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© Janne Olsen Frenvik, 2016
Series of dissertations submitted to the
Faculty of Mathematics and Natural Sciences, University of Oslo
No. 1790
ISSN 1501-7710
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
Contents
1 Acknowledgement ...................................................................................................... 12 List of abbreviations ................................................................................................... 33 List of papers .............................................................................................................. 64 Abstract ...................................................................................................................... 75 Thesis at a glance ....................................................................................................101 Introduction ..............................................................................................................132 Background ..............................................................................................................14
2.1 History of Radiopharmaceuticals .......................................................................142.2 Production and development of radiopharmaceuticals at Institute of Energy Technology (Kjeller, Norway) and the history of Xofigo® ............................................162.3 Targeted radionuclide therapy ...........................................................................172.4 Choice of radionuclide for therapy .....................................................................202.5 Radiolabelling considerations ............................................................................222.6 High versus low LET radiation in targeted radionuclide therapy .......................252.7 Radiation induced cell deaths ............................................................................302.8 Clinical radioimmunotherapy .............................................................................312.9 Radionuclide purity of radiopharmaceuticals .....................................................382.10 Targeted alpha therapy and in vivo generators ..............................................392.11 Selected methods for column separation and purification of thorium(IV) in literature and the established method for purifying decayed 227Th at Bayer ...............432.12 Targeted Thorium Conjugates (TTCs) and the 227Ac-227Th-223Ra technology platform 45
2.12.1 Product life cycle and manufacture of TTCs at Bayer .............................452.12.2 Preclinical and clinical studies of 227Th ....................................................482.12.3 Ingrowth of 223Ra and toxicological data ..................................................50
3 Aim ...........................................................................................................................514 General experimental presentation ..........................................................................52
4.1 Sorption materials ..............................................................................................524.2 High purity germanium gamma-ray spectroscopy .............................................544.3 Statistical methods ............................................................................................55
4.3.1 Design of Experiments; variables and tested range ...................................554.3.2 Determination of significant main and two-interaction variables and predictive ability of models .......................................................................................56
5 Additional data .........................................................................................................57
5.1 Sorption of 223Ra and daughters ........................................................................575.2 Batch method; sorption of 223Ra after 60 versus 180 minutes equilibration time 60
6 Discussion ................................................................................................................616.1 Important parameters for the development of an in situ purification method of 227Th …………………………………………………………………………………………616.2 Continuous removal of 223Ra during product shelf-life versus removal immediately prior to patient dose administration .........................................................646.3 Use of micro-spin columns ................................................................................676.4 Purification of decayed 227Th by the method in Paper II versus the established purification procedure at Bayer ...................................................................................696.5 PSA strong cation exchange resin packed on micro-spin columns; method development and material considerations ...................................................................716.6 Purification of decayed 227Th versus purification of decayed TTC ....................736.7 Purification methods for protein biotherapeutics like monoclonal antibodies and strategies for TTC purification .....................................................................................766.8 Purification of decayed 227Th; sorption of 223Ra versus other short-lived daughter nuclides ........................................................................................................806.9 Statistical models ...............................................................................................82
6.9.1 Model uncertainties .....................................................................................826.9.2 Future DoE application ...............................................................................836.9.3 Tested radioactivity levels and TTC product requirements .........................846.9.4 Statistical models and radiochemical purity of TTC ....................................84
7 Main conclusions and (summary of) suggestions for further research .....................858 References ...............................................................................................................889 Patents .................................................................................................................. 10910 Paper I-III ........................................................................................................... 109
Page | 1
1 Acknowledgement
The work presented in this thesis started at Algeta in 2012 through an industrial PhD
grant from the Research Council of Norway. The project was established as a
collaboration with the School of Pharmacy, University of Oslo. Algeta’s research and
development activities focused on alpha-particle emitting radiopharmaceuticals, and I
was very luck to start my career as a scientist within this exciting and important field.
My sincere gratitude goes to my supervisors Solveig Kristensen and Olav B. Ryan.
Thank you for supporting me, always believing in me and making this journey full of
memories and learning that I shall never forget.
I would like to thank my many good colleagues and friends at Research & Development
Algeta/Bayer; Dessi, Ellen, Lene, Kristine, Hanne, Hong, Katrine, Christine, Sara, Olav,
Åsmund, Jørgen, Liv-Ingrid, Jenny, Alan, Roger, Gro, Lars, Urs. And my new
colleagues at Technology Development, Bayer; Judit, Jan Roger, Georg and especially
my manager Dimitrios. I am so lucky to have such good colleagues and friends who
have supported and encouraged me through these years and hopefully many more to
come.
I would also like to thank Anne Kjersti Fahlvik, Executive Director at the Research
Council of Norway, who, as my mentor in 2011, encouraged me to leave my position as
a QP and pursue the dream of being a scientist. A decision I have never regretted.
Without Anne Kjersti and Lars Abrahamsen, who at the time had a managing position at
Algeta, the project would not have been initiated.
Page | 2
My sincere thanks also to my friends and family for encouraging and supporting me
during these years. Especially thanks to Heidi for making me food and showing me love
and support during times of heavy work load.
It feels as though a special chapter in my life is coming to an end, but I am very
thankful. I am also sure that many more interesting ones will be written thanks to all the
experiences and learnings I have gained.
Janne, September 2016
Page | 3
2 List of abbreviations
ALARA As Low As Reasonably Achievable
API Active Pharmaceutical Ingredient
Regression coefficient
cGMP current Good Manufacturing Practices
CI Confidence Interval
CT Computed Tomography
DOE Design of Experiments
DOTA 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
DMPS 2,3-dimercapto-1-propanesulfonic acid
DMSA Meso-2,3-dimercaptosuccinic acid
DSPG Distearoyl phosphatidylglycerol
DTPA Diethylenetriaminepentaacetic acid
DTPMP [[(Phosphonomethyl)imino]]bis[[2,1-ethanediylnitrilobis(methylene)]]tetrakis-
phosphonic acid
EDTA Ethylenediaminetetraacetic acid
EDTMP Ethylene diamine tetramethylene phosphonate
EPR Enhanced Permeability and Retention
FDA US Food and Drug Administration
H2O2 Hydrogen peroxide
HCl Hydrochloric acid
Page | 4
HEPES (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)
piperazine-N -(2-ethanesulfonic acid)
HNO3 Nitric acid
HPGe High Purity Germanium
HSE Health, Safety and the Environment
IE Ion Exchange
IFE Institute of Energy Technology (Institutt for energiteknikk)
iTLC instant Thin-Layer Chromatography
LET Linear Energy Transfer
mAb Monoclonal Antibody
MLR Multiple least square linear regression
MRI Magnetic Resonance Imaging
MVA Multivariate Analyses
MWCO Molecular Weight Cut-Off
p p-value
pABA para-aminobenzoic acid
PEG Poly-(ethyleneglycol)
PET Positron Emission Tomography
pI isoelectric point
pka acid dissociation constant
PLSR Partial Least Square Regression
Page | 5
PSA Propyl Sulfonic Acid
R Correlation coefficient
Rcf Relative centrifugal force
RCP Radiochemical Purity
RIC(s) Radioimmunoconjugate(s)
RNP Radionuclide Purity
RSD Relative Standard Deviation
SEC Size-Exclusion Chromatography
SD Standard Deviation
SPE Solid Phase Extraction
SPECT Single photon emission computed tomography
T t-value
TAT Targeted Alpha Therapy
TTC(s) Targeted Thorium Conjugate(s)
Page | 6
3 List of papers
Paper I
Development of separation technology for the removal of radium-223 from decayed thorium-227 in drug formulations. Material screening and method development
Janne Olsen Frenvik, Solveig Kristensen, Olav B. Ryan
Drug Dev. Ind. Pharm., 42 (2016) 1215-1224
Paper II
Development of Separation Technology for the Removal of Radium-223 from Targeted Thorium Conjugate Formulations
Part I: Purification of Decayed Thorium-227 on Cation Exchange Columns
Janne Olsen Frenvik, Knut Dyrstad, Solveig Kristensen, Olav B. Ryan
Drug Dev. Ind. Pharm., Ahead of print (2016), DOI: 10.1080/03639045.2016.1234484
Paper III
Development of Separation Technology for the Removal of Radium-223 from Targeted Thorium Conjugate Formulations
Part II: Purification of Targeted Thorium Conjugates on Cation Exchange Columns
Janne Olsen Frenvik, Knut Dyrstad, Solveig Kristensen, Olav B. Ryan
Submitted to Journal of Labelled Compounds and Radiopharmaceuticals (September 2016)
Page | 7
4 Abstract
This thesis comprises development of a new in situ purification method related to the
technology platform with Targeted Thorium Conjugates (TTCs) in Bayer. TTCs are
radiopharmaceuticals where the alpha-emitting radionuclide thorium-227 (227Th, 18.7
days half-life) is connected to a targeting moiety, e.g. an antibody. These products have
the potential to give efficient and specific therapy for various cancers dependent on the
type of targeting moiety. Long-lived daughter nuclide radium-223 (223Ra, 11.4 days half-
life) is formed from the radioactive decay of 227Th. This radionuclide will not be linked to
the targeting moiety and will thus not have the same targeting abilities as the TTC. It is
therefore necessary to have available a purification method with a standardized level of
radionuclide sorption and good selectivity between 223Ra and 227Th/TTC. Other
important aspects in developing a new purification method include user-friendliness, use
of non-hazardous materials, and minimization of the time and resources required for the
operation. In this thesis three studies have been conducted with subject of sorption and
separation of 223Ra and 227Th/TTC.
In the first study several materials were screened for their ability to retain 223Ra and 227Th. For selected materials both passive diffusional sorption by batch method
(materials as suspensions) and sorption on gravity columns were tested. The screening
matrix consisted of organic and inorganic materials, i.e. strontium and calcium alginate
gel beads, distearoyl phosphatidylglycerol (DSPG) liposomes, ceramic hydroxyapatite,
Zeolite UOP type 4A and cation exchange resins AG50W-X8 and SOURCE 30S. The
sorption of 223Ra by passive diffusion ranged from 31% to 95%, with the DSPG
liposomes demonstrating superiority at 95% sorption. Sorption on gravity columns by
the cation exchange resins and ceramic hydroxyapatite was shown to be immediate and
nearly quantitatively with minimal variation. The compatibility of the materials with
trastuzumab (used as a model antibody) by batch method was further tested, and
Zeolite UOP type 4A, AG50W-X8 resin and DSPG liposomes showed the lowest
interaction with 10% reduction of antibody concentration. Impact on measured
Page | 8
hydrogen peroxide level in solution (as an indication of the level of radiolysis in the
sample) by the presence of ceramic hydroxyapatite was further studied. The measured
H2O2 level formed during 14 days storage was significantly lower in samples with than
without ceramic hydroxyapatite.
Purification of decayed 227Th (227Th with presence of daughter nuclide 223Ra) by cation
exchange and column method was explored in the second study. The goal was to have
a good separation with high 223Ra and low 227Th sorption on micro-spin columns packed
with a selected cation exchange resin (propyl sulfonic acid (PSA), silica based). This
was studied through a design of experiments (DOE) with formulation and process
parameters interpreted by multivariate analyses and development of statistical
regression models. The purified 227Th was further tested for radiolabelling of a model
antibody-chelator conjugate (trastuzumab (for preparation of a TTC).
The cation exchange resin and micro-spin columns were further used to explore the
sorption of 223Ra from TTC (radiolabelled with decayed 227Th) in the third study. The
target was a good selectivity with high 223Ra and low TTC sorption. The DOE
formulation and process variables were equivalent to the second study, with the
exception of inclusion of sodium chloride concentration as an additional parameter.
The sorption of 223Ra can be high (>90%) on micro-spin columns both when purifying
decayed 227Th and TTC radiolabelled with decayed 227Th. The sorption was influenced
by formulation and process parameters, which can be utilized in order to obtain a high 223Ra and low 227Th/TTC sorption. However, the sorption of 223Ra from TTC is more
complex with a greater compromise between high 223Ra uptake (>90%) and low TTC
uptake (<25%) than sorption of free radionuclides where both a low 227Th sorption
(<3%) and high 223Ra sorption (>90%) could be obtained. In addition, stability issues of
the TTC must be taken into consideration when purifying TTC.
Page | 9
The development of a new in situ purification method for TTCs requires evaluation of
parameters relating to the TTC product, method as well as materials used. The column
method is concluded to be the most feasible due to the higher and less variable
sorption, as well as the perceived less complexity with regard to technology
development compared to the batch method. Other columns than the micro-spin
columns used in this thesis should, however, be tested, as their use involves a too high
risk of radioactive contamination.
For purification of decayed 227Th, the ion exchange procedure with formulation buffers is
judged to have the potential to be developed into a more user-friendly in situ purification
method compared to the established procedure at Bayer with multiple steps and use of
strong acids. The ion exchange purification of TTC is on the other hand more complex
and relatively high sorption of TTC was shown. For in situ purification of TTCs, other
methods utilizing size-exclusion chromatography is recommended for further
exploration.
The work in this thesis has resulted in three patent applications. The first patent (patent
application granted) comprises materials for sorption of 223Ra studied in Paper I, while
the two latter patents comprise the purification technology developed in Paper II and III,
respectively.
Page | 10
5 Thesis at a glance
Paper Objective Method Illustration Main findings/ conclusions
I) Development of separation technology for the removal of radium-223 from decayed thorium-227 in drug formulations. Material screening and method development
Screening
of materials
for their
ability to
sequester 223Ra as
well as
methods for
doing this.
Passive
diffusional
uptake of 223Ra with
materials as
suspensions/
by batch
method and
selected
materials on
gravity
columns.
All the materials
retained 223Ra by
passive diffusion
(31 to 95%). All
materials suitable
for assessment by
column method
retained 223Ra
almost
quantitatively
(~100%) and with
minimal variation
(RSD <1%).
The sorption was
significantly higher
compared to
passive diffusional
sorption for the
materials tested by
both methods.
Page | 11
Paper Objective Method Illustration Main findings/ conclusions
II) Development of Separation Technology for the Removal of Radium-223 from Targeted Thorium Conjugate Formulations Part I: Purification of Decayed Thorium-227 on Cation Exchange Columns
To study
the sorption
and
separation
of 223Ra
and 227Th
by ion
exchange
resin as
influenced
by
formulation
and
process
parameters
Sorption of 227Th and223Ra on
micro-spin
columns
packed with
PSA strong
cation
exchange
resin.
Statistical
experimental
design with
formulation
and process
parameters
interpreted by
the aid of
multivariate
data analysis.
The statistical
models for both
citrate and acetate
buffered
formulations show
the potential for
high sorption of 223Ra (>90%) and
low sorption of 227Th (<3%) by the
optimization of
formulation and
process
parameters.
Page | 12
Paper Objective Method Illustration Main findings/ conclusions
III) Development of Separation Technology for the Removal of Radium-223 from Targeted Thorium Conjugate Formulations Part II: Purification of Targeted Thorium Conjugates on Cation Exchange Columns
Study the
sorption
and
separation
of 223Ra
and TTC by
ion
exchange
resin as
influenced
by
formulation
and
process
parameters
Sorption of 223Ra and TTC
on micro-spin
columns
packed with
PSA strong
cation
exchange
resin.
Statistical
experimental
design with
formulation
and process
parameters
interpreted by
the aid of
multivariate
data analysis.
Evaluation of
radiochemical
purity of the
TTC.
The sorption of 223Ra and TTC was
a compromise
between low TTC
sorption (<25%)
and high 223Ra
sorption (>90%) in
both citrate and
acetate buffered
formulations.
Stability studies of
radiochemical
purity (RCP)
indicated that the
observed TTC
sorption may be
partly due to free 227Th, but RCP of
the TTC was
affected by
formulation
parameters.
Page | 13
1 Introduction
Several radiopharmaceuticals within targeted alpha-therapy (TAT) are investigated for
their potential use in cancer therapy [1-7]. Large cellular destruction may be achieved
by alpha-particles due to their high energy deposit within tissues [8, 9]. For eradication
of cells or tissue, the radionuclide needs to be located into or very close to the cancer
target, leading to a low eradication of normal health cells [10].
By using targeting moieties, like antibodies which are specific for an antigen expressed
by the tumor cells, targeting of the alpha-particle to tumor tissues may be achieved
(radioimmunotherapy). The likeliness of destruction of healthy tissues is thereby further
decreased by the specificity of the antibody when injecting the radiolabelled antibody
into the patient’s veins [11, 12].
Health authorities approved the first radiolabelled antibodies for the treatment of non-
Hodgkin’s lymphoma in 2002 and 2003 (Zevalin® and Bexxar®) [13]. However, these
radiopharmaceuticals emit beta-particles which have a longer range and lower energy
than alpha-particles. Xofigo® (radium Ra 223 dichloride) injection (Bayer Pharma AG),
which is used in the treatment of bone metastases in castration-resistant prostate
cancer, is the first TAT approved by the US Food and Drug Administration and in the
European Union (in 2013) [14].
Targeted Thorium Conjugates (TTCs) are currently explored as a new approach to TAT
in Bayer. In the approach the alpha-emitter thorium-227 (227Th) is combined with
targeting molecules like selected monoclonal antibodies. A chelator is necessary to
radiolabel the antibody with 227Th and prepare the TTC. Practically no unbound 227Th
will be present after radiolabelling due to the affinity of 227Th to the utilized in-house
octadentate hydroxypyridinone class derived chelator [15, 16]. However, radium-223
Page | 14
(223Ra) and other short lived progenies are formed from the decay of 227Th. These will
not be attached to the targeting moiety due to the high recoil energies which are
released during the radionuclide decay. 223Ra will thereby not have the same tumor
targeting as the TTC [17, 18]. The tolerance of 223Ra is known to be good from studies
conducted in the development of Xofigo® [19]. Preclinical TTC studies also indicate a
good tolerance of 223Ra (non-published in-house data). It is, however, necessary to
standardize the level of this radionuclide before i.v. injection both due to safety and
dose calculation considerations
In this thesis the development of a new in situ purification method to retain 223Ra, as
part of the TTC preparation procedure, has been explored. Materials have been
screened for sorption of 223Ra, methods for doing this have been developed and the
impact of formulation and process parameters on separation of 223Ra from 227Th and
TTC has been explored. The goal of the purification method is to achieve a high
separation and to maximize 223Ra sorption while limiting the sorption of 227Th, the latter
either as free radionuclide or radiolabelled antibody/ TTC (depending on the approach
for the TTC preparation).
2 Background
2.1 History of Radiopharmaceuticals
Two important breakthroughs for new treatments of malignant diseases were made by
Wilhelm Conrad Röntgen in 1895 and his discovery of X-rays and some months later by
Henry Becquerel when he discovered natural radioactivity [20]. In 1896 the first clinical
exploration of such treatments was performed by Emil Grubbé when he treated breast
cancer with X-rays [20, 21]. Marie Curie discovered radium in 1898, and this pioneering
work led to the growth of the field of radiation therapy in the early 1900s [20, 22].
Page | 15
Treatment was performed on a large range of diseases from skin and breast cancer to
epilepsy and syphilis [22]. Sealed sources of radium-226 and radon-222 were used
already in 1915. One way of using radon-222 (then referred to as radium emanation)
was through inhalation to treat diseases of the lungs. Radium salt dilutions were
prescribed either to be used internally or they were placed in metal tubes to be used
externally [23]. Efforts were thereafter put into understanding the underlying
mechanisms of how radiation could cause such distinct biological effects on cells ([24].
Remote source handling techniques and availability of reactor produced radionuclides
caused a more widespread use of radiotherapy by the 1950s [25]. Today, radionuclides
are used both for diagnostic and therapeutical purposes. Information about
physiological and biochemical processes can be provided by nuclear diagnostic
techniques such as gamma imaging (single photon emission computed tomography
(SPECT) and positron emission tomography (PET) [25]. Sodium iodine labelled with
iodine-131 (131I-NaI) is at present the most commonly used therapeutical radionuclide.
In radioactive iodine therapy, the radionuclide is administered as a capsule or in liquid
form and is used to treat thyroid-related diseases due to accumulation of iodine in the
thyroid gland [26, 27]. Bone metastasis is another major application of targeted
radionuclide therapy with the use of radionuclides like strontium-89 and samarium-153,
as these radionuclides accumulates in diseased bone [26]. Radioimmunotherapy
(molecular targeting radionuclide therapy) in which a radionuclide is chemically attached
to an antibody for targeting before the radiopharmaceutical is injected into the
bloodstream, has been developed for more than 30 years [12, 26]. The number of new
antibodies which have been studied in clinical trials have increased, and positive results
have been shown particularly in non-Hodgkin’s lymphoma with the marketing approval
of 90Y-ibritumomab tiuxetan (Zevalin®) and 131I-tositumomab (Bexxar®) regimens [28].
Page | 16
2.2 Production and development of radiopharmaceuticals at Institute of Energy Technology (Kjeller, Norway) and the history of Xofigo®
At the end of 1952, the first radioactive isotopes were supplied to a hospital in Oslo (i.e.
Iodine-131). The nuclear reactor at Institute of Energy Technology (IFE) outside of Oslo
was one of the first in operation in Europe. The new products were classified as
radiopharmaceuticals in Norway, this in contrast to other countries in Europe where they
were classified as radioactive chemicals. Production, control and distribution of the
radiopharmaceuticals were associated with IFE. The first molybdenum/technetium-99m
generator in Norway was constructed in 1966 following progress in the development of
technical detection equipment, data processing and nuclear medicine in general. The
Norwegian technology spread to several countries in Europe and Asia over the next 30
years. Norway was also involved in producing three important monographs on
radiopharmaceuticals in the European Pharmacopoeia [29].
Today, IFE’s main activities within nuclear technology and health are control and
distribution of radiopharmaceuticals to Norwegian hospitals. IFE is also a contract
manufacturer for production and distribution of Xofigo® (radium Ra 223 dichloride)
injection (Bayer Pharma AG), the first approved alpha emitting radiopharmaceutical [14,
30]. A state of the art production facility has been built at IFE for the global supply of
Xofigo® [31].
The history of Xofigo® originates from the work of Roy Larsen (nuclear chemist from the
University of Oslo) and Øyvind Bruland (professor of oncology, Norwegian Radium
Hospital). Based on their research on alpha-emitting cancer therapeutics, they funded
Anticancer Therapeutic Inventions AS in 1997. Their target was to develop a new
pharmaceutical treatment for cancer patients with bone metastasis. The company was
subsequently built through key appointments, scientific development and financing
Page | 17
events and changed name to Algeta before it was listed on the Oslo stock exchange in
2007. In 2014, Bayer completed the acquisition of Algeta [32].
2.3 Targeted radionuclide therapy
Currently, local cancer therapies to remove primary tumors and large metastases
consist primarily of surgery and external beam therapy. There is a limited success of
systemic cytostatic therapies for prevention of growth of distant metastasis due to the
toxicity experienced by normal tissues within curative doses [33, 34].
Figure 1 External beam radiation therapy (a) versus targeted radionuclide therapy (b) where the radiopharmaceutical is intravenously injected [35].
In targeted radionuclide therapy, normal tissues can be spared by local irradiation at
doses able to kill the tumors (Figure 1). Treatment of several indications such as thyroid
carcinomas, bone metastases and neuroendocrine tumors are currently conducted by
the aid of radiopharmaceuticals and systemic radiation therapy [36]. Extensive research
is ongoing for the use of bio-vectors like monoclonal antibodies (mAbs), peptide
conjugates or other chemical compounds to transport radionuclides to the cancer cells
through selective targeting [12, 26, 28, 37].
Page | 18
Paul Ehrlich was the first to postulate the concept of targeted therapy when he
conceived the idea of the “magic bullet” as a therapeutic agent that attacked disease by
locating specific cellular targets [38, 39]. With the development of targeted therapies
within cancer, largely based on the use of designer mAbs, his vision is realized. Two
antigen-binding sites linked together via a variable region to a constant region
(consisting of light and heavy chains) comprise the typical antibody, see Figure 2. The
antigen-binding sites recognize and bind various molecules of the immune system and
molecules that determine the antibody’s biodistribution [40]. By being able to deliver
toxins, drugs, enzymes or radionuclides through conjugation to mAbs, the selectivity of
the therapies may increase significantly, and new forms of therapies may be delivered
[41-43]. Development of rodent mAbs with a single specificity towards antigens was
made possible by the hybridoma technology developed by Milstein and Köhler [44].
Figure 2 Schematic structure of an antibody showing the antigen-binding sites with a variable and constant region as well as the light and heavy chains of the antibody [45].
Numerous biopharmaceutical companies have exploited this technology with designer
mAbs to develop vehicles for delivery of radionuclides, both to image and treat various
kinds of cancers [11]. A schematic illustration of the preparation and mode of a
radioimmunoconjugate is given in Figure 3. Initial imaging of the tumor volume by the
aid of a radionuclide antibody can serve as an important diagnostic tool. Planar imaging,
Page | 19
positron emission tomography (PET) and single photon emission computed tomography
(SPECT) can be used to image the tumor. Also hybrid imaging systems can be used by
incorporating PET or SPECT with computed tomography (CT) or, as more recently
developed, hybridization of PET with magnetic resonance imaging (MRI) instruments
[46, 47]. When an appropriate amount of antibody is shown to be retained by the tumor
site through the use of these imaging techniques, a therapeutic dose of the same
antibody labelled with a radionuclide possessing abilities to kill the cancer cells may be
given [48-51].
Figure 3 Schematic illustration of the preparation and mode of action of a radioimmunoconjugate. The antibody-chelator conjugate consisting of a monoclonal antibody coupled to a chelator (in green, see section 2.5 ) is radiolabelled with a radionuclide, forming the radioimmunoconjugate. After i.v. injection, targeting to antibody specific antigen (purple triangle) on tumor cells and decay of radionuclide with tumor cell toxic radiation (alpha-particle emission shown here) leads to the wanted cancer cell specific effect. Adapted with modifications from [37].
Page | 20
The early clinical development of mAbs was not successful mainly due to their murine
(mouse) origin. Immune-complexes and other non-specific complexes were formed
which led to low tumor uptake, and in addition toxic immunogenic responses were
occasionally seen in patients. These shortcomings have been overcome by the use of
chimeric, humanized or fully humanized mAbs [48, 52].
Another challenge with radioimmunoconjugates is in the treatment of solid tumors,
where in the majority of cases, the delivered radiation dose to the tumor has not been
able to give sufficient effect without significant side-effects. It is therefore a need for
gathering quantitative pharmacokinetic information of radioimmunoconjugates, and to
compare the radiation dose delivered to the tumor sites to the dose delivered to normal
tissues. This provides the calculation of percentage of injected radiation dose per gram
of tissue (e.g. kBq/kg of bodyweight) to limit the damage to normal tissue [53-55].
Radio-immunotherapy (RIT) history was established by antibodies labelled with beta-
emitters, but years with variable results delayed the acceptance of its clinical role in
various cancers [56]. The radiolabelling of molecules with alpha emitters has been
proposed in recent years [57]. The use of beta and alpha emitters will be presented
further in sections 2.6, 2.7 and 2.8.
2.4 Choice of radionuclide for therapy
In radionuclide therapy the choice of radionuclide and (radio)labelling method (see
section 2.5) is just as important as the choice of the targeting vector (e.g. mAb in RIT).
Not only must the radionuclide be stably attached to e.g. the mAb, but a variety of
biological and pharmacological factors must also be taken into account. The goal is to
develop a radionuclide antibody-conjugate which delivers a high radiation dose to
malignant cells while sparing healthy cells within organs and tissues [26, 58].
Page | 21
General requirements of the physicochemical properties of radionuclides is summarized
in the following bullet points [59, 60];
• To exert cytotoxic action the radionuclide should emit particulate radiation in
sufficient amounts: alpha-particles, beta-particles, Auger electrons or conversion
electrons
• It is undesirable to have a high level of high-energy gamma components which
gives whole-body irradiation, but it might be advantageous to have low
abundance photons (100-200 keV) for imaging and therapy monitoring
• The physical half-life of the radionuclide should match that of the targeting agent
depending on the in vivo pharmacokinetics, i.e. there should be sufficient
radioactivity left for the time the targeting agent is in systemic circulation and at
the time of binding to the targeted site
• It should be possible to produce the radionuclide with a sufficient amount of
radioactivity and with an acceptable radionuclide purity profile
• The production of the radionuclide should be cost-efficient
• The labelling of targeting vectors like proteins and peptides should be done with
high yields and under acceptably mild conditions to provide a conjugate that is
stable in systemic circulation
• Accumulation of the radiocatabolites (i.e. metabolites of the radiolabelled
conjugate) in normal organs or tissues should be limited and they should be
quickly removed from the body
Both the physical and biochemical characteristics are important when evaluating the
clinical suitability of a radionuclide or radiopharmaceutical. Physical half-life, energy of
the radiation(s), type of emissions, daughter product(s), radionuclide purity and method
of production are important characteristics. In addition, retention of radioactivity in the
tumor, tissue targeting, in vivo stability and toxicity are important biochemical aspects.
[26, 61]. The physical half-life (t1/2) of the radionuclide is important both with regard to
delivery flexibility and the physiological retention of the radiation dose. A long t1/2 will
give a good flexibility but will make the patient radioactive for a longer period. The
Page | 22
biological half-life (tb) of the nuclide within the patient’s organs or body is determined by
the targeting moiety (e.g. antibody). tb is also important with regard to delivery of the
radiation to the target. The effective half-life (te), which considers both the physical and
biological half-life is the most important factor when choosing a radionuclide for therapy
(i.e. (te)= t1/2tb/( t1/2+tb). If the daughter nuclide of the radionuclide is radioactive, the total
amount of absorbed dose will also depend on this nuclide and must be considered in
the treatment plan. This is the case for the alpha-emitter 227Th utilized in this thesis,
where the decay to 223Ra leads to a chain of radioactive decays and a different targeting
profile by 223Ra. [26, 61].
2.5 Radiolabelling considerations
Regarding the radiolabelling of the targeting vector with the radionuclide, some general
requirements apply regardless of the chosen labelling strategy [62]:
• The labelling procedure should have a maximized yield due to the high cost of
radionuclides which contribute significantly to the overall price of the
radiopharmaceutical
• The requirements for specific radioactivity of the conjugate should be met (i.e.
radioactivity per mass of conjugate)
• A high radiochemical purity (ref section 6.1) should be provided by the labelling
and purification methods
• Target specificity should be preserved by the labelling methods
• Acceptable stability of the conjugate during storage, distribution and in vivo
should be provided by the labelling method
• Labelling and purification procedures should be done without extensive manual
handling or under remote control when high radioactive doses are given during
handling
Page | 23
Selection of method for labelling also depends on the distribution strategy of the
radiopharmaceutical. There are two approaches; either labelling at a centralized
dispensary and shipment to hospitals (e.g. as for Bexxar®), or labelling at hospitals
immediately before patient treatment (e.g. as for Zevalin®). The two approached differ
with regard to delivery flexibility, training and facility requirements at the site of patient
dose administration, as well as influence of radiolysis on the radiolabelled conjugate.
When designing the radiolabelling procedure for the conjugate, radiolysis must be taken
into consideration as the high level of radioactivity in therapeutic applications may
destroy the functionality of the conjugate [63-65]. High radionuclide concentration is
often required during labelling but subsequent dilution of the radiolabelled conjugate
significantly reduces radiolysis [66, 67]. Freezing of radiolabelled proteins may also
reduce radiolysis [68, 69]. Formulation of radiolabelled proteins with addition of ascorbic
acid and/or human serum albumin or other scavengers of free radicals could also be a
way to reduce radiolysis during storage [70-73].
Labelling of proteins and peptides with radionuclides usually requires the use of a
chelator which is a ligand covalently bound to the targeting vector, e.g. peptide. The
attached chelator further non-covalently binds the radionuclide. The chelator therefore
needs to be bifunctional. Different groups of metals require different chelators, and the
stability of the radiometal-bifunctional chelator complex should be high since a number
of plasma proteins have chelating abilities and are present in much larger quantities
than the chelator. Either macrocyclic (e.g. derivatives of DOTA) or acyclic (e.g.
derivatives of DTPA) polyaminopolycarboxylate chelators are commonly employed for
labelling of radiolanthanides like 177Lu, 111In and 90Y (ref section 2.6). DOTA and
derivates gives stable attachment of radionuclides, but elevated temperatures are
needed for labelling which can affect the stability of the targeting moiety. DTPA
derivates can be used for labelling at ambient temperature, however, the labelling is
less stable than for DOTA [62]. For TTCs, labelling with the utilized in-house
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Page | 25
radioimmunoconjugate Zevalin®, where 111In labelled ibritumomab tiuxetin is used for
imaging and biodistribution data and 90Y labelled ibritumomab tiuxetin is used for the
subsequent therapy [80-82].
While cellular processing aspects are usually the main factors when choosing labelling
method for proteins like mAbs, the biological kinetics is of greater importance for smaller
molecules like peptides [83]. Shifts of excretion pathways from the liver to the kidneys
have been demonstrated by use of more polar or charged chelators [84, 85].
2.6 High versus low LET radiation in targeted radionuclide therapy
The choice of radionuclides should also be based on the optimal range and energy of
radiation in the tissues, as also based on the size of the tumors. The average energy
deposited by a particle per unit track length traversed is called the linear energy transfer
(LET, in units keV/μm). Two types of radiation induced cellular inactivation by high and
low LET have been proposed; 1) lethal events by high LET radiation 2) sub-lethal
damage that is due to accumulation of multiple events repaired at low doses. Higher
doses could, however, saturate the cellular repair mechanisms for low LET radiation
[86]. Table 1 lists some selected high and low LET radionuclides with potential use in
targeted radionuclide therapy.
Page | 26
Table 1 Selected high (alpha- and auger-emitters) and low (beta-emitters) LET radionuclides with potential use in targeted radionuclide therapy, their physical half-life and average energy by emission [58, 61, 87, 88].
High/low LET Radionuclide Physical half-life Eavg,keV
High LET
Alpha-emitters 211At 7.2 h 5868 212Bi 1.0 h 6051 213Bi 45.6 min 1390 212Pb 10.6 h 6050 225Ac 10 days 5915 227Th 18.7 days 5900 223Ra 11.4 days 5979
Auger-emitters 125I 60.2 days 0.7-30
111In 2.8 days 0.5-25 201Tl 73.1 h 2.7-77
Low LET
Beta-emitters 90Y 64.1 h 935 131I 8.0 days 181
177Lu 6.7 days 140 67Cu 2.6 days 141
186Re 3.7 days 329 188Re 153Sm
17.0 h
1.9 days
795
225
Particles with a LET >10–30 keV/ m are called high LET particles and they deliver a
much more localized and energetic radiation than low LET particles. High LET emitting
radionuclides studied in clinical cancer therapy emit alpha-particles which are charged
nuclei of two protons and two neutrons (i.e. particle identical to helium nucleus).
Depending on particle energy, the LET of alpha particles range from 25 to 230 keV/μm.
A series of articles by Barendsen and colleagues in the 1960s established the
radiobiology of alpha particles and demonstrated the characteristic features of alpha-
particle radiation [89-95].
Page | 27
The typical range of alpha-emitters in tissues is 10-100 μm, which is well matched with
micrometastases [96, 97]. There is an increasing interest in using alpha-emitters in
cancer therapy, and preclinical and dosimetric studies have indicated that they may be
promising as an alternative to beta-emitters in radioimmunotherapy (i.e. low LET
particles), and to treat minimal residual disease where a small number of leukaemic
cells are present in the recovering patient during or after treatment [1, 98-101].
Compared to low LET beta-emitters, alpha-emitters are more mutagenic and toxic, but
the ability to irradiate significantly less volumes of normal cells compensate for these
unwanted properties [10].
Supply limitations, expenses and low availability of radionuclides with the suitable
physical and chemical properties have slowed down the progress in application of the
most popular alpha-emitters 211At, 213Bi and 225Ac [1, 102]. Alpha-emitters that can be
prepared from long term operating generators have therefore been an issue of
significant research activity [103, 104]. Examples include 223Ra and 227Th, which can be
produced in large amounts from 227Ac (t½ = 21.7 years) in a long term generator [103]. 227Ac can be produced by neutron irradiation of 226Ra in reactors relatively easily and in
large amounts [105]. 223Ra and 227Th are studied in this thesis, as they are the selected
radionuclides for therapeutic development at Bayer. 223Ra is utilized in the first marketed
alpha-emitting radiopharmaceutical Xofigo ® (Bayer Pharma AG) while 227Th is part of
the extensive research program of Targeted Thorium Conjugates (TTCs). Unlike for 227Th, the exploration of 223Ra in radioimmunotherapy has been hindered by the
unavailability of complexing agents for radium isotopes. But like strontium, radium is a
natural bone seeker and this quality has been utilized in the Xofigo® treatment regime
where skeletal events from castration-resistant prostate cancer are treated [18, 30].
Photons (gammas and x-rays) and electrons (shell-electrons and beta-particles) give
low LET radiation [106-108]. The cell killing capacity of low LET radiation is well
characterized at high dose-rates given by external radiotherapy with photons (0.5 -2.0
Page | 28
Gy/min) but less is known when applied in targeted radionuclide therapy (0.01-1.0 Gy/h)
[108-111]. Beta-particles have a LET in the range of 0.1-1.0 keV/μm [88, 112].
Alpha- and beta-particles differ with regard to the mass, electrical charge, kinetic energy
and penetration range in biological tissue, see Table 2. The specific cell killing efficiency
is also much higher for alpha-particles, with an energy deposition in the order of 3500
more per track length for alpha-particles [8, 9, 97].
Table 2 Mass, electrical charge, kinetic energy and penetration range in biological tissue of alpha- versus beta-particles [97].
Alpha-particle Beta-particle
Mass 4.0 amu 5.5*10-4 amu
Electrical charge 2+ 1-
Kinetic energy 5-9 MeV 200 keV
Penetration range in biological
tissue
10-100 μm
( 1-10 cell diameters)
5 mm
( 500 cell diameters)
These differences and the different LET properties further mean that the relative
biological effectiveness1 of high LET radiation is greater, mostly due to the higher
likeliness of causing double strand breaks of DNA. The damage of normal cells is
minimized for alpha-particles due to the low effective range. The cytotoxic effects of
high LET radiation are much less dependent on dose rate2 and cell cycle as well as the
presence of oxygen. Low LET radiation causes mainly damage to DNA indirectly
through ionization of other molecules and generation of free radicals, including oxygen
radicals [3, 58, 113].
1 A measure of the capacity of a specific ionizing radiation to produce a specific biological effect. 2 Amount of radiation dose absorbed to tumor per unit time.
Page | 29
Beta-particles may, however, be advantageous in radio-immunotherapy since the long
range in tissues may be sufficient to irradiate tumor cells also adjacent to the tumor cells
with bound radiolabelled antibody. This effect is often referred to as the crossfire or
bystander effect [58, 114, 115]. Therefore, a relatively uniform radiation dose can be
given by beta-emitters despite an inhomogeneous distribution and inadequate uptake of
radiolabelled antibodies in the tumor [116]. The longer effective range of beta-emitters
may on the other side cause toxicity, as irradiation of normal bone marrow (which is
considered to be the dose limiting organ in radio-immunotherapy) has been observed in
clinical practice, e.g. with Zevalin® and Bexxar® [117, 118].
Other implications and differences between alpha- and beta-particle emitting radio-
immunoconjugates include the potential need for dilution of the radiolabelled antibodies
in alpha-therapy prior to administration. This is due to a great excess of antibody
binding sites on cancer cell membranes compared to the number of isotopes needed to
kill the cells [52]. In addition, due to the short penetration range, small cell clusters and
circulating isolated cancer cells may be a better application for alpha-emitters [5].
Besides alpha- and beta particles, nuclides which exert the Auger effect have been
explored in targeted radionuclide therapy. The Auger effect is caused by energy
instability of the atom due to vacancies in the inner electron shells. Emission of many
low energy electrons as well as characteristic x-rays is occurring when the energy
balance is regained [119]. Multiple high LET ionizations (4-26 keV/μm) with short
penetration length in biological tissue (<100 nm) is produced by most Auger electrons
[120].
Due to the short penetration length, the use of the Auger effect in targeted radionuclide
therapy has been challenging as there is a need to target the DNA in the tumor cells
[119]. As an example, 125I has a high number of Auger electrons and can be easily used
Page | 30
for radiolabelling of biomolecules [119]. The nucleoside analogue (DNA directed agent)
5-iodo-2 -deoxyuridine (IdUR) has been labelled with 125I and studies revealed that it is
very radiotoxic to mammalian cells [121].
2.7 Radiation induced cell deaths
Ionizing irradiation may cause damage both to the DNA and cell membrane, leading to
cell death. Single and double strand breaks in the DNA may occur within the nucleus,
while cell death pathways may be activated by the damage to cell membranes. Cell
death by radiation is, however, complex and is a field of continuous evolvement and
redefinitions [122].
Cell death is due to a number of mechanisms like apoptosis, autophagy, necrosis,
senescence and mitotic catastrophe [123-134] . The degree of damage determines the
pathway as well as the percentage of unrepaired double strand breaks in DNA, which
will be highest for high LET radiation [8].
DNA has been accepted as the primary molecular target for high LET alpha-particles
[135]. This was shown in the work of Soyland and Hassfjell , where the cytotoxicity was
defined by the path of an alpha-particle through the cells [136]. The cytotoxic effect was
not present when the pathway was through the cytoplasm but was present when the
alpha-particle went through the nucleus. The cytotoxicity was also correlated to the
actual distance traveled through the nucleus. Beta-particle emissions and Auger
electrons in the cell membrane or cytoplasm did not cause these high alpha-particle
effects [137]. Many kinds of DNA damage are likely to occur from high LET radiation,
including double-strand breaks, chromosomal rearrangement and cross-linking, and
they add to the high efficiency of cell killing. The overall effect of alpha-particle radiation
may, however, not be solely attributed to DNA damage, as generation of increased
Page | 31
amounts of intracellular reactive oxygen species and mitochondrial involvement have
been included to explain the observed effects. The reactive oxygen species can also
cause bystander effects with DNA damage in adjacent cells to those directly irradiated
[138, 139]. In summary, the therapeutic effect exerted by alpha-particle radiation is a
result of several complex molecular pathways, and cell death is due to the high dose
and subsequent irreparable DNA damage [8].
2.8 Clinical radioimmunotherapy
There has been a considerable evolvement of clinical applications of targeted
radionuclide therapy during the last 30-40 years. Major advances in biochemistry and
the understanding of biological processes, as e.g. cancer development, have made it
possible to identify biochemical pathways and proteins that are much more abundant on
cancer cell surfaces than on healthy cells [140]. The use of new specific cancer
therapies, like radioimmunoconjugates, allow for the opportunity to efficiently treat
metastatic tumors which is a great challenge in cancer therapy [12]. The focus in this
section will be on clinical advances in radioimmunotherapy, but first some words about
the clinical effect of Xofigo®.
Following i.v. injection, Xofigo® (223Ra dichloride) targets selectively osteoblastic bone
metastases. The treatment was the first targeted alpha therapy to show improved
overall survival in controlled clinical studies and has a good safety profile for bone
metastases in castrate-resistant prostate cancer patients [14]. The most widely used
pain palliation for localized metastases remains to be external beam radiation therapy
[141, 142]. The metastases are usually multiple and widely distributed, and therefore
the use of external beam therapy may have limited effect [141-143] . There is no
targeting vector in the targeted alpha therapy with 223Ra dichloride. However, 223Ra is a
calcium-mimic and naturally self-targets bone metastases by being incorporated in the
bone matrix of metabolically active bone as radium cation. Tumor cells are thereby
Page | 32
killed by the short-range, high energy alpha-irradiation, and the progressive growth of
osteoblastic metastases may be stopped [10, 144-146]. Bone-seeking
radiopharmaceuticals based on beta-emitters are also commercially available, i.e.; 89Sr
dichloride (Metastron, GE Healthcare) and 153Sm-EDTMP (Quadramet, Schering AG).
Bone marrow toxicity is, however, a concern with these products due to the mm-range
of the emitted beta-particles. This limits their use when dose escalation and/or repeated
treatments are desired [96, 144, 147].
The use of monoclonal antibodies and radioimmunoconjugates requires the selection of
suitable antigens on the surface of cancer cells to obtain the desired targeting and
therapeutic effect [148, 149]. Hematopoietic differentiation antigens (e.g. CD20 and
CD33), cell surface differentiation antigens (e.g. prostate-specific membrane antigen
(PSMA), growth factor receptors (e.g. epidermal growth factor receptor (EGFR), and
angiogenesis and stromal antigens (e.g. vascular endothelial growth factor receptor
(VEGFR) are among the categories of tumor antigens that have been identified in a
variety of cancers [150].
Within haematologic malignancies, successful clinical results have been shown for
radioimmunotherapy of lymphomas. Targeting against several antigens in lymphoma
have been studied [151]. Two beta-particle emitting radiolabelled antibodies that target
CD20, namely 131I-tositumomab (Bexxar®, GlaxoSmithKline) and 90Y-ibritumomab
tiuxetan (Zevalin®, Spectrum Pharmaceuticals B.V.) have been approved for clinical
use for the treatment of non-Hodgkin’s lymphoma patients which are either relapsed or
that do not respond well to the effect of rituximab (chimeric anti-CD20 antibody) and
chemotherapy [13]. These drugs are so far the very highlight of successful radiolabelled
antibodies in cancer therapy. Zevalin® contains the monoclonal mouse antibody
ibritumomab together with the chelator tiuxetan and the radioactive isotope of 90Y (for
therapy) or 111I (for imaging). The drug is approved in Europe, the United States, Asia
and Africa. Bexxar® also contained a mouse monoclonal antibody (tositumomab) which
Page | 33
was covalently bound to the radionuclide 131I. The marketing approval of Bexxar® was,
however, withdrawn and sales discontinued in 2014 due to decline in usage [152]. Like
for Zevalin®, Bexxar® treatment included assessment of biodistribution prior to
treatment through a trace labelled infusion for calculation of the right therapeutic dose
[153].
There are several reasons why radioimmunotherapy is an attractive approach for
haematological malignancies including; identification of cell surface antigens that are
not expressed in other tissues, availability of good quality antibodies, the high
radiosensitivity of leukaemias and lymphomas, and the inherent immunosuppressive
nature of the diseases which reduces the risk of formation of human anti-mouse
antibodies [28]. Research has supported the antibodies’ immune effector function
(especially for anti-CD20) and the natural radiosensitivity of lymphomas as being partly
responsible for the success of these therapies [154-157].
The major side effect of radioimmunotherapy is hematologic toxicity, which depends on
prior treatment and bone marrow involvement [158, 159]. Good hematologic status and
minimal bone marrow involvement are recommended for patients to use Zevalin® due
to the risk of myelosuppression [28]. However, when comparing to patients that have
received chemotherapy the incidence may not be higher and some studies show that
administration of lower adjusted activities may give a better safety profile [160, 161].
Compared to the administration of unlabeled anti-CD20 antibody alone, both Zevalin®
and Bexxar® have shown higher response rates3 and more durable effects for complete
responses4 [162-166]. In addition, an increased tolerance and improvement of response
3 The percentage of patients whose cancer shrinks or disappears after treatment. 4 The disappearance of all signs of cancer in response to treatment.
Page | 34
compared to standard chemotherapy has been shown with the use of Bexxar® [167-
169].
Radioimmunotherapy for lymphomas is less frequently used than chemotherapy
regimens despite the documented safety and efficacy, and this led to Bexxar® no longer
being marketed [28]. Several factors seem to have contributed to this limited adoption
by the medical community; myelodysplasia concerns, multiple novel competing targeted
agents being available, and perhaps most importantly; the need for haematologists and
oncologists to refer patients to third parties with license to handle radioactivity to
administer the drugs and not being able to sell it directly to patients [170].
The story for radioimmunotherapy of solid tumors is not so successful as for
haematologic malignancies and radioimmunotherapy of lymphomas, and responses
have been infrequent. Reasons for this include insufficient doses of radioactivity being
delivered to the tumor cells, solid tumors’ lower sensitivity to radiation, and difficulties of
uniform penetration of the solid tumors by antibodies [171, 172]. Variable uptake in
epithelial tumors have been shown in studies and reasons include; size of the tumors,
vascularity status, histological type, necrosis extent, and expression of antigen in
addition to the factors already mentioned [173-176]. Some promising results with
response improvement and survival without progression have, however, come from
loco-regional infusion of radioimmunoconjugates particularly in ovarian cancer and
glioma with 131I, 177Lu and 90Y-labelled antibodies [177-183]. In these approaches,
injection is done directly into the body compartment containing the tumor and is not
suited for all cancer treatments [28].
Page | 35
Other approaches and possible solutions to increase the therapeutic index of
radioimmunoconjugates (i.e. the ratio of absorbed radiation dose to the tumor divided by
the dose absorbed by radiosensitive tissues, e.g. bone marrow and kidney) include [28];
• dose fractionation with multiple injections to achieve higher administered total
doses and expected bone marrow recovery between treatments
• addition of chemotherapy and combination with unlabelled antibodies
• normalization of tumor vasculature or selective improvement of tumor vascular
permeability
• reduction of the circulating half-life of the radioimmunoconjugate by using
smaller antibody moieties
• pre-targeting with unlabelled antibody
• use of alpha- or Auger electron-emitting radionuclides with a higher LET and
shorter radiation range in tissues
Dose fractionation has shown to be feasible for both solid tumors and lymphoma [184-
186]. In preclinical studies, chemotherapy in sub-therapeutic doses has been able to
enhance the effect of radioimmunotherapy [187-192]. Chemotherapy often delays
cancer cell growth, and the cells are arrested in a radiation sensitive phase [193-195].
Several studies also show improvement of the radioimmunotherapy when the unlabelled
antibody has an anti-tumor effect in itself and/or increase radiosensitivity of the tissue
[196]. For example, in EGFR-positive tumors a combination with unlabelled antibodies
has enhanced the radiosensitivity of tumors [197-201]. Increased interstitial pressure
and hindering of uniform tumor penetration of radioimmunoconjugates may be
counteracted by normalization of tumor blood flow through therapies like anti-VEGF
therapy [202, 203]. Enhancing the vascular permeability may also increase therapeutic
efficiency by increasing the amount of the radioimmunoconjugate reaching the tumor
cells [204].
Page | 36
Use of smaller forms of antibodies (e.g. F(ab’)2 or Fab’) and molecularly engineered
sub-fragments of antibodies have been explored to reduce the circulation time. The
more rapid blood clearance of these agents may improve the tumor/blood distribution
ratio [205]. Even though there is a lower fraction of injected activity which is delivered to
the tumor with antibody fragments, the approach may still be attractive due to the
possibility of administrating higher doses of radioactivity with reduced hematological
toxicity. Care must on the other hand be taken regarding the possibility of renal toxicity
of the smaller antibody fragments which are cleared from the blood through the kidneys
[206-208]. This applies especially for radionuclides which are metals, as radioiodinated
fragments do not show retention in the kidneys and a good therapeutic index may be
achieved with these fragments [209, 210].
In pretargeted radioimmunotherapy, the slow phase of distribution of the antibody is
separated from the administration of the therapeutic radionuclide. In these multistep
strategies, the reactive antibody (not radiolabelled) is allowed to accumulate and
localize to the solid tumor site without the rest of the body being exposed to radioactivity
from a circulating radioimmunoconjugate [211-218]. A radiolabelled moiety with low
molecular weight which has a high affinity for the accumulated antibody is then
administered and, because of its small size, the radiolabelled moiety rapidly penetrates
the solid tumor and binds to the antibody. Unbound molecules of the small moiety are
rapidly cleared from circulation. In some approaches an agent that clears the non-
radioactive antibody from the circulation has been administered prior to the small
radioactive moiety in order to prevent complexation [214, 215, 219, 220]. Other
approaches to pretargeting that has been studied include the development of haptens
and bispecific antibodies and modifications thereof [212, 221, 222]. Encouraging results
have been shown in pilot pretargeting clinical trials both with solid tumors and
lymphoma, but care must be taken to possible targeting to normal tissues [223-225].
Page | 37
The low potency and long range of beta-particle emitting radionuclides often used in
radioimmunotherapy may have contributed to reasons why the treatment of solid tumors
in humans has not reproduced the encouraging results from preclinical studies. The low
LET restricts the potency for killing of the cancer cells by commonly employed beta-
emitters as 131I, 90Y, 177Lu, 186Re or 188Re, and a “cross-fire” effect of the bone marrow
from the radioimmunoconjugate in the systemic circulation (due to the long range)
reduces the radioactivity dose that can be administered [4, 28]. The high LET and
higher cytotoxicity as well as shorter range of alpha- and Auger electron-emitting
radionuclides may both increase the potency and reduce unwanted radiation effects on
non-target normal tissue. The indication for these therapies may, however, be restricted
to smaller volumes of tumors and single cells (<1 cm diameter) while larger tumor
volumes may be more feasible for beta-emitters [226].
Clinical studies with radioimmunotherapy including the alpha-particle emitting
radionuclides 225Ac, 211At, 212Bi, 213Bi, 212Pb and 227Th have been conducted (see Table
1 p.26 for half-lives and section 2.12.2 for 227Th details) and some will be mentioned
here [4, 7]. As discussed earlier, there has been a limited use of alpha-emitters due to
suboptimal physical properties (e.g. safety of daughter nuclides or inappropriate
(physical) half-life), chemical properties (e.g. difficulties in labelling to antibodies) and
limited availability [88]. A phase I trial of malignant gliomas treated with a 211At
radioimmunoconjugate showed promising results with low toxicity and no toxicity
leading to dose-limitations [227]. 212Pb decays to 212Bi and radioimmunoconjugates
labelled with 212Pb have been studied as in vivo generators of 212Bi (ref. section 2.10)
[88]. 212Pb-trastuzumab (anti-HER2) with intra peritoneal administration for ovarian,
pancreatic and colon tumors following pretreatment with infusion of unlabeled antibody
have been studied in a phase I trial revealing minimal toxicity [228].
Radioimmunotherapy of acute myeloid leukemia has been studied with 213Bi-labelled
lintuzumab in a phase I study where the radioimmunoconjugate accumulated at the
sites of leukemia and 93% and 78% of the patients experienced reductions in leukemic
Page | 38
blasts in blood and bone marrow, respectively, without any significant kidney uptake of 213Bi [229].
As mentioned in section 2.6, proximity to nuclear DNA is necessary for Auger electrons
to exert lethal DNA damage, and some preclinical studies have taken advantage of
nuclear translocation sequence peptides to direct antibodies labelled with 111In to the
cell nucleus [230-233]. To the knowledge of the author, no clinical studies have been
performed with Auger electron emitting radionuclides, but preclinical studies have
reported potential for use of this technology in therapy [4].
2.9 Radionuclide purity of radiopharmaceuticals
The radionuclide purity (RNP) of radiopharmaceuticals is an essential parameter in the
quality control of such preparations [234]. According to the European Pharmacopoeia,
radionuclide purity is defined as the ratio between the radioactivity of the base
radionuclide and total radioactivity of the radioactive compound. Absolute radionuclide
purity is attained if no other radionuclides besides the one of interest is present [234]. A
common method to analyze the radionuclide purity is to use high purity germanium
(HPGe) gamma-ray spectrometry to determine the amount and identity of radionuclides
present prior to patient treatment [234-236].
Page | 39
2.10 Targeted alpha therapy and in vivo generators
While targeted alpha therapy with radionuclides like 211At decay via single alpha particle
emission, others decay in a cascade of several alpha particle emissions, e.g. 225Ac, 212Pb (Figure 5), 227Th and 223Ra (Figure 6).
Figure 5 Decay scheme of 225Ac (left) and 212Pb (right) (adapted from [88])
Page | 40
Figure 6 Decay scheme of 227Th and 223Ra (adapted from [88] and Paper II)
Alpha-particle emitters decaying via such cascades are called in vivo generators [237].
The radiotherapeutic efficacy of targeted alpha therapy could be enhanced by the use of
such in vivo generators, as the potentially delivered radiation dose may be dramatically
increased [17, 237]. This is utilized in the use of Xofigo® for metastatic bone cancer,
where four alpha particles are emitted in the decay chain of the calcium mimetic 223Ra
[144]. The daughter products of 223Ra either have a short half-life (see Figure 6) or a
high affinity for bone like the mother nuclide 223Ra (i.e. 211Pb). A different mechanism for
both delivery and retention of the daughters would be required for targeting to other
sites than metastatic bone cancer [17].
Page | 41
One of the major challenges with in vivo generators is retaining the daughter
radionuclides at the target site [237]. The radiochemistry of alpha emitters may be
complicated as the daughter nuclides often have a radically different chemical behavior
from the mother nuclide. Also, the radiolabelled targeting moiety may be damaged by
the high LET radiation of recoiling daughter nuclides in the decay chain, leading to the
daughters not being attached to the moiety [237, 238]. Stabilization of the targeting
moiety in vitro could be achieved by addition of radical scavengers to such preparations
with high specific activity5, but both radionuclide chemistry and physical decay
properties of the daughters must be assessed. When the half-life of daughter nuclides is
very short (ns-s) recoil release may not be a problem as these nuclides will decay
before any significant distribution away from the in vivo location of the mother nuclide.
However, for nuclides like 227Th, with the daughter nuclide 223Ra which has a (in this
application) long half-life of several days (t1/2=11.4 days) and an inherent targeting
ability (i.e. to bone), the therapeutic impact of the daughter nuclide must be assessed,
unless targeting to bone is also desired for the 227Th preparation. 213Bi from the decay
chain of 225Ac and 212Bi from 212Pb is known to accumulate in the kidneys, which is of no
potential therapeutic benefit like the bone targeting of 223Ra [239-241]. Methods to limit
the level of such in vivo generated daughters in the preparation or to control their
biodistribution most likely has to be developed as damage to healthy tissue may occur,
especially when applied systemically [238]. For 223Ra, the lack of appropriate
bifunctional ligands (and thereby possibilities for radiolabelling) limits the ways of
controlling the biodistribution of this radionuclide as well as its use in receptor-targeted
therapy [18].
Recoil energy and recoil charge are important parameters in the alpha particle decay
physics [238]. The law of momentum conservation depicts the released kinetic energy in
the decay of an alpha particle emitting radionuclide, and the energy is distributed among
the daughters according to their masses. The released kinetic energy of alpha particle
decay is much higher than that of a covalent chemical bond (i.e. ~100 keV versus 1-10
5 Activity (in Bq) per mass unit of targeting moiety
Page | 42
eV). This means that to stabilize the alpha emitter in a molecular structure, thousands of
chemical bonds must be hit before it loses its energy (e.g. in a polymer or crystallite
structure) [238]. Additional damage may be caused by the recoil charge of the alpha
emitter through electronic interaction with the surrounding molecules [238]. In 227Th
decay and the recoils of 223Ra, high charge values may be reached and neutralization of
these charged radionuclides is done through ionizing and excitation of molecules along
their paths [242]. Monte-Carlo based computer codes can be used to calculate both the
recoil atom and alpha particle range in tissues [243].
Several approaches to ensure proper targeting and/or utilize the radiation dose of in
vivo generated daughter products of alpha emitters have been studied, and some will
be briefly mentioned here. Carriers based on nanomaterials have been developed and
examples include NaA nanozeolites labelled with radium radionuclides [18], polymer
vesicles for incorporation of 225Ac [244], sterically stabilized liposomes as carriers of 212Pb [245], lanthanum phosphate nanoparticles as carriers for 223Ra and 225Ra [246],
labelling of 225Ac to core-shell structured gold coated lanthanide phosphate
nanoparticles [17, 237], polymersomes as carriers of 225Ac, trapping of 225Ac in
fullerenes [247] and liposomes loaded with 225Ac [248, 249]. Chelators have also been
incorporated into nanomaterial carriers as a mean to further reduce daughter migration.
Studies include the use of DOTA and DTPA chelators in a polymer to trap the daughters
of 225Ac [250], and indium-DTPA-tagged liposomes as carriers of an in vivo 212Pb/212Bi
generator [251]. Three general approaches apply to stop recoil release from
nanomaterial carriers; 1) reduction of the recoil energy in the material due to the particle
size of the material, 2) stabilization and trapping of the radionuclide in a depot of
carriers after passing through several of the carriers, and 3) stopping the radionuclide
by time through selection of radionuclide with appropriate physical half-life [238]. The
size of the nanoconstruct has been shown to influence both the bioavailability and
passive targeting through the enhanced permeation and retention effect [252]. Specific
uptake or elimination issues may also be influenced by shape [253]. Biocompatibility
and biodistribution may be influenced by e.g. poly-(ethyleneglycol) (PEG) coating of the
Page | 43
surface to reduce immune response and by connecting antibodies to the surface for
targeting [238]. Another approach, besides the use of nanomaterial carriers, is the use
of pharmacological agents to alter pharmacokinetics of daughter nuclides. An example
of this is oral co-administration of the chelating agents DMPS or DMSA prior to i.v.
injection of 225Ac to reduce renal uptake of 213Bi [254].
2.11 Selected methods for column separation and purification of thorium(IV) in literature and the established method for purifying decayed 227Th at Bayer
There are several descriptions of the use of polymeric resins on columns for removal of
radionuclides in literature with scopes ranging from drinking water purification to
production of radionuclide raw materials to be used in pharmaceutical preparations
[255-258].
Both ion exchange and extraction chromatography is commonly employed for isolation
of thorium(IV). Several of the approaches utilize the fact that thorium(IV), and not Ac(II)
and Ra(II), in nitric acid (HNO3) solutions has a high affinity for anion-exchange and
extraction chromatography resins due to complexation to nitrate and formation of
negatively charged complexes. The complexes are formed over a wide range of HNO3
concentrations, but distribution coefficients for anion exchange resin is maximized at 7-8
M HNO3 [241, 242]. Ion exchange resins AG MP-1M and AG1-X8 (Bio-Rad
Laboratories, Inc.) and extraction chromatographic resins TEVA, TRU and UTEVA
(Triskem International) are commonly used, and elution of thorium(IV) from the resins is
done with a lower concentration of HNO3 or with hydrochloric acid (HCl) at various
concentrations [257-260]. Purification of thorium(IV) from daughter products has also
been described by the use of anion exchange Dowex1-X8 resin (The Dow Chemical
Company) and HCl [261]. The use of chelating anion ion exchange resins like Dowex A-
1 (The Dow Chemical Company) and Zeo-Karb 226 (Permutit Company) have in
Page | 44
addition been described for the separation of thorium(IV), and strong acids were used in
these processes as well [262, 263].
Examples in literature of the use of cation exchange and extraction chromatography
also exist. Thorium(IV) has been retained on resins AG50W-X8 (Bio-Rad Laboratories,
Inc.) and DOWEX 50W-X8 (The Dow Chemical Company) before being eluted with
H2SO4 [264, 265]. More interestingly for the aim of this thesis are studies with the
elution of thorium(IV) from cation exchange resins without strong acids. In one study of
the separation of actinium(II) from thorium(IV) and radium(II), thorium(IV) was eluted
with citric acid from AG50W-X8 resin. The procedure did however include several other
steps with the use of strong acids and extraction chromatography resin DGA (Eichrom)
[266]. Cation exchange resins DOWEX 50W-X8, AG50W-X8 and Merk I have in another
study been used for the sorption of calcium (II), barium(II) and radium(II) with desorption
by the aid of tartrate, EDTA and citrate, but the pH (>8) was high for desorption of
radium(II) and no separation of thorium(II) from radium(II) was studied [267].
The established purification procedure for decayed 227Th (227Th with presence of
daughter nuclide 223Ra) at Bayer utilizes anion exchange resin and strong acids in a
multiple-step procedure. This bind/elute mode by the use of strong acids like HNO3 and
HCl assures a high RNP of the product and the method is robust with regard to this
parameter. This is due to the fact that thorium(IV) and not radium(II) forms negatively
charged complexes with nitrate that is retained by the anion exchange column. By initial
sorption of the negatively charged thorium nitrate complex, proceeding elution of
unbound radium(II) from the column and final elution of the thorium by disruption of the
nitrate complex, both a high separation efficiency and high robustness of the method
regarding the RNP parameter are achieved (in-house data, not shown).
Page | 45
2.12 Targeted Thorium Conjugates (TTCs) and the 227Ac-227Th-223Ra technology platform
227Th (t1/2=18.7 days) studied in this thesis is part of the 227Ac decay chain (t1/2=21.8
years). A long-term 227Ac generator is capable of generating 227Th in large amounts over
several decades [268, 269]. Thermal neutron irradiation of 226Ra is an efficient method
of producing significant quantities of 227Ac and is the production method used in Bayer
[270].
2.12.1 Product life cycle and manufacture of TTCs at Bayer
Figure 7 shows the product life cycle and manufacture of TTCs at Bayer. A 227Ac
generator is first stored for in-growth of daughter nuclide 227Th to be utilized in
manufacture of the TTCs. A purification of 227Th by the aid of acids, solvents and solid
phase exctraction (SPE) resins is then conducted (named purification #1) before
shipment to radiopharmacies/sites of patient dose preparation. At these sites a second
purification of 227Th (named purification #2) is done prior to further shipment and/or
administration of patient dose.
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Page | 47
In Bayer the 227Ac generator is harvested for 227Th followed by a first purification of 227Th
from any traces of 227Ac and 223Ra (and other nuclides, 227Th purification #1 in Figure 7).
In the current manufacturing process, it is inevitable that there is a certain storage
period of 227Th after this initial purification, since 227Th will be shipped for further use to
other sites where labelling of the monoclonal antibody will be performed. The time for
transportation and storage will lead to 227Th decay and formation of 223Ra and daughters
(i.e. decayed 227Th). Thus a second purification of 227Th (227Th purification #2 in Figure
7) is done at these sites before complexation to the targeting moiety and administration
of the patient dose. This second purification is the subject explored in the studies of this
thesis.
Theoretical calculations of the 227Th dose could be done. However, without this second
purification of 227Th, the composition may become more toxic, have a reduced safe
storage period as well as possibly change the therapeutic window (i.e. doserelation
between therapeutic effect and adverse effects) in undesirable ways. Most important is
to obtain a reproducible situation with a defined time for measurement of 227Th activity
and theoretical zero 223Ra activity (as given by the radionuclide purity of the purification
method). This leads to standardization of the level of generated 223Ra and daughters
from the defined time interval from the second purification until administration of the
dose (i.e. the shelf life of the pharmaceutical preparation). The relation between efficacy
and adverse effects due to radiation from 227Th and daughters will therefore be
standardized within the time frame from the second purification of 227Th to patient
injection. In other words, it leads to an independence of the storage time of 227Th for the
therapeutical window of the preparation.
In this thesis, an alternative method for purification of decayed 227Th by the use of
formulation buffers and with fewer steps than the established strong acid based method
has been explored (Paper II). The advantage and necessity of a second purification of 227Th also applies if one was to ship 227Th already radiolabelled to the antibody-chelator
Page | 48
conjugate to the sites of patient administration. Such a purification approach, where the
second purification and removal of 223Ra is performed with the presence of TTC, has
also been studied in this thesis (Paper III). The removal of 223Ra is in the current
process at Bayer done shortly before administration of the dose. Continuous removal of 223Ra during storage of 227Th, either as free radionuclide or labelled to a targeting
moiety, may be another option which has also been explored in this thesis (Paper I). In
the approach with continuous removal of 223Ra, the preparation must contain materials
capable of sorption which are in contact with the radionuclides during storage.
2.12.2 Preclinical and clinical studies of 227Th
Several studies have revealed 227Th as an attractive nuclide for targeted alpha therapy,
especially by labelling of monoclonal antibodies for cancer therapy [271]. Rituximab, an
anti-CD20 monoclonal antibody used for the treatment of lymphoma, has been labelled
with 227Th. 60% of the treated mice showed complete regression of human lymphoma
xenografts without significant toxicity in examined tissues [272, 273]. Another study
concluded that 227Th-rituximab injection was more effective per absorbed radiation dose
unit (dose rate) than the beta-emitting radioimmunoconjugate 90Y-tiuexetan-ibritumomab
(Zevalin®) or treatments with external beam X-rays [274], see Figure 8.
Page | 49
Figure 8 Dose rate (amount of radiation dose absorbed to tumor per unit time) as a function of time for Zevalin® and 227Th-rituximab [274].
Xenografts with breast cancer expressing HER2 in mice have been treated with 227Th-
trastuzumab, and the therapy was well tolerated and dose dependent growth inhibition
was shown [268]. A TTC which targets the CD33 receptor for treatment of acute
myeloid leukemia has been studied both in vitro and in vivo. In vitro the TTC induced
cytotoxicity independent of multiple drug resistance phenotype in CD33 positive cells. In
vivo, antitumor activity was shown in a subcutaneous xenograft mouse model using HL-
60 cells. Dose-dependent significant survival benefit was in addition demonstrated in a
disseminated mouse tumor model, which supported the further development of the TTC
[275]. Exploitation of the possible synergistic effect of the bone targeting daughter
nuclide 223Ra has also been investigated in a study where 227Th was complexed to
DTPMP and used to target bone metastasis. Selective accumulation and long term
retention of the 227Th complex as well as 223Ra was demonstrated [276]. In 2016 the
Targeted Thorium Conjugate BAY1862864 injection (Bayer) reached a Phase I clinical
trial in Sweden and UK. Patients with relapsed or refractory CD22-positive non-
Hodgkin’s lymphoma is at present included in the study which is investigating safety and
tolerability, safety profile, maximum tolerated dose and tumour response [277].
Page | 50
2.12.3 Ingrowth of 223Ra and toxicological data
The TTC BAY1862864 injection (described in previous section), has a shelf life of
maximum 48 hours from the time of the second purification of 227Th and radiolabelling to
the antibody-chelator conjugate. Within this time frame the dose has to be administered
[277]. Studies of safety and toxicology have therefore been performed with an ingrowth
of 48 hours of 223Ra. No observable differences have been found in any of the toxicity
end points between animals of the high dose group, or in animals treated with a range
of different antibody-chelator conjugate doses (in-house data, not shown). These data
also include the in vivo generated 223Ra. They support that a therapeutic treatment
window exists, in which a therapeutically effective amount of 227Th can be administered
after 48 hours product shelf life without generating an amount of 223Ra sufficient to
cause unacceptable myelotoxicity. This is important given the strong bone-targeting
ability of 223Ra. When released from 227Th in vivo, 223Ra has been shown to accumulate
in the skeleton where the risk of myelotoxicity is significant [278].Other studies of 223Ra
show that it is rapidly cleared from the systemic circulation and is either concentrated in
bone or excreted via the renal or intestinal routes [19, 99]. The amount of 223Ra
generated in vivo from 227Th decay will depend on the physical half-life of 227Th and the
biological half-life of the TTC. Ideally, the TTC will have a rapid tumor uptake, strong
tumor retention and a short biological half-life in normal tissues.
Page | 51
3 Aim
The aim of this thesis was to develop a new in situ purification method for
standardization of the amount of the 227Th daughter nuclide 223Ra in the pharmaceutical
preparation prior to intravenous injection of a targeted antibody labelled with 227Th
(TTC). Purification is required to obtain a therapeutic window of the preparation which
only depends on the amount of 227Th present, and may be necessary to assure safety of
the product with a standardized amount of non-targeted 223Ra present. Increased user-
friendliness, minimization of the number of steps and use of non-hazardous materials
were important aspects in the development of a new purification procedure.
Materials were screened for their ability to retain both 223Ra and 227Th (either as free
radionuclide or as radiolabelled antibody-chelator conjugate, i.e.TTC). Methods for
separation with a good selectivity and high 223Ra and accompanying low 227Th/TTC
sorption were explored.
The impact of formulation and process parameters was explored as part of the
development of the purification method, by use of statistical factorial design.
Page | 52
4 General experimental presentation
4.1 Sorption materials
Table 3 shows the sorption materials with selected physico-chemical characteristics
studied in this thesis [18, 279-287]. A broad range of materials, both organic and
inorganic, were studied; DSPG liposomes, calcium and strontium alginate gel beads,
Zeolite UOP type 4A, ceramic hydroxyapatite, as well as strong cation exchange resins
AG50W-X8, SOURCE 30S and PSA modified silica resin. The expected mechanism for 223Ra sorption was ion exchange for all materials except the DSPG liposomes, for which
ionic attraction was the expected mechanism. The particle size of the materials ranged
from 0.3 μm for the DSPG liposomes to ~2000-5000 μm for the Zeolite UOP type 4A.
Pore size, which is another important parameter for sorption, also exhibited a great
range for the materials; from 0.0004 μm for the Zeolite to 0.2 μm for the SOURCE 30S
resin. The DSPG liposomes was the only material for which no porous diffusion was
expected. The materials were tested by the batch and/or column method, i.e. passive
diffusion by having the materials as suspensions or packed on columns.
Page | 53
Table 3 Studied sorption materials with selected physico-chemical characteristics. For some materials the particle size is given as an interval without standard deviation (SD, *). B=batch method, C=column method, NA= not applicable, NN= not known, ¤ no systematic pore structure is part of the material IE= ion exchange, Y=yes, N= no.
Sorption material
Method tested (B/C)
Particle size±SD
(μm)
Pore size (μm)
Expected mechanism of 223Ra sorption
Functional group
Counter ion
Resin backbone material
DSPG
liposomes B 0.3±0.2 NN¤
Ionic attraction
(Zeta potential=
-85 mV)
(R-O)2PO-
O-No IE NA
Calcium
alginate gel
beads
B/C 607 ±
445 NN¤ IE R-COO- H+ NA
Strontium
alginate gel
beads
B 434 ±
383 NN¤ IE R-COO- H+ NA
AG50W-X8
strong cation
exchange
resin
B/C
63–150
(wet
bead)*
0.1 IE R-SO2O- H+
Styrene divinyl-
benzene
copolymer
SOURCE 30S
strong cation
exchange
resin
B/C 30 0.2 IE R-SO2O- Na+
Rigid
polystyrene/
divinyl benzene
polymer
Zeolite UOP
type 4A B
~2000–
5000* 0.0004 IE AlO3O- Na+ NA
Ceramic
hydroxy-
apatite
B/C 80 ± 8 0.08-0.1 IE R-O2PO2- H+ NA
PSA strong
cation
exchange
resin
C 45 0.006 IE -C3H6-
SO2O-H+ Modified silica
Page | 54
4.2 High purity germanium gamma-ray spectroscopy
For measurement of radionuclide sorption by materials, high purity germanium (HPGe)
gamma-ray spectroscopy and a HPGe detector (GEM15-P) from Ortec (Oak Ridge, TN)
were used. A GMX model HPGe-detector from Ortec (Oak Ridge, TN) was used for
radiochemical purity analyses by iTLC. For both detectors Gammavision software was
used (from Ortec, Oak Ridge, TN). Radionuclides with gamma energies ranging from
approximately 30 to 1400 keV are identified and quantified by the detectors.
Gammavision calibration wizard and a mixed gamma source (Eckert and Ziegler, GA)
were used for calibration. Table 4 shows the gamma peaks (keV) used for 227Th and 223Ra measurements and their abundance (%).
Table 4 Gamma peaks (keV) and their abundance (%) used for measurement of 227Th and 223Ra by HPGe gamma-ray spectroscopy
Gamma peaks (keV) used for 227Th measurement and
abundance (%)
Gamma peaks (keV) used for 223Ra measurement and
abundance (%)
235.96 (12.90) 323.87 (3.99)
256.23 (7.00) 338.28 (2.84)
329.85 (2.90) 445.03 (1.29)
286.09 (1.74) 269.46 (13.90)
304.50 (1.15) 154.21 (5.70)
334.37 (1.14) 144.24 (3.27)
299.98 (2.21)
Samples analyzed by the HPGe-detectors were placed in a fixed, calibrated distance
from the detector before being counted.
Page | 55
4.3 Statistical methods
4.3.1 Design of Experiments; variables and tested range
In paper II and III, two-level factorial Design of Experiments (DOEs) with formulation
and process parameters were studied, as given in Table 5 and Table 6. The use of
citrate or acetate buffered formulations and the impact of buffer concentration, pH,
presence of free radical scavenger (pABA) and chelator (EDTA), resin amount, and
sodium chloride concentration (only in Paper III) were evaluated as variables. The
effects were interpreted by the aid of multivariate data analysis. The Unscrambler
version 9.8 (Camo Software AS, NJ) was used for statistical analysis while the pooled
standard deviation was calculated in Excel (Microsoft, Albuquerque, NM).
Citrate and acetate buffers were studied due to their chelating abilities and
pharmaceutical applicability [288-292]. Citrate is also a free radical scavenger which
may reduce the level of radiolysis in the formulation [293]. I.v. compatibility and effective
pH range of the buffers determined the pH values in the DOEs. In addition a large
enough range in pH to see effects on sorption was sought. The buffer capacity was
maintained at all buffer concentrations. pABA and EDTA are included in TTC
formulations at Bayer and were therefore included also in the DOEs. pABA is a free
radical scavenger which may aid the TTC stability, while the chelator EDTA may
compromise the desirable uptake sorption of 223Ra. However, EDTA has been shown to
have stabilizing effects on TTC formulations at Bayer [293, 294]. Sodium chloride
concentration was included to vary the ionic strength of the respective formulations,
which is known to influence ion exchange chromatography [295]. The presence of
sodium chloride may also impact the stability and conformation of the TTC [296, 297].
All excipients included in the DOEs are listed in the inactive ingredient list from the US
Food and Drug Administration (FDA) for approved drug products suitable for i.v.
injection.
Page | 56
Table 5 Design of Experiments (DOE) studied in Paper II (purification of decayed 227Th) with variables and range. W=with, wo=without
citrate/acetate buffer
Table 6 Design of Experiments (DOE) studied in Paper III (purification of TTC) with variables and range. W=with, wo=without
(2 mg/ml) (2 mM)
4.3.2 Determination of significant main and two-interaction variables and predictive ability of models
Partial least square regression (PLSR) and multiple least square linear regression
(MLR) were used to determine the correlation between variables. Full cross validation,
by keeping one sample out, was used for testing of the predictive ability of the models
and significant variables. The significance of variables was tested by both PLSR and
MLR, and variables that were found insignificant by both were rejected. The difference
between model predicted response (sorption, %) and measures response (sorption, %)
was used to define the predictive ability of the models. To estimate the uncertainty of
Page | 57
replicated samples, pooled standard deviation was used which is not impacted by errors
from model adaption.
5 Additional data
5.1 Sorption of 223Ra and daughters
The focus in this thesis has been the sorption of 223Ra and not the daughters of 223Ra.
In the decay schedule of 223Ra, the first three of four alpha-emissions occur within 5 s
(see Figure 6 p. 40). The lifetime of these progenies in the formulation once 223Ra is
removed will therefore be very limited. 211Pb is the daughter with the longest half-life, i.e.
36 minutes, but within the time frame from purification to sterile filtration, quality control
testing and readying of the dose for patient administration, the remaining level of this
radionuclide will be significantly reduced. The data in Paper I, II and III have therefore
not comprised the daughters of 223Ra. The data presented in this section are from
gamma-ray spectra already utilized for calculations of 223Ra and 227Th/TTC sorption in
the respective papers. 227Th, 223Ra, 219Rn, 211Pb and 211Bi were the nuclides detected by
HPGe gamma-ray spectroscopy (performed as described in section 4.2).
Table 7 shows the activity of the radionuclides (in kBq) retained by the materials tested
by the batch method in Paper I.
Page | 58
Table 7 Average radionuclide activity (kBq) detected by HPGe gamma-ray spectroscopy in the respective materials by the batch method as in Paper I
Material
Average 227Th
activity in material
(KBq)
Average 223Ra
activity in material
(KBq)
Average 219Rn
activity in material
(KBq)
Average 211Pb
activity in material
(KBq)
Average 211Bi
activity in material
(KBq)
DSPG liposomes 56.5 75.7 74.6 75.8 76.5
Calcium alginate gel beads 76.2 107.1 105.2 104.1 105.5
Strontium alginate gel beads 65.9 107.1 105.2 104.1 105.5
AG50W-X8 cation exchange resin* 75.1 123.7 120.3 123.6 125.8
SOURCE 30S cation exchange resin 68.6 123.1 120.0 123.0 124.9
Zeolite UOP type 4A 70.7 105.0 103.1 105.3 107.9
Ceramic hydroxyapatite 87.4 127.7 125.8 127.5 130.9
n=3 (except* n=1)
Table 8 and Table 9 show the activity of radionuclides retained by the PSA strong cation
exchange resin and activity remaining in the eluate, respectively, for randomly selected
DOE formulations studied in Paper II (purification of decayed 227Th).
Table 8 Activity of radionuclides (kBq) detected by HPGe gamma-ray spectroscopy in the PSA cation exchange resin in selected DOE formulations after purification of decayed 227Th (from Paper II)
Formulation buffer
pABA+ EDTA
pH Buffer conc.
(M)
Resin amount
(mg)
227Th activity in resin (KBq)
223Ra activity in resin (KBq)
219Rn activity in resin (KBq)
211Pb activity in resin (KBq)
211Bi activity in resin (KBq)
citrate w 4.75 0.065 22.5 4.8 137.5 119.5 124.7 128.3
w 4.75 0.065 22.5 8.6 149.4 136.6 160.2 130.6
acetate w 4.75 0.065 22.5 24.0 447.7 406.5 393.0 402.9
w 4.75 0.065 22.5 22.4 389.6 356.1 347.8 337.7
Data for 2 parallels per DOE setting is shown, W=with, Buffer conc.=buffer concentration
Page | 59
Table 9 Activity of radionuclides (kBq) detected by HPGe gamma-ray spectroscopy in the eluates for selected DOE formulations after purification of decayed 227Th (from Paper II)
Formulation buffer
pABA+ EDTA
pH Buffer conc.
(M)
Resin amount
(mg)
227Th activity
in eluate (KBq)
223Ra activity
in eluate (KBq)
219Rn activity in
eluate (KBq)
211Pb activity
in eluate (KBq)
211Bi activity
in eluate (KBq)
citrate w 4.75 0.065 22.5 145.7 10.9 4.4 26.1 26.5
w 4.75 0.065 22.5 159.2 7.2 2.3 24.3 26.6
acetate w 4.75 0.065 22.5 369.2 2.2 ND ND ND
w 4.75 0.065 22.5 333.7 ND ND ND ND
Data for 2 parallels per DOE setting is shown, W=with, Buffer conc.=buffer concentration
The activity of radionuclides retained by the PSA resin and the activity in the
corresponding sample eluate for randomly selected formulations studied in Paper III
(purification of TTC), are shown in Table 10 and Table 11.
Table 10 Activity of radionuclides (kBq) detected by HPGe gamma-ray spectroscopy in the PSA cation exchange resin in selected DOE formulations after purification of TTC (from Paper III)
Formulation buffer
pABA+ EDTA
pH
Sodium chloride conc. (%
w/w)
Buffer conc.
(M)
Resin amount
(mg)
227Th activity in resin (KBq)
223Ra activity in resin (KBq)
219Rn activity in resin (KBq)
211Pb activity in resin (KBq)
211Bi activity in resin (KBq)
Citrate w 5 0.45 0.050 30.0 100.0 271.5 275.4 195.2 194.4
w 5 0.45 0.050 30.0 100.1 273.5 274.0 199.3 201.9
Acetate w 5 0.72 0.075 22.5 63.6 285.5 245.5 169.9 163.1
w 5 0.72 0.075 22.5 63.5 258.1 237.6 180.4 163.2
Data for 2 parallels per DOE setting is shown, W=with, Sodium chloride conc.= sodium chloride concentration, Buffer
conc.=buffer concentration
Page | 60
Table 11 Activity of radionuclides (kBq) detected by HPGe gamma-ray spectroscopy in the eluates for selected DOE formulations after purification of TTC (from paper III)
Formulation buffer
pABA+ EDTA
pH
Sodium chloride conc. (%
w/w)
Buffer conc.
(M)
Resin amount
(mg)
227Th activity
in eluate (KBq)
223Ra activity
in eluate (KBq)
219Rn activity
in eluate (KBq)
211Pb activity
in eluate (KBq)
211Bi activity
in eluate (KBq)
Citrate w 5 0.45 0.05 30 328.5 62.4 53.5 148.4 165.9
w 5 0.45 0.05 30 342.1 81.4 57.5 172.9 177.2
Acetate w 5 0.72 0.075 22.5 188.8 5.7 4.2 125.4 133.0
w 5 0.72 0.075 22.5 190.3 5.5 9.1 137.3 143.2
Data for 2 parallels per DOE setting is shown, W=with, Sodium chloride conc.= sodium chloride concentration, Buffer
conc.=buffer concentration
5.2 Batch method; sorption of 223Ra after 60 versus 180 minutes equilibration time
Table 12 shows data for 223Ra sorption after 60 and 180 minutes, respectively,
equilibration time by the batch method studied in Paper I (materials as suspensions).
The source of 223Ra activity was decayed 227Th in 0.05 M HCl (named “(B)” in Paper I,
~19 days in-growth). The equilibration time is an important parameter to consider
regarding the feasibility of the approach with continuous removal of 223Ra in
development of an in situ purification method. It is undesirable to have an unsuitable
time dependence for sorption (and desorption) as this may impact the robustness of the
method and put restrictions on product shelf-life. The results in Table 12 show that there
is no significant difference in the average percentage sorption of 223Ra after 60 and 180
minutes equilibration time for any of the tested materials.
Page | 61
Table 12 Average percentage 223Ra sorption after 60 and 180 min equilibration time by the batch method studied in Paper I for the respective materials. SD=standard deviation (%)
Material
Average % 223Ra sorption after 60
min equilibration
(SD)
Average % 223Ra sorption after 180
min equilibration (SD)
DSPG liposomes 99.9 (0.1) 99.9 (0.1)
Calcium alginate gel beads 63.6 (13.5) 71.6 (4.6)
Strontium alginate gel beads 92.3 (4.1) 88.8 (4.0)
AG50W-X8 cation exchange resin 76.4 (1.3) 84.3 (1.9)
SOURCE 30S cation exchange
resin 85.1(7.0) 84.0 (5.2)
Zeolite UOP type 4A 60.7 (3.1) 69.8 (4.2)
Ceramic hydroxyapatite 77.2 (5.9) 80.6 (3.1)
6 Discussion
6.1 Important parameters for the development of an in situ purification method of 227Th
Important parameters in developing an in situ purification method for standardizing the
amount of 223Ra from radioactive decay of 227Th prior to i.v. injection of a 227Th labelled
mAb include parameters related to the finished drug product, the purification method as
well as the sorption materials used.
Page | 62
Important parameters for the finished drug product include:
• Radiochemical purity (RCP); the relationship between 227Th present in a bound
form (i.e. as TTC) to free 227Th.
• Radionuclide purity (RNP); the ratio of 223Ra to 227Th activity (in Bq) in the drug
product.
• Radioactive concentration (RAC); the activity of 227Th (in Bq) per volume of drug
product.
• pH; the pH of the drug product should comply with i.v. injection and stability of
the formulation and TTC.
• Aggregate level and antigen binding affinity; the aggregate level in the drug
product may compromise the stability of the formulation, patient safety as well as
impact the antigen binding affinity of the TTC.
• Sterility and endotoxin level; TTCs must be prepared as sterile and endotoxin
level controlled pharmaceutical preparations compatible with i.v. injection.
• Formulation; the formulation of the TTC must be stable and within specifications
at the time of i.v. injection.
For the purification method, important parameters include:
• Efficient and reproducible removal of 223Ra; the sorption of 223Ra must be
acceptable with regard to the resulting RNP of the drug product.
• Acceptable yields of 227Th/TTC; the sorption of free 227Th or TTC (depending on
the approach for purification) must be acceptably low and robust. Loss of 227Th
leads to a lower amount of 227Th which can be utilized for labelling of the
antibody-chelator conjugate and formation of the API (Active Pharmaceutical
Ingredient), while loss of TTC is loss of the API of the drug product.
• Good selectivity between 223Ra and 227Th/TTC; a simultaneous high 223Ra and
low 227Th/TTC sorption by the method for an efficient separation.
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• Conservation of product quality and i.v. injection compatibility; the formulation,
sterility and stability of the TTC must not be compromised by the utilized
purification method.
• Minimization of the number of steps and handling of the product; it is desirable to
develop a purification method that requires the minimum amount of resources.
The risk of operation failure will decrease with decreasing complexity of
operation, as well as reduce the radiation exposure of the operators according to
principle of ALARA (As Low As Reasonably Achievable).
• Avoid or minimize the use of hazardous materials; if the use of strong acids in the
purification method could be avoided it would be better concerning HSE (Health,
Safety and the Environment) issues.
• Conformance to current Good Manufacturing Practices (cGMP) regulations;
TTCs are pharmaceutical preparations, thus manufacture (including the
purification), testing and materials must comply with the regulations of cGMP in
the respective market for clinical testing or sale.
• Radiosafety; not only must the TTC drug product be protected from
contamination during manufacture but the operators and staff involved in
manufacture and testing of the product must be protected from the radiation from 227Th and progenies.
• Possibility for automation; it would be of a great advantage if the purification
method could be automated since this would most likely lead to a higher level of
operation compliance as well as reduce the radiation exposure of operators.
• Supply of disposable, one-time use kit; the ability to discard materials utilized in
operation of the purification method after one-time use is a demand for TTCs due
to the radiation exposure and long half-life of 227Th, contamination risk, as well as
demands for bioburden level during operation.
Page | 64
Important parameters for the materials utilized in the purification method include:
• Efficient and reproducible high 223Ra and low 227Th/TTC sorption.
• Compatibility with the method employed, composition of the drug product and
use in preparation of a sterile radiopharmaceutical for i.v. injection.
• Stability; both physico-chemical stability as well as stability in the presence of
radioactivity.
• Quality and conformance to cGMP regulations for use in preparation of sterile
radiopharmaceutical preparation.
• Financially acceptable to dispose after one time use due to contamination and
radiation safety issues.
Not all parameters have been thoroughly analyzed in this thesis, but have been part of
the decision process regarding which materials and methods that should be tested. This
will form the basis for the discussion of the results and methods.
6.2 Continuous removal of 223Ra during product shelf-life versus removal immediately prior to patient dose administration
Preparation of a ready to use TTC product in which 223Ra was continuously retained by
a material present in the formulation during the shelf-life of the product could increase
the user-friendliness and reduce the amount of work which has to be done at sites of
patient dose preparation. The stability of the TTC product may also benefit from this
strategy since continuous sorption of 223Ra could reduce the degree of radiolysis in the
formulation compared to shipment of TTC to be purified immediately before patient dose
administration.
In Paper I the aim was to study potential sorption materials and aspects regarding
passive diffusional sorption of 223Ra generated from decay of a TTC product [298]. A
batch method where the sorption materials were present as suspensions in the
Page | 65
formulation was used to study the passive diffusional approach. The sorption of 223Ra
was studied by an experimental set up with two sources of radioactivity; (A) 223Ra in
formulation or (B) 223Ra from decay of 227Th as free radionuclides in formulation. In a
TTC product there will be a very limited amount of free 227Th due to the near
quantitative labelling yield of the utilized chelator (as discussed in section 2.5), and as
controlled by analyzes of RCP. The data with radioactivity source (A) will therefore form
the basis of this discussion.
The level of hydrogen peroxide in solution (as an indication of the level of radiolysis in
the formulation) was in Paper I studied during 14 days storage of 227Th with and without
the presence of ceramic hydroxyapatite. Results showed a significantly lower level of
hydrogen peroxide in the samples with than without ceramic hydroxyapatite, and
supported the potential for a reduced radiolysis in formulations by use of the passive
diffusional approach for 223Ra sorption. However, sorption of 223Ra on columns was
revealed as being almost quantitative and with minimal variation for the cation exchange
resins and ceramic hydroxyapatite tested. The sorption of 223Ra by the batch method for
the same materials was not so efficient and showed a significantly higher variation.
The DSPG liposomes did, in contrary to the other tested materials, demonstrate a
superiority of sorption by passive diffusion at 95±3% 223Ra sorption. The superiority of
the DSPG liposomes by the batch method is likely due to a different sorption
mechanism for the liposomes compared to the other materials, with sorption to active
groups on the liposome surface without any diffusion through pores. For porous
materials like some resins, ion exchange occurs by diffusion and in the stages of film
and particle diffusion. In film diffusion ions move through the thin liquid film at the
surface of the resin. Particle diffusion is on the contrary the diffusion within the pores of
the resin [299]. The reduction of model antibody trastuzumab concentration (as an
indication of TTC sorption) in the samples with the DSPG liposomes was also among
the lowest of the studied materials with a 10% reduction. However, the immediate high
Page | 66
and robust sorption of the column method together with a perceived increased
complexity of product development for a purification approach with continuous removal
of 223Ra, including extensive compatibility and stability testing, led to the DSPG
liposomes and passive diffusion approach not being further explored. In addition,
concerns regarding the stability of the DSPG liposomes in the presence of radioactivity
were raised.
The higher sorption of radium(II) by column method compared to batch method has also
been demonstrated in other studies. One study of the sorption of radium(II) and
actinium(II) on soils concluded that the sorption of radium(II) on columns showed
distribution coefficients several times higher for the utilized column method compared to
the batch method [300]. Another study of the biosorption of thorium(IV) from aqueous
solution also concluded with a higher sorption capacity of the column method under the
same pH conditions as in a batch method [301].
The results from the batch method presented in Paper I are, however, after 60 minutes
equilibration and may have been improved by a longer equilibration time, as would be
the case for a TTC with sorption material present in the formulation during the shelf-life
of the preparation. Section 5.2, therefore presents data for 223Ra sorption for an
extended equilibration time for the materials studied in Paper I. As can be seen in Table
12 on page 61, there is no significant difference in sorption of 223Ra after 60 and 180
minutes equilibration. Moreover, as part of the exploration of H2O2 formation in the
presence and absence of ceramic hydroxyapatite, 227Th sorption was measured after 90
minutes and 227Th and 223Ra sorption after 14 days equilibration. The results revealed
no desorption of 227Th, and 223Ra sorption was 99±5% after 14 days. Saline was
however used as the sample matrix, and further studies will have to include sorption
data in the formulation of the drug product.
Page | 67
Additional studies with the use of Slide-A-Lyzer MINI Dialysis Device (Thermo Scientific,
10 000 molecular weight cut-off (MWCO) were performed as a mean to create an
experimental set up with a separate compartment for the TTC and sorption material
during passive diffusional uptake of free radionuclides (data not shown). This could lead
to a reduced loss of the TTC by sorption due to the size exclusion by the membrane
between the compartments. The studies were, however, not successful in reproducing
the 223Ra sorption results presented in Paper I, even after 18 hours equilibration. The
method was thus abandoned, although further studies with optimization of the
experimental set up could have given different results.
Sorption of 223Ra and TTC in relevant drug product formulation, and material as well as
formulation stability and compatibility would be important to study for the timeframe of
the TTC product shelf-life if the approach with continuous sorption of 223Ra was to be
further explored.
6.3 Use of micro-spin columns
The studies of 223Ra, 227Th and TTC sorption by cation exchange resin in Paper II and
III were performed by packing of the resin on micro-spin columns and proceeding
centrifugation for flow through of the sample. In Paper I, gravity columns were used for
packing of the sorption materials studied by the column method.
With gravity columns the resin bed has to be wetted before application of the sample,
and elution of the sample is performed by application of an additional volume which
pushes the sample volume through the resin bed. Depending on the resin bed volume,
sample volume will be lost on the column without an applied elution volume sufficient to
push the sample volume through the resin bed. With the sought flow through mode of
TTC this would mean loss of non-retained TTC in sample volume on the column. The
Page | 68
procedure also implies that the sample is diluted by the liquid present on the column
from equilibration and by the added elution volume (depending on how much of the
elution volume that is retained on the column).
A minimization of the number of steps was desirable when developing new methods of
purification in this thesis and other columns were thereby sought. In the developed
procedure with the micro-spin columns, the sample was applied and eluted by spinning
without the addition of further fluid in an additional step. Also the resin bed was dry both
before sample application and after elution, which meant no dilution of the sample. In
addition, the micro-spin column procedure gave sorption results with minimal data
variation compared to initial testing of other columns, and was thus seen as suitable for
the experimental set up. Performance may however be improved by optimization of
contact time, resin bed height and flow rate, but have not been studied.
The utilized relative centrifugal force was already used in the preparation of antibody-
chelator conjugates at Bayer and was therefore judged to be compatible with TTC
stability. The use of the micro-spin columns does, however, require further studies and
analyses regarding several aspects relating to product quality, process and sorption
material parameters as well as radiation safety issues. In these regards, concerns were
raised regarding TTC product quality and RCP as influenced by centrifugation of the
columns (as discussed in Paper III). In addition, the relatively high risk of radioactive
contamination during operation, due to the loose fitting of the columns in Eppendorf
tubes during centrifugation, make the use of micro-spin columns on sites of patient dose
administration less likely. Thus other forms of disposable columns should be sought in
further method development.
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6.4 Purification of decayed 227Th by the method in Paper II versus the established purification procedure at Bayer
Like in many of the described studies in section 2.11, the established purification
method of thorium(IV) at Bayer utilizes anion exchange resin and strong acids in a
multiple-step procedure. The use of strong acids like HNO3 and HCl requires that the
acids be removed (e.g. by evaporation) before further use of the 227Th in a
pharmaceutical preparation suitable for i.v. injection. Part of the aim of the method
development in this thesis has therefore been to develop separation methods in which
buffers and other excipients compatible with i.v. injection are used. Similarly, extraction
chromatography is not viewed as a desirable approach, since the stationary phase is in
a liquid form with the functional groups of the resin not being covalently bound to the
inert support. This leads to an increased risk of leachables from the stationary phase
which can contaminate the pharmaceutical preparation. Also, in order to minimize the
steps and handling of the product on site of patient dose administration, a flow through
mode with binding of impurity 223Ra instead of a bind/elute mode of 227Th (e.g. as
thorium nitrate complex) was sought in this thesis. The established purification method
of 227Th at Bayer differs on several aspects from the purification of 227Th by the aid of
formulation buffers studied in Paper II. An analysis of the impact on some of the
product, method and process parameters given in section 6.1 will be discussed in this
section.
As described in section 2.11, the established purification method in Bayer assures a
high and reproducible RNP of the product. With the flow through mode with cation
exchange and formulation buffers, the separation of 227Th and 223Ra is relying more on
tuning of the formulation and process parameters as presented in Paper II. The
separation is, however, done in a one-step procedure instead of in several steps. As
discussed in Paper II, there is a potential for high 223Ra (>90%) and low 227Th (<3%)
sorption from both citrate and acetate buffered formulations, but the robustness of the
Page | 70
separation must be explored with clinically relevant sample settings of radioactivity
levels, volumes and applicable drug product formulation.
An efficient separation of 223Ra and 227Th by flow-through mode means not only a high
sorption of 223Ra and high RNP, but also a low sorption of 227Th and following
acceptable yield from the purification process. Both the citrate and acetate buffered
formulations show potential for a low 227Th sorption, but as for the 223Ra sorption, the
robustness and clinically relevant parameters must be explored.
The development of a more user-friendly and less time consuming purification method
have been among the main aims of this thesis. The purification of decayed 227Th (227Th
with presence of daughter nuclide 223Ra) on cation exchange resin by the aid of
formulation buffers has the potential to fulfill these measures. As discussed in Paper II,
no strong acids in multiple-steps is utilized, and the buffered formulations have the
potential to be used directly in the drug product without the need for e.g. heat to remove
strong acid residues in the purified 227Th eluate. Residues of strong acids may cause a
risk to the final product quality. The use of strong acids also causes HSE concerns at
the site of patient dose administration. The more time consuming and complex the
purification procedure, the more effort is required regarding training of operators and the
more resources is required for patient dose preparation. Radiation safety is of course
also of concern, and the principle of ALARA should be followed. Time for radiation
exposure is a great contributing factor in the total radiation dose that is given to the
operators.
Ready to use 227Th produced by the flow through method does, however, put some
constraints on the further use of the eluate with concern to radiolabelling of the
antibody-chelator conjugate. By adding 227Th in buffered formulation to the antibody-
chelator conjugate for labelling, the antibody-chelator conjugate formulation will be
Page | 71
diluted. The composition of the final drug product must be within given specifications,
and both the composition of the eluate and antibody-chelator conjugate must comply to
this. It is therefore important to study the impact of the ion exchange procedure on the
buffered formulation, and to formulate the antibody-chelator conjugate according to
quality standards and i.v. injection compatibility as to achieve a finished drug product
within specifications.
6.5 PSA strong cation exchange resin packed on micro-spin columns; method development and material considerations
The studies in Paper II and III are both conducted by the aid of packing PSA strong
cation exchange resin (Macherey Nagel) on micro-spin columns (Thermo Fischer). The
conclusion to use the PSA strong cation exchange resin packed on micro-spin columns
was based also on additional studies of 223Ra sorption to those in the first publication
(data not presented). Initially, the separation of 223Ra and 227Th/TTC was hypothesized
to be done efficiently either by size exclusion (223Ra and TTC separation), cation
exchange (223Ra and 227Th/TTC separation), chelation (223Ra and 227Th/TTC separation)
or by a combination of these. In the additional studies, other materials and methods for
purification were explored and will be only briefly mentioned here.
It was concluded from the study presented in Paper I that the column method was more
efficient than the batch method for all materials tested by both methods, and a selection
of other cation exchange resins than those presented in the paper were packed on
gravity columns. Micro-spin columns were explored as a mean to reduce the number of
steps for purification and to increase the robustness of the separation (see section 6.3).
The decision to use the PSA strong cation exchange resin on micro-spin columns was
based both on the qualities of the resin as well as the method. The sorption of both
Page | 72
223Ra and 227Th/TTC was shown to be reproducible and immediate, and after
equilibration of the column, the sample could be applied and centrifuged through the
resin bed without further volume additions. The PSA strong cation exchange resin has a
good mechanical strength, and is biocompatible and stable. It has a hydrophilic surface,
which may reduce the risk of TTC sorption to the surface of the resin if hydrophobic
surfaces of the antibody and chelator are exposed during purification. The resin is
stable between pH 2-8, which is within the range of the pH in DOEs in both Paper II and
III. The 60 Å pore size is also below the cut off of TTCs based on monoclonal
antibodies. The purification method could, in other words, benefit both from size
exclusion and ion exchange mechanisms. The ion exchange mechanism is also known
to be affected by several formulation parameters, and these could therefore be
explored. The scope of studying the influence of formulation parameters on
radiochemical separation was the main reason why size exclusion chromatography
(which in its pure form is known to be less influenced by formulation parameters) was
not explored as a mean to purify TTCs in this thesis (ref. section 6.7).
In addition to testing of cation exchange resins, Empore 3M radium chelating disks
(solid phase extraction with chelator as functional group) were mounted in spin columns
and tested for sorption of 223Ra and flow through of 227Th (data not shown). Several
steps were required prior to sample application with acidification of the disks and a
proceeding rinse with water to minimize the amount of acid ending up in the eluate.
Without the removal of acid from the disk, the pH of the buffered antibody-chelator
conjugate would, after addition of the acid containing eluate, be too low for a successful
labelling reaction. The method was therefore abandoned.
Page | 73
There are several excellent references for ion exchange and sorbent extraction
chromatography [295, 299, 302-304].Important ion exchange material parameters,
which should be further explored as part of the development of an in situ purification
method for TTCs, include:
• particle size
• pore size and surface area
• functional group
• nature and cross-linking of backbone material and swelling characteristics
• physico-chemical properties of the surface
• counter ion
• capacity and selectivity
• stability issues
Several process parameters should also be explored in upcoming studies and include
the influence of:
• applied sample volume
• flow rate
• bed height and amount of material
• wet versus dry resin bed prior to sample application
6.6 Purification of decayed 227Th versus purification of decayed TTC
As discussed in Paper II and III, purifying the ready to use TTC drug product instead of
decayed 227Th (227Th with presence of daughter nuclide 223Ra) to be used in
radiolabelling of the antibody-chelator conjugate, puts different demands on the
purification procedure. Less handling will be required on the sites of patient dose
administration. This may increase the robustness of drug product preparation, decrease
Page | 74
radiation safety issues and reduce the time and resource demands on the sites.
However, decreased delivery flexibility and higher demands on logistical frameworks
are likely with shipment of radiolabelled product to the sites of patient dose
administration, due to constraints on the shelf-life of the drug product.
Of particular concern with the strategy of purification of decayed TTC is the physico-
chemical stability and preservation of biological activity of the TTC during the shelf-life
of the drug product. The purification procedure may also have an impact on these
parameters. If no radiolabelling is to be performed on the site of patient dose
administration, the level of 223Ra and daughters will be increasing in the formulation
within the time-frames that is expected for a shelf-life of a TTC drug product (i.e.
maximum 96 hours is expected according to in-house data). Results from the studies in
Paper III showed a potential for 223Ra sorption >90% and TTC sorption <25% from both
acetate and citrate buffered formulations. The TTC sorption was thus relatively high
despite the hydrophilic surface of the PSA resin and size exclusion of the TTC from the
resin pores. This may be due to the higher complexity of protein ion exchange
chromatography as compared to ion exchange of ions, as discussed in Paper III and
section 6.7. Concerns were also raised regarding the RCP of the TTC (antigen binding
affinity and aggregate level of the TTC were not tested). Indications were given of a
decreased RCP of the TTC in some of the formulations and may be due both to
radiolysis and the purification procedure itself. The inclusion of radical scavengers like
pABA and other excipients to reduce radiolysis, aggregation and conserve binding
affinity of the TTC must be further explored, as well as the impact of the purification
procedure on both product quality and composition.
In the conducted studies, the level of 223Ra in the preparation was significantly higher
than what can be expected from decay of 227Th during a TTC shelf-life (21 days in-
growth). The TTC was labelled with decayed 227Th instead of freshly purified 227Th, but
the time of TTC exposure to radioactivity was less than during the shelf-life of a TTC
Page | 75
(i.e. purified immediately after radiolabelling). Antibody-chelator conjugates based on
other mAbs than traztuzumab may also give different results. The strategy with labelling
of TTC with decayed 227Th immediately before purification (instead of labelling prior to
shipment to sites of purification and patient dose administration) may be further
explored as a purification and labelling strategy to minimize the time of radiation
exposure to the TTC. More handling and radiolabelling at the site of patient dose
administration will however be required for this approach.
Regarding the impact of the formulation and process parameters studied in the DOEs of
TTC (Paper III) and decayed 227Th purification (Paper II), differences can be found in
the models with citrate versus acetate buffered formulations, as well as between the
models of purification of TTC versus decayed 227Th. The TTC sorption studied in Paper
III showed similar profiles and range of sorption from citrate and acetate buffered
formulations, but additional borderline effects were significant in the citrate model. The
impact of DOE variables were, however, much smaller for TTC than 223Ra sorption in
the citrate buffered model. This is seen as reflecting the strong complexation ability of
citrate and following impact on the ion exchange of 223Ra [305]. For acetate buffered
formulations, the impact of the DOE was more equivalent for the two statistical models
of TTC and 223Ra sorption.
Differences between citrate and acetate buffered formulations were also seen in the
models of 223Ra and 227Th sorption, studied in Paper II. Resin mass was the only
significant variable for 227Th sorption from citrate buffered formulations. Several
variables as well as interactions were on the other hand significant for 223Ra sorption.
The lower complexation ability of 223Ra may have led to the larger impact of the DOE
variables, while for 227Th the strong complexation to citrate may have overruled the
effect of other formulation parameters [289, 290]. The acetate buffered models showed
the complete opposite effects. 227Th sorption from acetate buffered formulations
involved a complex correlation structure with impact of the pH, pABA+EDTA and resin
Page | 76
amount variables as well as interaction variables. The sorption of 223Ra from acetate
formulations was however only influenced by the presence of pABA+EDTA. This is
likely due to weaker complexation of 223Ra (a calcium(II) mimetic) to acetate than to
citrate, as observed for calcium [288, 305, 306]. In acetate buffered formulations,
EDTA+pABA was the only significant variable, and the presence of EDTA was likely to
control the sorption of 223Ra.
6.7 Purification methods for protein biotherapeutics like monoclonal antibodies and strategies for TTC purification
In the production of biotherapeutics like monoclonal antibodies, several liquid
chromatography steps are used and may include hydrophobic or affinity
chromatography, size exclusion as well as ion exchange [307, 308]. With regard to ion
exchange, many parameters regarding operation and chromatography require
consideration. The ion exchange sorption of proteins depends amongst others on the
composition of the protein samples including buffer, pH and other
excipients/substances, as well as properties of the ion exchange material, flow rate and
sample load [307]. In the studies of TTC purification in Paper III (as discussed in the
previous section), the effects of certain formulation and process parameters on the
sorption of TTC to the selected strong cation exchange resin were studied. A relatively
high TTC sorption was found even though the resin surface properties and pore size
were seen as optimized for minimal TTC sorption to the ion exchange resin. I this
section, aspects regarding ion exchange of protein biotherapeutics like monoclonal
antibodies are further discussed, to highlight its complexity compared to ion exchange
of radionuclides. Also, an alternative strategy of the application of size exclusion
chromatography for TTC purification is discussed.
In ion exchange chromatography of inorganic ions like 223Ra/radium(II) and 227Th/thorium(IV), the charge of the species is said to determine the selectivity between
Page | 77
and separation of the species in the given liquid phase and solid phase resin [303, 309].
For proteins like monoclonal antibodies, however, the overall charge, charge density
and surface charge distribution will influence their interaction with the ion exchange
material. Proteins are amphoteric and consist of multiple charged groups with different
acid dissociation constant (pKa) values. A unique combination of net charge versus pH
exists for these molecules, and at the pH of the isoelectric point (pI) the protein is said
to have no net charge. At pH below the isoelectric point the protein has a net positive
charge and affinity for a cation exchanger is expected, while at a pH above its pI affinity
for an anion exchanger is assumed to prevail [310, 311].
The interaction between the functional groups of the ion exchange material and proteins
has, however, been shown to be more complex than for inorganic ions or smaller
molecules. Examples of sorption of proteins close to the isoelectric point are known.
Minor hydrophobic interactions and uneven surface charge distribution, which can only
be fully understood by using three-dimensional structures, are thought to explain the
phenomenon [312]. From studies of protein sorption to ion exchange resins, Noh et al.
reported that sorption to the ion exchange surfaces were poorly predicted by pI of the
proteins and that “energy-compensating interactions between water, protein and ion-
exchange surfaces” were more important [313]. In addition, the pore size, surface
characteristics and other physical and chemical properties of the surface of the material
have to be taken into consideration when studying protein sorption [307, 312, 314].
Interactions between formulation parameters like pH and salt concentration can also be
expected, as e.g. in a study by Gao et al. where a salt-independent sorption of bovine
serum albumin was observed close to the pI of the protein, while at other pH levels high
salt concentration did not favour sorption [315]. Chromatographic retention models are
thus also less straight forward for proteins and can be either stoichiometric or non-
stoichiometric [310].
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The results of TTC sorption presented in Paper III support the complexity of protein ion
exchange as well as impact of formulation parameters, especially in the citrate buffered
formulations. In the statistical citrate buffered model, a decreased pH increased TTC
sorption which may be correlated to an increased net positive charge of the TTC and
binding to unbonded silanol groups on the resin surface. In addition, a decreased buffer
and sodium chloride concentration led to a lower TTC sorption (borderline significant
effects).
Renewed interest has been gained for size-exclusion chromatography (SEC) with the
increasing number of developed protein biotherapeutics [316]. Due to the risk of
compromising both efficacy and safety of these products, aggregation is of particular
concern [317]. SEC can be used to investigate both purity and aggregation of protein
based products [316, 318]. No interaction between the analyte and the solid phase is
present in pure SEC, and separation of molecules in the sample is based solely on size
(i.e. hydrodynamic volume) with little influence from sample composition [316]. Instead
flow rate, length of the column, mass of stationary phase and sample load may be
manipulated [318]. Interactions with the solid phase may, however, occur which require
actions regarding chromatographic parameters. Further, the hydrodynamic volume of
the protein may be influenced by the sample composition [316, 318].
There are several descriptions of the use of one-time disposable, open column SEC for
purification of monoclonal antibodies after radiolabelling. The main purpose of the SEC
purification of these products is the removal free radionuclides after the radiolabelling
procedure due to varying yields of radiolabelling of e.g. 225Ac, 89Zr, 111In, 211At and 117Lu
with the used chelators [319-324]. SEC has also been used in the early development of
TTCs where a derivate of DOTA (with poor labelling yield) was utilized [105]. Poor
labelling yields are no longer a challenge for TTC development due to the utilized in-
house octadentate chelator (see section 2.5). However, a purification procedure is
needed for the purpose of removal of the long lived daughter nuclide 223Ra which is not
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radiolabelled to the antibody-chelator conjugate [15, 16]. SEC is nonetheless judged as
a possible advantageous purification procedure also for the current TTCs which should
be further explored. Reasons include [325]:
• RCP: molecules can be separated based on size and a high RCP of the collected
TTC eluate for further use can be achieved by the method
• RNP: 223Ra and progenies can be separated from the TTC eluate
• Aggregation and mAb fragments may be separated from TTC eluate dependent on
among others size exclusion limit of the stationary phase material and column length
• The TTC eluate may contain TTC in the final drug product formulation given that the
SEC column is equilibrated with this formulation
• Pure SEC is less likely to influence the composition of the eluate compared to ion
exchange
• Pure SEC is not influenced by formulation parameters and may be used for different
TTC formulations given that the hydrodynamic volume of the TTC is not significantly
affected by the formulation
• No use of strong acids for radiochemical separation of 227Th and 223Ra is required
• TTC can be purified in situ directly after radiolabelling or by purification of decayed
TTC. This means less handling on sites of patient dose preparation compared to a
separate purification of decayed 227Th before radiolabelling
• If pure SEC purification can be developed, the procedure may be more gentle
regarding TTC stability and conservation of biological activity, as e.g. binding
properties
For the application of an in situ purification method of TTCs, one-time disposable, open
column SEC is seen as most applicable. Separation of TTC and 223Ra may in this
application be largely controlled by column, void and applied sample volumes. Thus, the
RAC of the TTC sample and impact on radiolysis, as well operational robustness of the
Page | 80
method regarding volumes, column length, stationary phase material particle size, as
well as size exclusion limit of the material must be explored.
6.8 Purification of decayed 227Th; sorption of 223Ra versus other short-lived daughter nuclides
As discussed in Paper II and III and presented in section 5.1, the rationale for focusing
on removal of 223Ra and not the other nuclides in the decay chain of 227Th is the short
half-life of the other daughter nuclides (see Figure 6, page 40). Within 5 seconds of the
decay of 223Ra the first three alpha emissions have occurred (219Rn, 215Po). 211Pb
decays via beta emission and has a half-life of 36.1 minutes which is the longest of the 223Ra daughters. From 211Pb decay, the daughter 211Bi is generated which decay further
by alpha emission within 2.2 minutes. 211Pb is in other words the daughter with the
longest half-life. However, after removal of 223Ra, half of the amount of 211Pb has
decayed after ~36 minutes. Even though the product will be purified in situ at the site of
patient dose administration, the sterile filtration, quality control testing and readying of
the dose for administration is likely to be performed within a time frame that results in a
very limited amount of the daughters of 223Ra remaining in the preparation. Also, as
presented in section 2.12.3, the preclinical toxicological data with 48 hours in-growth of 223Ra and daughters has not revealed any safety risks.
Section 5.1 shows sorption data for 227Th, 223Ra, 219Rn, 211Pb and 211Bi as detected by
HPGe-gamma spectroscopy, by use of the materials tested by the batch method in
Paper I and the PSA strong cation exchange resin studied in Paper II and III. The data
in Table 7 (p.58), showing the activity (in kBq) of the radionuclides in the materials from
passive diffusional sorption, indicate an equal level of sorption of 223Ra and daughters in
the respective materials after 60 minutes equilibration. The activity of 223Ra and the
daughters 219Rn, 211Pb and 211Bi in the respective materials are judged to be equal by
means of the method. If only 223Ra was retained by the materials, the activity of the
Page | 81
daughter nuclides would be lower. This is because a new equilibrium would have to be
established from the 223Ra activity in the material for the activity of the daughters to be
equal to that of 223Ra (after one half-life of 223Ra, i.e. 11.4 days). The activity of 223Ra
and daughters in the supernatant was also on the same level, which further support an
equal sorption of the radionuclides (data not shown). Thus, the levels of 223Ra sorption
presented in Paper I are likely to be representative also of the sorption of the daughters
of 223Ra.
Section 5.1 also shows data for randomly selected DOE samples tested with the
column method and the PSA strong cation exchange resin in Paper II and III. In these
studies, the samples were centrifuged through the resin bed almost immediately after
application of the sample to the micro-spin columns. The data differ for the purification
of decayed 227Th (Table 8 and Table 9) to the purification of radiolabelled decayed 227Th
(Table 10 and Table 11). For decayed 227Th, the activity levels of 223Ra and daughters
(kBq) detected in the PSA resin in both citrate and acetate buffered formulations are
judged to be equal by means of the method, and sorption of both 223Ra and daughters is
thus likely (Table 8). For the acetate buffered formulations, the sorption was almost
quantitative and no daughters of 223Ra were detected in the eluate (Table 9). For the
presented citrate buffered formulation, a lower level of sorption of both 223Ra and
daughters were achieved, and some activities could be detected in the eluate at the
time of measurement (Table 9).
Table 10 and Table 11 in section 5.1 show the activity of radionuclides (in kBq) in the
PSA resin and in the corresponding eluates for randomly selected DOE samples from
the purification of TTC, as presented in Paper III. There is a likely lower sorption of both 211Pb and 211Bi compared to 223Ra and 219Rn from the presented citrate and acetate
buffered samples. As can be seen in Table 10, the activity of both 211Pb and 211Bi in the
resin was lower than the activity of 223Ra (and 219Rn). The corresponding activity levels
of 211Pb and 211Bi in the eluates in Table 11 was also significantly higher than for 223Ra
Page | 82
(and 219Rn), especially from the acetate buffered samples. In contrast to the samples in
Paper II, the DOE for purification of TTC in paper III included sodium chloride as a
variable and may explain why there was a lower sorption of 211Pb and 211Bi from the
TTC samples. However, the impact of formulation (especially buffer type) and process
parameters must be further explored to explain the trends indicated by these data.
Nonetheless, the observed higher activity of 211Pb and 211Bi in the eluate after TTC
purification is judged to pose no risk to patient safety due to the short half-life of these
radionuclides, and toxicological data show no increased toxicity at significantly higher
levels of radionuclide activities (ref. section 2.12.3).
6.9 Statistical models
In Paper II and III the separation of 223Ra and 227Th/TTC, respectively, were studied by
the aid of the DOEs presented in section 4.3.1 and the statistical methods presented in
4.3.2. Statistical models for 223Ra and 227Th or TTC sorption in citrate and acetate
buffered formulations were elaborated, and analyses of the impact of formulation and
process parameters for both purification of decayed 227Th (paper II) and decayed TTC
(paper III) were conducted.
6.9.1 Model uncertainties
The predictive ability of multivariate models has been tested by cross validation in
various ways to evaluate the robustness of radionuclide sorption. However, the true
performance and reproducibility of sorption by micro-spin columns will be dependent on
a greater number of runs than tested in this thesis. The influence of significantly higher
concentrations of the radionuclides and less experienced users may affect the optimal
variable combination and reproducibility presented in Paper II and III. In some models
the variation suggested slightly wider confidence intervals (CI) for sorption than wished
Page | 83
for. Examples include the sorption of 227Th from acetate buffered formulations in Paper
II with a ±16% 95% CI, and sorption of 223Ra from citrate buffered formulations in paper
III with a ±15% 95% CI. Whether the variations represented the true standard
distributed variation, any laboratory error, or important but non-studied variables will
need further testing.
6.9.2 Future DoE application
The further use of micro-spin columns may require slight update of variable effects and
optimal variable conditions. Testing of other formulation and process variables and
levels than performed in Paper II and III, may further optimize the sorption and
separation between radionuclides as well as the reproducibility. The effect of different
alternative resin materials (type, pore size, particle size, packing etc.) and their masses
in combination with clinically relevant levels of radioactivity of radionuclides and TTC
should be retested and fine-tuned to find the optimal operational settings. The DOE
concept should be applied so that any new variables are tested together with the ones
already studied (pH, buffer type and concentration, resin amount, inclusion of
pABA+EDTA and/or sodium chloride), to understand the impact on the optimal sorption
conditions. Evaluating the effect of a newly introduced variable normally requires testing
on more than one level and consideration of the interplay between variables which often
occurs. A structured fractional testing may in many situations be the only reliable way to
get a proper understanding of the potential for new variables in question. It is not
sufficient to consider the type of materials or excipients only (i.e. qualitative
considerations). To accurately define sorption, it is necessary to find the right levels of
variables (i.e. quantitative considerations).
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6.9.3 Tested radioactivity levels and TTC product requirements
The radioactivity levels tested in this thesis are significantly lower than those that will be
relevant in a clinical setting, but the principle of ALARA and amount of radioactivity
available for the studies were the determining factors. The target activity level of 227Th in
Paper II and III were, respectively, seven and six times lower than that of the lowest
tested clinical dose of 227Th. However, for both purification methods some single
samples with clinically relevant 227Th activities have been tested which were well fitted
into the statistical models (data not shown). The radioactivity levels of the radionuclides
and TTC that are to be purified in a clinical setting should be defined and further tested.
The requirements regarding radionuclide purity, radiochemical purity and other
parameters of a TTC drug product (ref section 6.1), as given by internal Bayer
standards and regulatory requirements, should then be evaluated together with these
clinically relevant process runs. These TTC product parameters will largely affect the
requirements of the method as well as selection of DOE variable conditions.
6.9.4 Statistical models and radiochemical purity of TTC
The RCP testing of TTC has not been performed as systematically as the micro-spin
column formulation and process DOE, even though some clear trends of a lower RCP
with decreased pH and absence of pABA+EDTA seem to exist (as discussed in Paper
III and in section 6.6). The reason behind the limited testing of RCP is the fact that the
variation in RCP became more evident by time. Further process runs where both
sorption and RCP are analyzed will provide an even better foundation for selecting an
optimal variable combination maintaining both sorption and RCP.
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7 Main conclusions and (summary of) suggestions for further research
The main aim of developing a new in situ purification method was to achieve a high and
reproducible sorption of long lived daughter nuclide 223Ra together with a low sorption of 227Th, either as free radionuclide or as TTC. The method should ideally also include a
minimal amount of steps, be user-friendly and utilize non-hazardous materials. The
work in this thesis has resulted in three patent applications, as listed in section 9.
From the sorption material and method screening in Paper I, it was concluded that the
column method was more applicable than the batch method. This was due to the higher
and less variable sorption for materials tested by both methods, and the perceived less
complexity of product development by choosing the column method. A batch method
could in theory involve less product handling and a simpler purification method for those
performing the purification at sites of patient dose administration. However, risk of
compromising product and sorption material quality and stability, and the risk of
variation of 223Ra sorption during the shelf-life of the preparation were seen as negative
factors for the batch method. If the batch method was to be further explored, the DSPG
liposomes showed superiority with a higher and much less variable sorption of
radionuclides combined with a low sorption of the model antibody trastuzumab
compared to the other materials tested by this method. The potential for reduced
radiolysis in the formulation by the presence of sorption material during shelf-life of the
preparation was also supported by the study of H2O2 reduction in Paper I.
The micro-spin columns packed with PSA strong cation exchange resin based on silica
were chosen for the further studies in Paper II and III. The reproducible sorption
observed by the use of the micro-spin columns made them a suitable testing platform
for the DOE studies of formulation and process parameters. The use of the micro-spin
columns was, however, judged to involve a too high risk of radioactive contamination for
them to be used on sites of patient dose administration and other columns should be
Page | 86
tested. In addition, their use warrants that the impact of centrifugation on TTC stability is
further explored.
The ion exchange procedure with formulation buffers studied in Paper II is seen as
having the potential to be developed further into a more user-friendly purification
procedure of decayed 227Th (227Th with presence of daughter nuclide 223Ra) compared
to the established procedure at Bayer with the use of strong acids and multiple steps.
The statistical model for 227Th sorption with citrate buffered formulations showed a
reproducible low sorption, while the acetate buffered model for 223Ra sorption was more
robust than the corresponding citrate model. A low 227Th sorption (<3%) and high 223Ra
sorption (>90%) could, however, be obtained from both the citrate and acetate buffered
formulations. Parameters relating to the finished TTC product, the purification method
and sorption materials must, however, be further explored and include:
• Robustness of the method regarding RNP
• Robustness of the method regarding 227Th yield
• Testing of clinically relevant levels of radioactive concentrations (and volumes) of 223Ra and 227Th
• Impact on formulation stability and excipients
• Impact on sorption material
• Optimization of the ion exchange process and resin characteristics
The ion exchange purification of TTC as studied in Paper III is more challenging
compared to the purification of free radionuclides, both with regards to the tested RCP
and desirable separation with high 223Ra and low TTC sorption. A compromise had to
be made with a relatively high TTC sorption (<25%) to achieve an accompanying
acceptably high 223Ra sorption (>90%).
Page | 87
The statistical models presented in both Paper II and III are based on a limited number
of process runs. They should be further studied and developed regarding robustness
and predictive ability, as required by further product and method development and
applicable regulatory guidelines.
Purification of TTC, manufactured either by in situ radiolabelling at sites of patient dose
administration or shipped from centralized sites, has the potential to be more user-
friendly and less labor intensive than purification of decayed 227Th. Ion exchange was
studied in this thesis since evaluation of formulation parameters was to be part of the
studies. However, purification by size-exclusion may have a larger potential for TTC
purification in this setting than ion exchange and should be studied further. Reasons
include the increased complexity of ion exchange chromatography for proteins like
monoclonal antibodies compared to free inorganic ions like radionuclides, less (or no)
impact of formulation parameters on the separation, a more gentle separation with
regard to TTC stability, and minimization of the number of handling steps with elution of
purified product in the finished drug product formulation.
The further development of an in situ purification method for preparation of TTC drug
product should be evaluated against parameters relating to the ready to use TTC
product, the purification method itself as well as the utilized sorption materials, as
outlined in section 6.1.
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9 Patents
• International Publication Number WO 2014/195423 A1, International publication
date 11 December 2014, Pharmaceutical preparation, Inventors Janne Olsen
Frenvik, Olav B. Ryan, Alan Cuthbertson, Assignee Algeta ASA, Norway, 2014
• United Kingdom Patent Application No. 1600161.2, Decayed thorium
purification, Inventors Janne Olsen Frenvik, Olav B. Ryan, Assignee Bayer AS,
filed January 2016
• United Kingdom Patent Application No. 1600158.8, Thorium complex
purification, Inventors Janne Olsen Frenvik, Olav B. Ryan, Assignee Bayer AS,
filed January 2016
10 Paper I-III