showed clearly an extra peak. The 1:3 and
1:10 modifications did not qualitatively show an extra peak, but a small RT peak shift and a
broader peak width were observed. The absence of the complex peak could be the result of
a low detection limit or the low affinity of the complex, as SEC is a method to detect high
affinity complexes.
4.3.4. Comparison of the modified somatropin structures
Table 35 gives an overview of the weighed means of the KD values over the different
channels. The calculations are enclosed in Attachment 10.
Table 35: Weighted mean of the KD values of (modified) somatropin.
KD Somatropin
(nM))
KD 1:1 NOTA-somatropin
(nM)
KD 1:3 NOTA-somatropin
(nM)
KD 1:10 NOTA- somatropin
(nM)
KD 1:10 NOTA- somatropin
(nM) (repetition)
Weighted mean 1 512 713 490 1725 365
Out of the duplication experiments with the SAW biosensor, we detected variability between
measurements. For example, two times 1:10 NOTA-modified somatropin was investigated
for its binding to hGHAb, resulting in two different overall calculated affinities (KD1: 1 725
nM, KD2: 365 nM). The experimental conditions were different: lower concentrations were
excluded from the first 1:10 experiment (KD 1 725 nM). However, this experimental
difference may not influence the calculated affinity. In addition, the standard error on the KD
values for each channel were relative high for the 1:10 NOTA-somatropin (KD 1 725 nM) and
somatropin (KD 1 213 nM) experiments.
The 1:1, 1:3 and 1:10 NOTA-somatropin samples seem to have at least similar affinities for
the hGHAb (in the higher nM affinity range). However, the calculated affinity of somatropin
for hGHAb seems to be low (KD in µM range, which is unusual for antibody interactions). This
could indicate that the NOTA-bifunctional chelator has strong affinity for the hGHAb
immobilized chip (either specific or aspecific). Since the high variability, we suggest to
optimize the experimental conditions to improve the reproducibility of measurements.
Figure 25 represents the risk analysis of the variability in affinities (KD values), based on the
Ishikawa diagram. Attention must be payed to the operational quality of the system
42
(equipment). During experiments care must be taken for any airbubble present in tubings or
injector syringes, which can cause aberrant signals and therefore, variability between
experiments. The flow cells are formed by pressing the sensor chip against a set of meandric
channels on the surface of the fluidic cell, so that the chip can easily be exchanged. Since the
fluidic cell is under constant pressure during experiments, it is recommended to replace the
fluidic cell once a year. Maintenance on a regular basis is necessary to remove absorbed
proteins or analytes and micro-organisms from tubings, pumps, fluidic cell and autosampler.
The researcher (personnel) should be trained and qualified to perform experiments with the
SAW biosensor. The presence of a quality system including standard operating procedures
(SOP) is therefore very important.
The quality of the materials is crutial, since they can influence the results obtained with
bioassays [84]. The purity of the analyte and ligand samples should be as high as possible.
When new running buffer is prepared, minimal variation in pH, salt or buffer concentration
can influence the baseline and hence the experimental variability. When buffers are
exchanged, the system should be equilibrated for at least one hour to stabilize the baseline.
The sensor chip was chosen to be classified seperatly (not under materials), since the
different manipulations that can influence the measurement variability. For example, before
modifying the chip with a carboxymethylated dextran hydrogel, the chip has to be of plain
gold. Therefore the cleaning and etching procedure is crucial to obtain a homogeneous
carboxymethylated dextran hydrogel. Environmental conditions like room temperature and
humidity can also influence the variation in affinity.
44
Figure 25: Risk analysis of the KD determination.
45
4.4. SOMATROPIN DERIVED PEPTIDES
4.4.1. Choise of peptides
The DruQuaR laboratory has rationally developed somatropin derived peptides based on the
3D structure of hGH in complex with the soluble part of the hGHR or growth hormone
binding protein (GHBp) (PDB 3HHR [16]). Figure 26 shows the different peptides that were
tested with hGHAb.
A. B.
C. D.
Figure 26: Somatropin derived peptides situated in the growth-hormone receptor complex. One molecule growth hormone binds two receptor molecules sequentially l [82-83], including binding site I
(blue), binding site II (green) and a third binding site between both receptors. The red marked structure represents the orientation of the somatropin derived peptide P0326 (A), P0320 (B), P0318
(C) and P0355 (D). The peptides P0368 and P0389 are similar to P0355, but extended with a few extra amino acids.
4.4.2. QC analysis somatropin derived peptides
Before starting experiments, the peptides were analyzed by UPLC-PDA to control if the
requested 95% purity was provided by the supplier. This quality control is absolutely
necessary, since studies have proved that the quality of peptides supplied by the
manufacturer sometimes can be unacceptable and insufficient for experiments [84]. A
reporting threshhold of 0.1% for impurities in peptides obtained by chemical synthesis was
46
applied (Ph. Eur.) (Attachment 11). The purity of the peptides was determined by using a
C18 reversed phase column. The somatropin derived peptides P0355 and P0389 showed a
too low retention (k’ 1.56 and 0.47 respectively). Therefore, the analysis was repeated with a
HILIC column where sufficient retention was obtained (Table 36).
Table 36: Purity of the somatropin derived peptides by UPLC analysis.
Peptide Purity (C18) (%) Purity (HILIC) (%)
P0326 98.2 -
P0320 44.1 -
P0318 89.7 -
P0368 81.4 -
P0355 - 62.8
P0389 - 91.7
Insufficient quality for most of the peptides was detected. Experiments performed with
these peptides consequently have to be interpreted carefully because by-products and
impurities can have a strong inpact on bioassay results, in this case the binding affinity (KD)
to hGHAb [84].
4.4.3. SAM5 binding experiments
4.4.3.1. Feasability calculations
Because of the presence of airbubbles, another sensor chip (including a new immobilization
procedure) was used to perform the somatropin derived peptide experiments. The total
bound ligand from the hGHAb immobilization and SAM5 feasibility calculations are shown in
Table 37. All peptides exceeded the limit of 0.5 pg/mm2 to obtain a detectable signal.
Table 37: Totatal surface bound hGHAb and maximal analyte signal.
Total
bound ligand (°)
Analyte signal P0320
(pg/mm2)
Analyte signal P0326
(pg/mm2)
Analyte signal P0318
(pg/mm2)
Analyte signal P0368
(pg/mm2)
Analyte signal P0389
(pg/mm2)
Analyte signal P0355
(pg/mm2)
Channel 1 7.70 4.44 4.31 2.58 1.09 0.32 0.12
Channel 2 8.35 4.82 4.68 2.80 1.18 0.34 0.13
Channel 3 7.19 4.15 4.02 2.41 1.02 0.30 0.11
Channel 4 2.88 1.66 1.61 0.96 0.41 0.12 0.05
Channel 5 3.36 1.94 1.88 1.13 0.48 0.14 0.05
47
4.4.3.2. SAW binding experiments
Figure 27 shows the binding of peptide P0320 to the immobilized hGHAb. No significant
increase in phase shift was detected during the association (0-300s) so this peptide was
considered as a non-binder. Steady state analysis was impossible, as well as for peptides
P0318, P0368 and P0355 (Figures 28, 29 and 30). The binding of these peptides can be
influenced by their quality and the presence of impurities.
Figure 27: Sensorgram of P0320 binding to hGHAb in channel 1.
Figure 28: Sensorgram of P0318 binding to hGHAb in channel 1.
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
-200 0 200 400 600 800
Ph
ase
sh
ift
(°)
Time (s)
100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000 nM800 nM
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
-200 0 200 400 600 800 1000
Ph
ase
shif
t (°
)
Time (seconds)
0 nM100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000 nM800 nM
48
Figure 29: Sensorgram of P0368 binding to hGHAb in channel 1.
Figure 30: Sensorgram of P0355 binding to hGHAb in channel 2.
Figure 31 shows the binding of peptide P0326 to the immobilized hGHAb. An increase in
phase signal was observed with increasing P0326 concentrations. The binding is
characterized with fast on- and off- rates; therefore, the KD will be determined by plotting
the phase signal during association (at 200s) against analyte concentration. In Table 38, the
calculated binding affinities for each channel are summarized. R2 values of more than 0.85
were obtained for fitting. The spiking in the sensorgrams can be due to physical or chemical
influences and will be investigated later.
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-200 0 200 400 600 800 1000
Ph
ase
sh
ift
(°)
Time (seconds)
0 nM100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000 nM800 nM
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
-200 0 200 400 600 800 1000
Ph
ase
shif
t (°
)
Time (seconds)
0 nM100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000 nM800 nM
49
Figure 31: Sensorgram of P0326 binding to hGHAb in channel 2.
Table 38: Calculated affinities of peptide P0326.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
KD (nM) 29 077
± 37 719 18 391
± 15 946 20 771
± 19 316 114 574
± 436 612 28 027
± 32 574
Figure 32 shows the binding of peptide P0389 to the immobilized hGHAb. An increasing
phase signal was observed with increasing P0389 concentrations. The binding is
characterized with fast on and off rates; therefore, the KD will be determined by plotting the
phase signal during association (at 200s) against analyte concentration. Table 39 represents
the calculated affinities for each channel. For all channels, R2 of more than 0.75 were
obtained for fitting.
Figure 32: Sensorgram of P0389 binding to hGHAb in channel 1.
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-200 0 200 400 600 800 1000
Ph
ase
shif
t (°
)
Time (seconds)
0 nM100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000800 nM
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
-200 0 200 400 600 800 1000Ph
ase
sh
ift
(°)
Time (seconds)
0 nM100 nM200 nM300 nM400 nM500 nM600 nM700 nM800 nM800 nM900 nM1000 nM2000 nM3000 nM4000 nM6000 nM8000 nM10000 nM800 nM
50
Table 39: Calculated affinities of peptide P0389.
Channel 1 Channel 2 Channel 3 Channel 4 Channel 5
KD (nM) 3 909
± 1 751 2 859
± 1 277 3 046
± 1 393 2 711
± 1 217 3 739
± 1 732
Lower affinities for P0326 (higher µM range) were calculated than for P0389 (µM range).
According to the R2 values, the fitting of P0326 was better than the fitting of P0389. The
theoretical molecular weight of P0389 is 1 478 Da and of P0326 is 2 231 Da. Since SAW is a
microbalance technology, higher signals would be expected from the P0326 peptide
interaction. The phase signal of channel 1 of the 10 000 nM P0389 injection at 200s
amounted for 0.086° and for P0326 0.145°. A large variance was observed in the KD values of
P0326. The system seemed to be more robust for the experiment with P0389. However this
data should be interpreted carefully.
Table 40 gives an overview of the weighted KD values of all somatropin derived peptides.
Only 2 somatropin derived peptides gave possible interaction to hGHAb, however this was
an interesting experiment to investigate the capacity of the SAW biosensor to detect
interactions with low molecular weight peptides.
Table 40: Weighted means and SEM of the calculated affinities for P0326 and P0389.
Peptide P0326 P0389
KD (nM) 29 144
± 25 384 3 250 ± 539
4.4.4. SEC experiments
4.4.4.1. SEC analysis of P0320
Only two somatropin derived peptides showed possible binding to the hGHAb. However,
since some ambiguity arised with peptide P0320, a SEC confirmation experiment was
performed. Figure 33 represents a typical chromatogram obtained by injecting 8.75 µM
P0320, showing a second related impurity peak. Table 41 represents the corresponding
results.
51
Figure 33: SEC chromatogram of P0320.
An average retention time of 8.55 minutes was found for the main compound, what
corresponds to 21 244 Da according to the calibration model. The large deviation to the
molecular weight of 3 746 Da from the peptide P0320 could be attributed to errors in the
calibration model, but also on the different hydratation or shape effects which influence the
separation by SEC. The extra peak showed deviation in peak values, which was due to a
variable integration of the peak. However, this impurity was identified as the 44.1% purity as
determined with C18 UPLC analysis. Considering the DAD spectra, this are peptides as well
and can be related products of the P0320.
Table 41: SEC results of P0320.
RT Area Height
P0320
1 8.553 42 480 2 432
2 8.550 39 780 2 500
3 8.553 39 189 2 376
4 8.551 40 699 2 433
Average 8.55 40 537 2 435
SD 0.0015 1 437 51
Extra peak
1 9.640 24 931 713
2 9.587 19 888 615
3 9.547 12 210 344
4 9.490 17 097 535
Average 9.57 18 532 552
Mean 0.06 5 317 156
52
4.4.4.2. SEC analysis of mixtures of hGHAb and P0320
Figure 34 represents a chromatogram of the two injections of the sample consisting of 0.875
µM hGHAb and 8.75 µM P0320. The results of the SEC analyses with samples including both
hGHAb and P0320 were represented in Table 42.
Figure 34: SEC chromatogram of hGHAb and P0320.
Table 42: SEC results of hGHAb and P0320.
RT Area Height
Extra peak
1 6.163 47 189 1 369
2 6.224 55 324 1 490
Average 6.19 51 257 1 430
Mean 0.04 5 752 86
P0320
1 8.552 42 394 2 477
2 8.534 42 398 2 417
Average 8.54 42 396 2 447
SD 0.013 3 42
Extra peak
1 9.588 14 716 485
2 9.736 17 897 547
Average 9.66 16 307 516
Mean 0.10 2 249 44
Comparing both experiments indicated that no statistic significant difference between the
AUC and peak heights of both experiments was observed, what confirms the results of the
SAW experiment. Further investigation of the other peptides is necessary, but interaction
might not be visible due to a lacking sensitivity of SEC to detect interactions on the
micromolar level.
53
5. CONCLUSION AND PERSPECTIVES
The first goal of this project was to develop a selective immobilization procedure for the
ligand hGHAb. The used immobilization procedure is responsible for the ad random ligand
binding and has major influences on the observed phase signal and on the integrity of the
ligand immobilized chip. We have calculated that 15-35 % of the surface bound ligand
remained active, however this amount was sufficient to perform binding experiments.
The second goal was to develop robust operational conditions; however, an overall
relatively large variability in the results was observed. For example the calculated affinity
(KD) between channels for the somatropin experiment varied from 1 276 to 1 900 nM. The
standard error on the KD was high with relative standard errors ranging from 32 to 68%. In
addition to the within-measurement variability (between-channel), the between-
measurement variability was high as well, as demonstrated for the 1:10 NOTA-somatropin
sample (KD1: 1 725 nM and KD2: 365 nM). Therefore, the operational conditions should be
further optimized to improve reproducibility: attention must be paid to the quality of the
operations, of the materials and of the chip.
The third goal contained the quantitative binding characterization of NOTA-modified
somatropin and multiple somatropin derived peptides. Since the overall variability, it is
difficult to make biomedical relevant conclusions about the binding characteristics.
Nevertheless, our data indicates that the binding of the multiple modified forms are at least
similar than unmodified somatropin. The experiments with somatropin derived peptides
have to be interpreted even more carefully, because of the presence of impurities in almost
all peptide samples, which can have a major influence on the KD. Again, our pilot data
indicate a possible interaction between two of the peptides and hGHAb.
For the last goal, the observed binding in the SAW experiments was confirmed with size
exclusion chromatography. Binding of hGHAb was observed with somatropine and 1:1
NOTA-somatropin, but not for 1:3 and 1:10 NOTA derivates.
As a final conclusion, we can state that the SAW biosensor is a very promising instrument to
act as a functional quality characterization tool in the drug discovery process, but further
improvements, especially in robustness, are absolutely necessary.
54
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Attachments
LIST OF ATTACHMENTS
Attachment 1: Certificates of analysis of the somatropin derived peptides.
P0318
P0320
P0326
P0355
P0368
P0389
Attachment 2: Certificate of the Acquity UPLC BEH 300 C18 column.
Attachment 3: Certificates of Acquity UPLC BEH HILIC column.
Attachment 4: Certificate of the Phenomenex BioSep-SEC-S 2000 column.
Attachment 5: Data analysis of somatropin to hGHAb using GraphPad.
Attachment 6: Data analysis of 1:1 NOTA-somatropin to hGHAb using GraphPad.
Attachment 7: Data analysis of 1:3 NOTA-somatropin to hGHAb using GraphPad.
Attachment 8: Data analysis of 1:10 NOTA-somatropin to hGHAb using GraphPad.
Attachment 9: Data analysis of duplication 1:10 NOTA-somatropin to hGHAb using
GraphPad.
Attachment 10: Calculation of weighted means of the GraphPad analysed data.
Attachment 11: European Pharmacopoeia 7.0: reporting, identification and
qualification of organic impurities in active substances.
Attachments
Attachment 1: Certificates of analysis of the somatropin derived peptides.
P0318
Attachments
Attachments
P0320
Attachments
Attachments
P0326
Attachments
Attachments
P0355
Attachments
Attachments
P0368
Attachments
Attachments
P0389
Attachments
Attachments
Attachment 2: Certificate of the Acquity UPLC BEH 300 C18 column.
Attachments
Attachment 3: Certificate of the Acquity UPLC BEH HILIC column.
Attachments
Attachment 4: Certificate of the Phenomenex BioSep-SEC-S 2000 column.
Attachments
Attachment 5: Data analysis of somatropin to hGHAb using GraphPad.
Experiment performed on 12/10/2012.
Table A: Data after fitting in GraphPad.
Conc (nM) Kobs channel 1 Kobs channel 2 Kobs channel 3 Kobs channel 4 Kobs channel 5
75 0.009996 0.01181 0.01332 0.0155 0.01145
100 0.008529 0.01002 0.01001 0.01226 0.01043
150 0.01224 0.01317 0.01464 0.01376 0.01223
200 0.01459 0.01536 0.0176 0.01708 0.01387
250 0.01635 0.01801 0.01988 0.01865 0.01508
300 0.01611 0.01723 0.01834 0.01787 0.01495
500 0.009229 0.009475 0.008896 0.009791 0.01144
500 0.008621 0.009028 0.008178 0.00893 0.01083
550 0.0206 0.02127 0.02428 0.02381 0.01779
600 0.0208 0.02159 0.0245 0.02378 0.01799
750 0.02248 0.02383 0.02558 0.02515 0.01868
900 0.01737 0.01831 0.01909 0.01921 0.01726
1000 0.01732 0.01906 0.01941 0.01911 0.01818
500 0.01088 0.01089 0.01049 0.01119 0.0118
Attachments
Table B: Statistical analysis of the non-linear regression on the association curves of somatropin to hGHAb interaction.
Channel 1
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1)
403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9675 0.9793 0.9876 0.986 0.9863 0.9863 0.9934 0.9932 0.9839 0.9835 0.9838 0.9821 0.9799 0.9525
Abs SS (2) 0.005913 0.00505 0.009485 0.01634 0.02194 0.02572 0.01692 0.01479 0.04889 0.05418 0.06763 0.06785 0.1439 0.05335
Sy.x (3) 0.003831 0.00354 0.004852 0.006368 0.007378 0.007989 0.00648 0.006059 0.01101 0.01159 0.01295 0.01298 0.0189 0.01151
Channel 2
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9681 0.9748 0.9853 0.9866 0.9862 0.986 0.9927 0.9925 0.9865 0.9867 0.9849 0.9859 0.9838 0.9762
Abs SS (2) 0.005196 0.005175 0.008381 0.01126 0.01563 0.01764 0.01215 0.0106 0.02745 0.02899 0.04169 0.03512 0.07213 0.01828
Sy.x (3) 0.003591 0.003583 0.00456 0.005285 0.006227 0.006615 0.00549 0.00513 0.008254 0.008482 0.01017 0.009335 0.01338 0.006735
Channel 3
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9488 0.9645 0.983 0.9858 0.9869 0.9876 0.9933 0.9927 0.987 0.9877 0.9876 0.9858 0.9841 0.9812
Abs SS (2) 0.003022 0.002816 0.004533 0.005903 0.007648 0.007888 0.005615 0.00514 0.01411 0.01425 0.01866 0.01894 0.03982 0.00735
Sy.x (3) 0.002738 0.002643 0.003354 0.003827 0.004356 0.004424 0.003733 0.003571 0.005916 0.005946 0.006805 0.006856 0.00994 0.004271
Channel 4
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9504 0.9651 0.9829 0.9877 0.9897 0.9893 0.9935 0.9938 0.9886 0.9899 0.9882 0.9876 0.9848 0.9863
Abs SS (2) 0.002216 0.002205 0.003467 0.003911 0.004625 0.005293 0.004562 0.003661 0.009899 0.009167 0.01423 0.01339 0.03109 0.004754
Sy.x (3) 0.002345 0.002339 0.002933 0.003115 0.003388 0.003624 0.003365 0.003014 0.004956 0.004769 0.005942 0.005763 0.008784 0.003435
Channel 5
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 402 402 402 402 402 402 402 402 402 402 402 402 402 402
R2 0.9707 0.9787 0.9856 0.9882 0.9891 0.9887 0.9922 0.9925 0.9895 0.9902 0.9902 0.9899 0.9886 0.9901
Abs SS (2) 0.014 0.01303 0.01723 0.01945 0.02261 0.02669 0.02476 0.02123 0.03704 0.03734 0.04565 0.04477 0.08264 0.01864
Sy.x (3) 0.005902 0.005694 0.006547 0.006956 0.0075 0.008148 0.007848 0.007267 0.009599 0.009638 0.01066 0.01055 0.01434 0.006809
(1) Degrees of freedom (2) Absolute sum of squares (3) Standard error or estimate
Attachments
Table C: Outlier method Grubb’s test on data somatropin to hGHAb interaction.
Concentration Channel 1 Channel 2 Channel 3
(in nM) Kobs-y G Kobs-y G Kobs-y G
75 -0.001241934 0.384598548 -0.000517 0.17284615 -0.000282216 0.052123748
100 -0.002916833 0.785494519 -0.002512 0.624406775 -0.00379777 0.701426696
150 0.00037837 0.003229661 0.0002268 0.004641038 0.000421122 0.077778886
200 0.002312572 0.466191247 0.0020057 0.397892677 0.002970014 0.548544898
250 0.003656774 0.787933191 0.0042445 0.904516736 0.004838907 0.893718749
300 0.003000976 0.63096449 0.0030534 0.634988882 0.002887799 0.533360169
500 -0.005543215 1.414133183 -0.006346 1.491945888 -0.008200632 1.514610408
500 -0.006151215 1.559661219 -0.006793 1.593094548 -0.008918632 1.647220954
550 0.005411987 1.208052771 0.0050379 1.084039465 0.00677226 1.250798254
600 0.005196189 1.156400412 0.0049468 1.063423304 0.006581153 1.215501737
750 0.005628796 1.259947083 0.0059535 1.291217516 0.006427829 1.187183813
900 -0.000728598 0.261728889 -0.0008 0.236947118 -0.001295494 0.239270368
1000 -0.001610194 0.472743502 -0.000872 0.253288272 -0.001797709 0.332026725
500 -0.003892215 1.018957545 -0.004931 1.171754939 -0.006606632 1.220207608
Average (Kobs-y) 0.000364876 0.0002473 -2.72599E-18
St Dev 0.004177889 0.0044192 0.005414351
Table C: Outlier method Grubb’s test on data somatropin to hGHAb interaction (continued).
Channel 4 Channel 5
Kobs-y G Kobs-y G
0.001359604 0.282857036 -0.000198199 0.089157442
-0.002059388 0.428442629 -0.001400853 0.630155632
-0.000917374 0.1908538 3.38399E-05 0.015222451
0.002044641 0.425374434 0.001308533 0.588626599
0.003256655 0.677526317 0.002153226 0.968600793
0.002118669 0.440775672 0.001657919 0.745793274
-0.007392273 1.537915263 -0.00331331 1.490449729
-0.008253273 1.717040827 -0.00392331 1.76485036
0.006268742 1.304171756 0.002671383 1.201687158
0.005880756 1.223453833 0.002506076 1.127325882
0.006176799 1.285043761 0.002100154 0.944727392
-0.000837157 0.174165237 -0.000415767 0.187027377
-0.001653128 0.343922828 -0.000226381 0.101834637
-0.005993273 1.246862226 -0.00295331 1.328508373
-9.91271E-19 -2.47818E-18
0.004806684 0.002223027
The critical G value (P=0.05) for a sample size 14 is 2.5073. The outliers as identified by the Grubb’s test and Dixon’s test are shown in blue.
Attachments
y = 0.0000089x + 0.0106142 R² = 0.2925078
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 1
y = 0.0000086x + 0.0117099 R² = 0.2616435
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 2
y = 0.0000082x + 0.0129856 R² = 0.1648493
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 3
y = 0.00000716x + 0.01360342 R² = 0.15959901
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 4
y = 0.0000073x + 0.0111002 R² = 0.4803981
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 5
Attachments
Attachment 6: Data analysis of 1:1 NOTA-somatropin to hGHAb using GraphPad.
Experiment performed on 19/03/2012.
Table D: Data after fitting in GraphPad.
Conc (nM) Kobs channel 1 Kobs channel 2 Kobs channel 3 Kobs channel 4 Kobs channel 5
75 0.6948 0.01385 0.01374 0.01249 0.01331
100 0.01295 0.01436 0.01406 0.01394 0.01559
150 0.01453 0.0166 0.01605 0.01507 0.01726
200 0.01472 0.01657 0.01546 0.01438 0.01855
250 0.01947 0.02091 0.01975 0.01761 0.02027
300 0.02463 0.02415 0.02259 0.01968 0.0218
500 0.02473 0.02425 0.02236 0.01989 0.02347
500 0.0265 0.02573 0.02346 0.02095 0.02374
550 0.03654 0.03146 0.02937 0.025 0.02567
600 0.03412 0.03042 0.02686 0.02295 0.0255
750 0.04526 0.03442 0.03069 0.02571 0.02751
900 0.04263 0.03475 0.03185 0.02664 0.02819
1000 0.04322 0.03536 0.03193 0.02724 0.02864
500 0.2397 0.04318 0.03517 0.02624 0.02493
Attachments
Table E: Statistical analysis of the non-linear regression on the association curves of 1:1 NOTA-somatropin to hGHAb interaction.
Channel 1
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1)
403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.6968 0.9612 0.965 0.969 0.9606 0.9562 0.9627 0.9604 0.9579 0.9591 0.959 0.9617 0.9632 0.96
Abs SS (2) 0.07377 0.02902 0.0326 0.06871 0.08559 0.1095 0.1638 0.1611 0.1616 0.1756 0.1882 0.2177 0.2196 0.1056
Sy.x (3) 0.01353 0.008486 0.008994 0.01306 0.01457 0.01649 0.02016 0.01999 0.02002 0.02087 0.02161 0.02324 0.02334 0.01619
Channel 2
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 402 402 402 402 402 402 402 402 402 402 402 402 402 402
R2 0.9634 0.9709 0.9762 0.9795 0.9775 0.9762 0.9799 0.9774 0.9772 0.9778 0.9782 0.9789 0.9804 0.9741
Abs SS (2) 0.0105 0.01788 0.01755 0.03267 0.0351 0.04145 0.05913 0.06176 0.06028 0.0645 0.06843 0.08032 0.07878 0.0485
Sy.x (3) 0.005111 0.006669 0.006608 0.009015 0.009344 0.01015 0.01213 0.0124 0.01225 0.01267 0.01305 0.01414 0.014 0.01098
Channel 3
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9615 0.9725 0.9768 0.9813 0.9817 0.9796 0.9824 0.9803 0.9794 0.981 0.9803 0.9808 0.9818 0.9768
Abs SS (2) 0.004507 0.007604 0.007393 0.01446 0.01302 0.01644 0.02543 0.02647 0.0269 0.02808 0.03189 0.03927 0.03985 0.02208
Sy.x (3) 0.003344 0.004344 0.004283 0.00599 0.005683 0.006387 0.007944 0.008105 0.00817 0.008347 0.008896 0.009872 0.009944 0.007403
Channel 4
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9656 0.9762 0.9831 0.9867 0.987 0.9867 0.9882 0.9865 0.9838 0.9863 0.9847 0.9859 0.9872 0.9826
Abs SS (2) 0.00329 0.005051 0.004195 0.007784 0.007192 0.008327 0.01318 0.014 0.01681 0.0159 0.01983 0.02286 0.02233 0.01332
Sy.x (3) 0.002857 0.00354 0.003227 0.004395 0.004224 0.004546 0.00572 0.005894 0.006459 0.006281 0.007014 0.007531 0.007444 0.00575
Channel 5
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9819 0.9845 0.9875 0.9868 0.9881 0.9882 0.9884 0.9876 0.9872 0.9879 0.9872 0.9875 0.9879 0.9873
Abs SS (2) 0.01662 0.02719 0.02684 0.04687 0.0442 0.04787 0.06815 0.06839 0.0714 0.07314 0.08256 0.09289 0.09289 0.05495
Sy.x (3) 0.006422 0.008214 0.008161 0.01078 0.01047 0.0109 0.013 0.01303 0.01331 0.01347 0.01431 0.01518 0.01518 0.01168
(1) Degrees of freedom (2) Absolute sum of squares (3) Standard error or estimate
Attachments
Table F: Outlier method Grubb’s test on data 1:1 NOTA-somatropin to hGHAb interaction.
Concentration Channel 1 Channel 2 Channel 3
(in nM) Kobs-y G Kobs-y G Kobs-y G
75 0.515717168 11.39368064 -0.002194 0.818485766 -0.0017846 0.499734445
100 -0.16716867 0.267282047 -0.002229 0.825043133 -0.0020092 0.562623964
150 -0.167660346 0.27567791 -0.001078 0.606822303 -0.0011083 0.310363149
200 -0.169542022 0.307809422 -0.002197 0.819037317 -0.0027875 0.780578494
250 -0.166863698 0.262074346 0.0010536 0.202615926 0.00041335 0.115750216
300 -0.163775374 0.209338107 0.0032044 0.205224219 0.00216419 0.606036123
500 -0.171962078 0.349134317 -0.001052 0.601919588 -0.0024225 0.678362383
500 -0.170192078 0.318909786 0.0004278 0.321283002 -0.0013225 0.370329911
550 -0.162223754 0.182842639 0.0050686 0.558709236 0.00349837 0.979646621
600 -0.16671543 0.259542526 0.0029394 0.154978714 -0.0001008 0.028225403
750 -0.161790459 0.175443677 0.0036719 0.293876669 0.00046172 0.12929387
900 -0.170635487 0.32648143 0.0007344 0.263128261 -0.0016458 0.460865674
1000 -0.174188839 0.387158492 -0.000834 0.560513348 -0.0037441 1.048459171
500 0.043007922 3.321694698 0.0178778 2.987574039 0.01038753 2.908815764
Average (Kobs-y) -0.151516178
0.0021221
-1.363E-18
St. Dev. 0.058561703
0.0052737
0.00357105
Table F: Outlier method Grubb’s test on data 1:1 NOTA-somatropin to hGHAb interaction
(continued).
Channel 4 Channel 5
Kobs-y G Kobs-y G
-0.001816144 0.902130522 -0.003195437 2.08053433
-0.00077696 0.385938509 -0.00130677 0.850831692
-0.000468594 0.232764142 -0.000419435 0.273092292
-0.001980228 0.983635867 8.78993E-05 0.057230834
0.000428138 0.212668605 0.001025234 0.667525007
0.001676505 0.832767875 0.001772568 1.154111043
-0.00140003 0.695435017 0.000311906 0.203080396
-0.00034003 0.168902678 0.000581906 0.37887617
0.002888336 1.434719263 0.00172924 1.125900389
1.67025E-05 0.008296614 0.000776575 0.505624145
0.000311801 0.154880682 0.000438578 0.28555597
-0.0012231 0.607548627 -0.001229419 0.800469165
-0.002266367 1.125769406 -0.00234475 1.52665624
0.00494997 2.458791729 0.001771906 1.153679765
1.363E-18
-2.8499E-18
0.002013172
0.001535873
The critical value of G (P= 0.05) for a sample size 14 is 2.5073. The outliers as identified by the Grubb’s test and Dixon’s test are shown in red.
Attachments
y = 0.0000380x + 0.0098868 R² = 0.9023988
0.0000
0.0200
0.0400
0.0600
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 1
y = 0.0000251x + 0.0134994 R² = 0.9195805
0.0000
0.0100
0.0200
0.0300
0.0400
0.0500
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 2
y = 0.0000213x + 0.0132950 R² = 0.9110390
0.0000
0.0100
0.0200
0.0300
0.0400
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 3
y = 0.00001643x + 0.01307369 R² = 0.85081169
0.0000
0.0100
0.0200
0.0300
0.0400
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 4
y = 0.0000157x + 0.0153314 R² = 0.8988969
0.0000
0.0100
0.0200
0.0300
0.0400
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 5
Attachments
Attachment 7: Data analysis of 1:3 NOTA-somatropin to hGHAb using GraphPad.
Experiment performed on 17/03/2012.
Table G: Data after fitting in GraphPad.
Conc (nM) Kobs channel 1 Kobs channel 2 Kobs channel 3 Kobs channel 4 Kobs channel 5
75 0.008914 0.01333 0.0003836 0.0082 0.007643
100 0.008832 0.01122 0.003959 0.00612 0.008473
150 0.007325 0.008683 0.006018 0.007252 0.008843
200 0.007258 0.008566 0.006703 0.007514 0.00982
250 0.00864 0.01035 0.008434 0.00909 0.01112
300 0.009204 0.01104 0.009294 0.0101 0.01227
500 0.01223 0.01441 0.01232 0.01302 0.01523
500 0.01244 0.01438 0.01287 0.01373 0.01482
550 0.01265 0.0152 0.01315 0.01453 0.0151
600 0.01344 0.01611 0.01453 0.0159 0.0166
750 0.0132 0.01581 0.01354 0.01438 0.01768
900 0.01499 0.01707 0.01541 0.01572 0.01573
1000 0.02071 0.02333 0.01959 0.01982 0.02042
Attachments
Table H: Statistical analysis of the non-linear regression on the association curves of 1:3 NOTA-somatropin to hGHAb interaction.
Channel 1
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM
Dgr. free. (1)
403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.8502 0.9387 0.9851 0.9942 0.9935 0.9939 0.9936 0.9938 0.9918 0.9919 0.9877 0.9824 0.9717
Abs SS (2) 0.004987 0.006251 0.008053 0.005404 0.006506 0.006111 0.01322 0.01184 0.01257 0.01553 0.03261 0.05552 0.1033
Sy.x (3) 0.003518 0.003938 0.00447 0.003662 0.004018 0.003894 0.005728 0.00542 0.005586 0.006208 0.008995 0.01174 0.01601
Channel 2
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.8197 0.9325 0.9854 0.9925 0.9914 0.9919 0.9925 0.9932 0.99 0.9899 0.9876 0.9848 0.9825
Abs SS (2) 0.002701 0.004165 0.005003 0.004383 0.005622 0.005143 0.009389 0.007802 0.009677 0.01235 0.02071 0.02889 0.04028
Sy.x (3) 0.002589 0.003215 0.003523 0.003298 0.003735 0.003572 0.004827 0.0044 0.0049 0.005537 0.007168 0.008467 0.009998
Channel 3
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM
Dgr. free. (1) 402 402 402 402 402 402 402 402 402 402 402 402 402
R2 0.7961 0.9353 0.9878 0.9935 0.9932 0.9933 0.9939 0.9949 0.9909 0.9899 0.9892 0.9869 0.9859
Abs SS (2) 0.001464 0.001673 0.002336 0.002156 0.002391 0.002114 0.003823 0.00291 0.004426 0.006552 0.009505 0.01373 0.01743
Sy.x (3) 0.001908 0.00204 0.002411 0.002316 0.002439 0.002293 0.003084 0.002691 0.003318 0.004037 0.004862 0.005844 0.006585
Channel 4
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.8374 0.9384 0.9847 0.9922 0.9902 0.9916 0.9923 0.9941 0.9897 0.9869 0.9881 0.9878 0.988
Abs SS (2) 0.001681 0.001644 0.002687 0.002279 0.003058 0.002261 0.004052 0.002717 0.0042 0.00736 0.008708 0.01062 0.01231
Sy.x (3) 0.002042 0.00202 0.002582 0.002378 0.002755 0.002369 0.003171 0.002597 0.003228 0.004273 0.004648 0.005132 0.005527
Channel 5
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9326 0.9718 0.986 0.9901 0.9894 0.9908 0.9913 0.9913 0.9907 0.9891 0.9883 0.9895 0.9888
Abs SS (2) 0.007103 0.008721 0.01291 0.01466 0.0188 0.01594 0.02505 0.02367 0.02259 0.03215 0.04523 0.03817 0.05328
Sy.x (3) 0.004198 0.004652 0.005659 0.006032 0.00683 0.006288 0.007885 0.007663 0.007487 0.008932 0.01059 0.009733 0.0115
(1) Degrees of freedom (2) Absolute sum of squares (3) Standard error or estimate
Attachments
Table I: Outlier method Grubb’s test on data 1:3 NOTA-somatropin to hGHAb interaction.
Concentration Channel 1 Channel 2 Channel 3
(in nM) Kobs-y G Kobs-y G Kobs-y G
75 0.001666586 1.667077701 0.00354 3.505167376 -0.0039261 2.260756458
100 0.001278801 1.368512216 0.0010209 2.072663229 -0.0007598 0.437507472
150 -0.000839771 0.262626538 -0.002334 0.164721603 0.00048109 0.277022579
200 -0.001518342 0.785074891 -0.003269 0.367045732 0.00034798 0.200373887
250 -0.000747914 0.191903529 -0.002303 0.182223019 0.00126087 0.726034486
300 -0.000795485 0.228529909 -0.002432 0.109370003 0.00130276 0.750154557
500 -0.000215771 0.217805969 -0.002334 0.164844849 0.00105631 0.608244739
500 -5.77078E-06 0.379489986 -0.002364 0.147784837 0.00160631 0.924946565
550 -0.000407342 0.070310549 -0.002362 0.148858539 0.0010682 0.615090165
600 -0.000228914 0.207686969 -0.00227 0.201112277 0.00163008 0.938637416
750 -0.002303628 1.389685123 -0.005025 1.365187708 -0.0018143 1.044685131
900 -0.002348342 1.42411172 -0.006219 2.044367075 -0.0023986 1.381158185
1000 0.002148515 2.038126021 -0.001595 0.585022159 0.00014519 0.083602851
Average (Kobs-y) -0.000498664
-0.002624
-1.268E-18
stdev 0.00129883
0.0017585
0.00173665
Table I: Outlier method Grubb’s test on data 1:3 NOTA-somatropin to hGHAb interaction
(continued).
Channel 4 Channel 5
Kobs-y G Kobs-y G
0.001190119 0.943289814 -0.001046208 0.852883426
-0.001217675 0.965130752 -0.000526358 0.429094157
-0.000741262 0.58752529 -0.000776657 0.633141213
-0.001134849 0.899483044 -0.000419956 0.342353591
-0.000214436 0.169962561 0.000259745 0.211748037
0.000139976 0.11094553 0.000789447 0.643567619
0.000437628 0.346864331 0.001268251 1.03389558
0.001147628 0.909611323 0.000858251 0.699657988
0.001292041 1.024073121 0.000517952 0.422241704
0.002006453 1.590317715 0.001397653 1.13938606
-0.001480308 1.173294367 0.000616757 0.502788482
-0.00210707 1.670066454 -0.00319414 2.603906423
0.000681756 0.540360635 0.000255262 0.208093339
-7.33921E-19
-2.0016E-19
0.001261668
0.001226672
The critical value of G (P= 0.05) for a sample size 13 is 2.4620. The outliers as identified by the Grubb’s test and Dixon’s test are shown in blue.
Attachments
y = 0.0000122x + 0.0058314 R² = 0.8852277
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 1
y = 0.0000133x + 0.0074343 R² = 0.8713845
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 2
y = 0.0000164x + 0.0030826 R² = 0.8914860
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 3
y = 0.00001311x + 0.00602650 R² = 0.90905246
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 4
y = 0.0000141x + 0.0073052 R² = 0.9764270
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 5
Attachments
Attachment 8: Data analysis of 1:10 NOTA-somatropin to hGHAb using GraphPad.
Experiment performed on 11/10/2012.
Table J: Data after fitting in GraphPad.
Conc (nM) Kobs channel 1 Kobs channel 2 Kobs channel 3 Kobs channel 4 Kobs channel 5
100 0.005574 0.004009 0.004681 0.005794 0.002198
150 0.01362 0.01476 0.01581 0.01374 0.01017
200 0.01557 0.01619 0.016 0.01541 0.01017
250 0.02244 0.02509 0.031 0.02179 0.01418
300 0.01636 0.01604 0.01476 0.01316 0.011
500 0.01845 0.01738 0.016 0.01377 0.01195
500 0.01946 0.01818 0.01623 0.01464 0.01195
600 0.02219 0.01972 0.01752 0.01603 0.01306
750 0.02485 0.02207 0.02013 0.01865 0.0144
1000 0.02429 0.02081 0.01906 0.01736 0.01427
500 0.1474 0.02332 0.01977 0.01575 0.01243
Attachments
Table K: Statistical analysis of the non-linear regression on the association curves of 1:10 NOTA-somatropin to hGHAb interaction. Channel 1
100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 600 nM 750 nM 1000 nM 500 nM
Degr. free. (1)
403 403 403 403 403 403 403 403 403 403 403
R2 0.856000 0.937500 0.924800 0.954600 0.948300 0.963600 0.957100 0.959900 0.969300 0.971000 0.936800
Abs SS (2) 0.002895 0.007656 0.015900 0.018930 0.038990 0.052810 0.064130 0.064000 0.071210 0.088410 0.072130
Sy.x (3) 0.002680 0.004359 0.006282 0.006853 0.009836 0.011450 0.012620 0.012600 0.013290 0.014810 0.013380
Channel 2
100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 600 nM 750 nM 1000 nM 500 nM
Degr. free. (1) 403 403 403 403 403 403 403 403 403 403 403
R2 0.837600 0.94900 0.955700 0.975600 0.973200 0.980200 0.976900 0.980000 0.98480 0.985700 0.966100
Abs SS (2) 0.001413 0.003751 0.005526 0.005639 0.01205 0.01737 0.02085 0.01937 0.02172 0.027630 0.0245700
Sy.x (3) 0.001873 0.003051 0.003703 0.003741 0.005467 0.006566 0.007192 0.006932 0.007341 0.008281 0.007808
Channel 3
100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 600 nM 750 nM 1000 nM 500 nM
Degr. free. (1) 404 404 404 404 404 404 404 404 404 404 404
R2 0.587100 0.926600 0.943800 0.952400 0.973400 0.984200 0.981500 0.982000 0.986900 0.987600 0.974400
Abs SS (2) 0.001878 0.003027 0.004248 0.006060 0.007197 0.008596 0.010280 0.010520 0.011930 0.015690 0.011600
Sy.x (3) 0.002156 0.002737 0.003243 0.003873 0.004221 0.004613 0.005045 0.005102 0.005434 0.006231 0.005359
Channel 4
100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 600 nM 750 nM 1000 nM 500 nM
Degr. free. (1) 404 404 404 404 404 404 404 404 404 404 404
R2 0.649000 0.945100 0.957400 0.965600 0.983000 0.989200 0.984700 0.98910 0.992600 0.991300 0.98100
Abs SS (2) 0.001666 0.001907 0.00278 0.003399 0.003738 0.004665 0.006873 0.00499 0.005399 0.008841 0.00705
Sy.x (3) 0.002031 0.002173 0.002623 0.0029 0.003042 0.003398 0.004124 0.003514 0.003656 0.004678 0.004177
Channel 5
100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 600 nM 750 nM 1000 nM 500 nM
Degr. free. (1) 404 404 404 404 404 404 404 404 404 404 404
R2 0.916400 0.963100 0.973500 0.985800 0.985600 0.990500 0.990300 0.990400 0.993300 0.99300 0.988300
Abs SS (2) 0.001440 0.006707 0.007579 0.00689 0.01165 0.01389 0.01473 0.01521 0.01513 0.02094 0.01519
Sy.x (3) 0.001888 0.004074 0.004331 0.00413 0.00537 0.005864 0.006039 0.006135 0.00612 0.007199 0.006132
(1) Degrees of freedom (2) Absolute sum of squares (3) Standard error or estimate
Attachments
Table L: Outlier method Grubb’s test on data 1:10 NOTA-somatropin to hGHAb interaction.
Concentration Channel 1 Channel 2 Channel 3
(in nM) Kobs-y G Kobs-y G Kobs-y G
100 -0.014738701 0.513986678 -0.009655 4.124500919 -0.0105725 1.791289814
150 -0.007514265 0.334837945 0.0007873 0.819130256 0.00024753 0.041938253
200 -0.006385829 0.306855439 0.0019083 0.464272748 0.00012856 0.021782031
250 -0.000337394 0.156868695 0.0104994 2.255173674 0.0148196 2.51086992
300 -0.007238958 0.328010992 0.0011404 0.70735728 -0.0017294 0.293005462
500 -0.008435215 0.357675316 0.0012445 0.674393035 -0.0017252 0.292304411
500 -0.007425215 0.33262973 0.0020445 0.421157275 -0.0014952 0.253335731
600 -0.006338344 0.305677921 0.0029666 0.129281263 -0.0008232 0.139467748
750 -0.006143037 0.300834776 0.0043897 0.321194543 0.00085994 0.145698831
1000 -0.010810859 0.416585597 0.0015849 0.566658847 -0.0017549 0.297328946
500 0.120514785 2.83997641 0.0071845 1.205882487 0.00204477 0.346443077
Average (Kobs-y) 0.005988567 0.003375 -3.154E-18
St dev 0.040326468 0.0031591 0.00590218
Table L: Outlier method Grubb’s test on data 1:10 NOTA-somatropin to hGHAb interaction
(continued).
Channel 4 Channel 5
Kobs-y G Kobs-y G
-0.007090913 1.997535272 -0.006434581 2.505344563
0.000530287 0.14938384 0.001126491 0.43860637
0.001875488 0.528331597 0.000715563 0.27860891
0.007930689 2.234102981 0.004314635 1.679930198
-0.001024111 0.288495621 0.000723707 0.281779716
-0.001713308 0.48264502 2.9994E-05 0.011678357
-0.000843308 0.237562948 2.9994E-05 0.011678357
-0.000102907 0.028989317 0.000318138 0.123868926
0.001542695 0.434582518 0.000425353 0.165613983
-0.001371302 0.386300725 -0.001759287 0.684989636
0.000266692 0.07512797 0.000509994 0.19856938
-1.97128E-18 -4.73106E-19
0.003549831 0.002568342
The critical G value (P=0.05) for a sample size 11 is 2.3547. The outliers as identified by the Grubb’s test and Dixon’s test are shown in blue.
Attachments
y = 0.0000158x + 0.0114211 R² = 0.6107206
0.0000
0.0100
0.0200
0.0300
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 1
y = 0.0000081x + 0.0146637 R² = 0.5473450
0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 2
y = 0.0000105x + 0.0111626 R² = 0.4597031 0.0000
0.0050
0.0100
0.0150
0.0200
0.0250
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 3
y = 0.00000501x + 0.01288435 R² = 0.55687816
0.0000
0.0050
0.0100
0.0150
0.0200
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 4
y = 0.00000440x + 0.01026988 R² = 0.52143752
0.0000
0.0050
0.0100
0.0150
0.0200
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 5
Attachments
Attachment 9: Data analysis of duplication 1:10 NOTA-somatropin to hGHAb using
GraphPad.
Experiment performed on 22/03/2012.
Table M: Data after fitting in GraphPad.
Conc (nM) Kobs channel 1 Kobs channel 2 Kobs channel 3 Kobs channel 4 Kobs channel 5
75 4.20E-07 0.001819 0.002199 0.002576 0.005347
100 0.004344 0.005949 0.005177 0.00551 0.009525
150 0.006284 0.007567 0.007159 0.007006 0.01305
200 0.001212 0.005516 0.005115 0.005628 0.01209
250 0.006827 0.008483 0.007852 0.008513 0.01645
300 0.00691 0.008443 0.007673 0.008087 0.01701
500 0.009186 0.01076 0.0104 0.01117 0.01933
500 0.009107 0.01083 0.01055 0.01149 0.01974
550 0.00866 0.01058 0.01047 0.01143 0.02003
600 0.009274 0.01117 0.01054 0.01158 0.02067
750 0.01065 0.01266 0.01212 0.0135 0.02208
900 0.01219 0.0146 0.0141 0.01573 0.02392
1000 0.01324 0.01545 0.01538 0.01619 0.02423
500 0.01507 0.01772 0.0195 0.02092 0.02504
Attachments
Table N: Statistical analysis of the non-linear regression on the association curves of duplication of 1:10 NOTA-somatropin to hGHAb interaction.
Channel 1
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1)
403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9828 0.9756 0.9436 0.9738 0.936 0.9424 0.9757 0.9708 0.9587 0.9738 0.9853 0.9917 0.991 0.9875
Abs SS (2) 0.04613 0.02289 0.03624 0.06099 0.07706 0.0925 0.08159 0.08864 0.1021 0.08169 0.0519 0.03757 0.04087 0.02457
Sy.x (3) 0.0107 0.007537 0.009482 0.0123 0.01383 0.01515 0.01423 0.01483 0.01591 0.01424 0.01135 0.009656 0.01007 0.007809
Channel 2
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 402 402 402 402 402 402 402 402 402 402 402 402 402 402
R2 0.9919 0.9853 0.9787 0.9866 0.9801 0.984 0.9917 0.9914 0.9888 0.992 0.9921 0.9917 0.9899 0.9864
Abs SS (2) 0.005732 0.008283 0.0093 0.0136 0.01452 0.01559 0.01657 0.01547 0.01702 0.01533 0.01731 0.02344 0.02939 0.01839
Sy.x (3) 0.003776 0.004539 0.00481 0.005816 0.006009 0.006228 0.006421 0.006204 0.006506 0.006175 0.006562 0.007636 0.008551 0.006764
Channel 3
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9922 0.9846 0.9805 0.9878 0.9792 0.9868 0.9912 0.9922 0.9912 0.9941 0.9926 0.9906 0.9859 0.9781
Abs SS (2) 0.003093 0.004192 0.0035 0.005907 0.005734 0.005397 0.007577 0.005989 0.005836 0.00511 0.007632 0.013 0.02066 0.01261
Sy.x (3) 0.00277 0.003225 0.002947 0.003828 0.003772 0.00366 0.004336 0.003855 0.003805 0.003561 0.004352 0.00568 0.00716 0.005594
Channel 4
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9938 0.9885 0.9844 0.9905 0.9868 0.9928 0.9928 0.9937 0.9931 0.9943 0.9931 0.9918 0.9882 0.9838
Abs SS (2) 0.001911 0.002552 0.002203 0.003319 0.002596 0.002165 0.004363 0.003351 0.003314 0.003586 0.005044 0.008248 0.01292 0.00683
Sy.x (3) 0.002178 0.002516 0.002338 0.00287 0.002538 0.002318 0.00329 0.002884 0.002868 0.002983 0.003538 0.004524 0.005662 0.004117
Channel 5
75 nM 100 nM 150 nM 200 nM 250 nM 300 nM 500 nM 500 nM 550 nM 600 nM 750 nM 900 nM 1000 nM 500 nM
Dgr. free. (1) 403 403 403 403 403 403 403 403 403 403 403 403 403 403
R2 0.9911 0.9892 0.9924 0.9872 0.9892 0.9886 0.9877 0.9887 0.9883 0.9876 0.9873 0.9869 0.9853 0.9867
Abs SS (2) 0.01085 0.01514 0.01059 0.03034 0.02313 0.02958 0.05324 0.04642 0.04308 0.05468 0.06068 0.0754 0.08755 0.04745
Sy.x (3) 0.005188 0.00613 0.005125 0.008677 0.007576 0.008567 0.01149 0.01073 0.01034 0.01165 0.01227 0.01368 0.01474 0.01085
(1) Degrees of freedom (2) Absolute sum of squares (3) Standard error or estimate
Attachments
Table O: Outlier method Grubb’s test on data duplication of 1:10 NOTA-somatropin to hGHAb interaction.
Concentration Channel 1 Channel 2 Channel 3
(in nM) Kobs-y G Kobs-y G Kobs-y G
75 -0.003450776 1.937018337 -0.00371 1.790517098 -0.0030379 1.084798224
100 0.000635017 0.164351418 0.0001152 0.050197119 -0.0003647 0.130213939
150 0.002059442 0.453651448 0.0011236 0.40859954 0.00100778 0.359871016
200 -0.003528132 1.970580098 -0.001537 0.801851477 -0.0016458 0.58769643
250 0.001571293 0.241862509 0.0008205 0.270686014 0.00048165 0.171992987
300 0.001138719 0.054185212 0.0001709 0.024840751 -0.0003069 0.109597875
500 0.00135242 0.146902108 4.967E-05 0.080012276 -1.818E-05 0.006493559
500 0.00127342 0.112627077 0.0001197 0.048165087 0.00013182 0.047070242
550 0.000310846 0.304996631 -0.00074 0.439233421 -0.0005578 0.199168512
600 0.000409271 0.262293655 -0.000759 0.448135483 -0.0010973 0.391843465
750 0.000238548 0.336364025 -0.001098 0.602230429 -0.001346 0.480652282
900 0.000231824 0.339281166 -0.000987 0.551593444 -0.0011947 0.426624298
1000 0.000250675 0.3311025 -0.001356 0.719534319 -0.0011339 0.404889322
500 0.00723642 2.699741139 0.0070097 3.086508252 0.00908182 3.24304366
Average (Kobs-y) 0.001013828
0.0002255
0
St. Dev. 0.002304885
0.002198
0.0028004
Table O: Outlier method Grubb’s test on data duplication of 1:10 NOTA-somatropin to hGHAb
interaction (continued).
Channel 4 Channel 5
Kobs-y G Kobs-y G
-0.002972253 1.011262784 -0.005712859 2.004031402
-0.000374658 0.127471724 -0.001974643 0.692691081
0.00044853 0.152605258 0.000670789 0.23530821
-0.001602282 0.54515165 -0.001168779 0.409999446
0.000609906 0.207511022 0.002311654 0.810912063
-0.000488906 0.166342663 0.001992086 0.698809883
-9.71535E-05 0.033054957 0.000793814 0.27846461
0.000222847 0.075820076 0.001203814 0.422289772
-0.000509965 0.17350779 0.000614247 0.215473461
-0.001032777 0.351386416 0.000374679 0.131434728
-0.001131213 0.38487761 -0.000854025 0.299585942
-0.000919648 0.312896116 -0.001652728 0.57976559
-0.001805272 0.61421585 -0.003101864 1.088112341
0.009652847 3.284231204 0.006503814 2.281493074
-1.73472E-18
1.23909E-18
0.00293915
0.002850683
The critical value of G (P= 0.05) for a sample size 14 is 2.5073. The outliers as identified by the Grubb’s test and Dixon’s test are shown in blue.
Attachments
y = 0.0000116x + 0.0023065 R² = 0.8115255
0.0000
0.0050
0.0100
0.0150
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 1
y = 0.0000118x + 0.0042091 R² = 0.8930930
0.0000
0.0050
0.0100
0.0150
0.0200
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 2
y = 0.0000118x + 0.0038016 R² = 0.9226857
0.0000
0.0050
0.0100
0.0150
0.0200
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 3
y = 0.00001304x + 0.00398535 R² = 0.94134953
0.0000
0.0050
0.0100
0.0150
0.0200
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 4
y = 0.0000173x + 0.0093675 R² = 0.8472319
0.0000
0.0100
0.0200
0.0300
0 200 400 600 800 1000 1200
Ko
bs
Concentration analyte (nM)
Channel 5
Attachments
Attachment 10: Calculation of the weighted mean of the GraphPad analysis data.
Table P: Calculation of the weighted mean of somatropin.
KD SE RSD weight (X-wm)2 (X-wm)2 * weight
channel 1 1276.37 588.855 46.14 0.69 201609 139109.281
channel 2 1424.19 706.3198 49.59 0.64 90715 58226.61832
channel 3 1579.337 1083.576 68.61 0.46 21328 9895.716343
channel 4 1899.995 1307.653 68.82 0.46 30491 14102.87696
channel 5 1519.302 483.6397 31.83 1.00 42468 42467.58191
Weighed mean 1511.711
som 263802.0746
som weights *(N'-1/N) 2.606689917
weighed SD 318.1225223
Table Q: Calculation of the weighted mean of 1:1 NOTA-somatropin.
KD SE RSD weight (X-wm)
2 (X-wm)
2 * weight
channel 1 259.8733 138.2474 53.20 0.21 2147707 445472.6541
channel 2 538.2794 67.8706 12.61 0.88 1409205 1233223.287
channel 3 623.196 77.5821 12.45 0.89 1214807 1076742.53
channel 4 795.5913 115.9327 14.57 0.76 864505 654625.5996
channel 5 979.4373 108.0732 11.03 1.00 556429 556428.9316
Weighed mean 713.6688
som 3966493.002
som weights *(N'-1/N) 2.98088946
weighed SD 1153.534029
Table R: Calculation of the weighted mean of 1:3 NOTA-somatropin.
KD SE RSD weight (X-wm)2 (X-wm)2 * weight
channel 1 550.6046 90.4539 16.43 0.41 1380095 572410.037
channel 2 560.2892 96.2198 17.17 0.40 1357434 538582.6449
channel 3 188.3956 59.984 31.84 0.21 2362318 505544.5702
channel 4 459.6262 67.4977 14.69 0.46 1602130 743360.2813
channel 5 516.5545 35.1967 6.81 1.00 1461257 1461256.529
Weighed mean 490.3784
som 3821154.062
som weights *(N'-1/N) 1.9916102
weighed SD 1385.144567
Table S: Calculation of the weighted mean of 1:10 NOTA-somatropin.
KD SE RSD weight (X-wm)2 (X-wm)2 * weight
channel 1 724.2986 250.785 34.62 1.00 1002162 998757.9745
channel 2 1808.001 650.5978 35.98 0.96 6826 6546.078126
channel 3 1062.321 455.4638 42.87 0.80 439646 353843.4559
channel 4 2571.057 887.1927 34.51 1.00 715171 715170.8723
channel 5 2336.167 811.9365 34.76 0.99 373062 370398.0731
Weighed mean 1725.379
som 2444716.454
som weights *(N'-1/N) 3.802594425
weighed SD 801.8151248
Attachments
Table T: Calculation of the weighted mean of 1:10 NOTA-somatropin.
KD SE RSD weight (X-wm)2 (X-wm)2 * weight
channel 1 316.1309 95.0929 30.08 0.51 1985980 1006158.216
channel 2 357.8182 67.4201 18.84 0.81 1870222 1512651.306
channel 3 322.2184 54.7485 16.99 0.90 1968860 1765897.049
channel 4 305.6636 46.5818 15.24 1.00 2015592 2015591.649
channel 5 553.7098 123.1743 22.25 0.69 1372809 940469.3216
Weighed mean 365.2577
som 7240767.541
som weights *(N'-1/N) 3.117937645
weighed SD 1523.907441
Anova test of the last three experiments
Anova: Single Factor
SUMMARY Groups Count Sum Average Variance
4 5 2275.4701 455.09402 23778.56882 5 5 3196.3773 639.27546 73637.36785 6 5 1855.5409 371.10818 10803.92004
ANOVA Source of Variation SS df MS F P-value F crit
Between Groups 188150.1904 2 94075.09519 2.607888184 0.114690594 3.885293835
Within Groups 432879.4268 12 36073.28557
Total 621029.6172 14
Attachments
Attachment 11: The reporting threshold limit for reporting, identification and
qualification of organic impurities in peptides obtained by chemical
synthesis.