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Page 1: SSe TTe RRe RRaadiuumm-22223 ffrrooomm …

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Page 2: SSe TTe RRe RRaadiuumm-22223 ffrrooomm …

© 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.

Page 3: SSe TTe RRe RRaadiuumm-22223 ffrrooomm …

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

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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

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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.

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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

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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

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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

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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)

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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)

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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

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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.

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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.

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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.

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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.

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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.

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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

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(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].

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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].

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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

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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].

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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,

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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].

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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].

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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

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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

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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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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.

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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].

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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

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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.

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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

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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

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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

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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

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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.

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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].

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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].

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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].

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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

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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].

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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])

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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].

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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

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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

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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

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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).

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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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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

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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.

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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].

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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.

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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.

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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.

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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

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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.

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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.

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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

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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.

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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

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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

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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.

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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.

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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.

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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

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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

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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.

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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

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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

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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

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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

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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.

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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

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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

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(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

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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

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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

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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

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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

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(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

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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

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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%).

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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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[323] C.J. Reist, C.F. Foulon, K. Alston, D.D. Bigner, M.R. Zalutsky, Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate, Nucl. Med. Biol., 26 (1999) 405-411. [324] P. Thakral, S. Singla, M.P. Yadav, A. Vasisht, A. Sharma, S.K. Gupta, C.S. Bal, Snehlata, A. Malhotra, An approach for conjugation of 177Lu-DOTA-SCN-Rituximab (BioSim) & its evaluation for radioimmunotherapy of relapsed & refractory B-cell non Hodgkins lymphoma patients, Indian J. Med. Res., 139 (2014) 544-554. [325] E. Kim, Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, in: Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, Society of Nuclear Medicine, New York, 2011, pp. 75-82.

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

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