IMPD preparation example 1: [11C]choline
Important notice:
[11C]choline is a well established radiopharmaceutical, whose preparation was first
reported 30 years ago. Since that time, several methods of preparation/purification
have been published and adopted by the various involved Small Scale
Radiopharmacies. Thus, [11C]choline may be prepared via [11C]CH3I obtained via the
classic, liquid phase route (by reduction of [11C]CO2 in THF, addition of HI and
subsequent distillation and transfer of [11C]CH3I into the dimethylaminoethanol
precursor) or via the more recent “gas phase” route (with initial formation of [ 11C]CH4,
and subsequent radical reaction with iodine). Even for the methylation reaction and
subsequent purification, the methods may be different. Finally, every applicant could
use different instrumentation (automated synthesis module, cyclotron, target, quality
control equipment).
The proposed example cannot account, for obvious reasons, for all of the
above mentioned methods and techniques. It includes information and experimental
data related to one of the possible preparation routes only.
It is of paramount importance to remember that, during the
preparation of the IMPD, every applicant should include the
specific
description
of
their
own
instrumentation,
radiosynthetic pathway, experimental conditions, methods,
data, etc. and also define their specifications with an
appropriate justification.
2.2.1.S DRUG SUBSTANCE
2.2.1.S.1.1 Nomenclature
Drug substance
IUPAC name: ethanaminium, 2-hydroxy-N,N,N-[11C]trimethylammonium chloride
CAS: (2-hydroxyethyl)-[11C]trimethylammonium chloride
Synonims: [11C]choline, [methyl-11C]choline, [11C]trimethylethanolamine
Radionuclide: C-11
2.2.1.S.1.2 Structure
Fig. 1 – Structure of [11C]choline
Molecular formula: C411CH14ClNO
Molecular weight: 137.62
Stereochemistry: the active substance does not contain chiral carbon atoms
CAS # 67-48-1 (referred to as choline chloride)
2.2.1.S.1.3 General Properties
Physico-chemical properties: [methyl-11C]choline structure includes an atom of the
positron emitting radionuclide C-11, whose characteristics are depicted in Table 1.
11
C decays to the stable isotope 11B, through the emission of β+ (99,8%), with a
physical half-life of 20,38 min.
1
Parent
Nuclide
11
C
6
T1/2
Decay
mode
20.38
min
β+
+ Emax Relative
intensity
960.2
KeV
99.759%
γKeV
Daughter
nuclide
511
11
B
5
Table 1 – C-11 decay scheme (data from F. Ajzenberg-Selove, Nuclear Physics
A506,1 (1990 - http://atom.kaeri.re.kr/cgi-bin/decay?C-11%20EC)
Physicochemical characteristics of [12C]choline chloride
Appearance
White
solid
crystals,
hygroscopic
Solubility in water
650 g/L
Solubility in other media
Highly soluble in methanol,
ethanol
LogP
-3.77
Melting point
244/247°C
pH
Choline chloride, at the
typical working concentration,
forms neutral, or close to
neutral solutions, in water (pH
= 6-7)
LD50 in rats (i.v. injection)
53 mg/Kg
Table 2 – List of selected physicochemical properties of “cold” choline.
As stated in the above Table 2 header, the listed properties refer to the “cold”, non
radioactive [12C]choline. As the difference with [11C]choline is only attributable to the
replacement of a C-11 atom with a C-12 atom (choline chloride), the physicochemical
properties reported in table 2 may also refer to the active substance [11C]choline. Due
to its inherent radioactive nature, most of the typical molecular structural
characterization test (e.g. MS, NMR) are not applicable with the radioactive
compound.
2.2.1.S.2.1 Manufacturer(s)
As stated above, the preparation of [11C]choline is usually a continuous process, and
the active substance is, as a rule, not isolated. Thus, the information related to the
manufacturer will be given in section 2.2.1.P.3.1.
2.2.1.S.2.2 Description of Manufacturing Process and Process Controls
Due to the high emission energy of 11C, combined with the need to use considerable
amounts of starting activity (typically >37 GBq) and the very short half-life of the
above radionuclide, [methyl-11C]choline, as well as most 11C labelled
radiopharmaceuticals, is prepared using fully automated radiosynthesis modules.
They are capable to perform all the necessary operations, from the transfer of the
radionuclide from the cyclotron, to the final formulation as an injectable solution of the
radiopharmaceutical. For these reasons, these radiopharmaceutical preparations are
considered as continuous processes carried out in closed systems. As a
consequence, the active substance, as well as intermediates or by-products, are as a
rule not isolated.
Radionuclide production
11
C is generated “in-target” in the form of [11C]CO2 by means of an accelerated
cyclotron proton beam via the nuclear reaction:
2
14
N(p,)11C
The beam, with a projectile energy usually of 18 MeV, is directed on a target loaded
with a gaseous mixture of 14N2 (99,5%) + O2 (0,5%). 11C “in-target” forming atoms
react with the available oxygen molecules, to yield [11C]CO2, representing a typical
example of the so called “hot chemistry”.
Radionuclide production is fully automated. Typical irradiation conditions are
strictly depending on the actual need and they are defined on a case by case basis.
However, beam current for clinically useful amount of [11C]choline are generally in the
range 30-40 A, while irradiation times are often in the range 40-60 min. The
radionuclide production is carried out in a target made of aluminium with a volume of
50 cm3.
Radiosynthesis of the intermediate [11C]CH3I (gas phase method)
[11C]choline, as well as numerous 11C labelled radiopharmaceuticals, is synthesized
via the formation of the useful intermediate [11C]CH3I, that may be prepared following
two different radiochemical pathways, depending on the physicochemical form of the
reactants. Indeed, they are defined as “gas phase” or “liquid phase (or wet)”
chemistry pathways. [11C]CH3I production via “wet chemistry” method will not be
described in the present document. In the [11C]CH3I “gas phase” preparation, the
cyclotron produced [11C]CO2, purified and concentrated, is mixed with hydrogen (H2)
and then delivered to a column loaded with a suitable nickel catalyst, at the
temperature of 300-350°C, with formation of [11C]CH4.
Water is then removed by passing the gaseous mixture through a column,
downstream to the catalyst, loaded with P2O5 ascarite (NaOH, adsorbed on a silica
pellet, 20-30 mesh); the latter plays the double role of efficiently absorbing the
generated moisture and the unreacted [11C]CO2, as well. [11C]CH4 is then purified and
concentrated by absorption on a suitable inert support (e.g. Carbosphere®), cooled
to low temperature (e.g. -175°C) using e.g. liquid nitrogen.
Heating the column, [11C]CH4 is rapidly released and transferred to a quartz
tube, at 100°C, where it is mixed with sublimated I2, under a helium stream; the two
reactants are then forced to re-circulate through a loop, which include a furnace
previously heated at 720°C, where [11C]CH3I is formed. The reaction mechanism is
as follows.
I2
I
+ 11CH3 H
11
CH3 + I2

2I
11
CH3
+ HI
CH3I
The by-product HI is absorbed by an additional ascarite column, while [11C]CH3I is
concentrated, at room temperature, on a column loaded with a suitable polymeric
matrix (e.g. Porapak® 50-80 mesh, Waters). As the radical reaction sequence above
described is not very efficient, a single passage through the furnace is usually not
sufficient to produce [11C]CH3I with high yield. To overcome this restriction, the
reactants are forced to re-circulate through the loop several times, until the maximum
[11C]CH3I activity is reached, by means of a suitable, low pressure, gas pump.
[11C]CH3I activity is monitored by a radioactivity sensor, located in close proximity to
the solid support column itself. At the end of the [11C]CH3I formation process, the
above column is heated to 200°C, thus allowing the release and transfer of the
3
desired intermediate, under a helium stream, to the reaction vial, previously loaded
with the DMAE precursor.
Radiosynthesis and purification of [11C]choline
Reaction vial has to be previously loaded with the DMAE precursor dissolved in
acetonitrile. The methylation reaction takes place at 70°C for 3 min. The reaction
schematic is as follows:
Fig. 2 – Radiosynthesis of [11C]choline
At the end of the above reaction, the mixture is purified through a cation exchange
column, which selectively trap [11C]choline. The desired product is then eluted using
an adequate volume of saline physiological solution, sterilized through a 0,22 μm
filter, and collected in a sterile, pyrogen free glass vial. With the aim to avoid
overpressure into the collection vial, a second, vented 0,22 μm filter should be
inserted through the rubber closure.
The overall synthesis time is 30 min. The radiochemical yield, calculated as
the ratio between the final [11C]choline and the [11C]CH3I activity, is in the range 2535%, corrected for decay. The radiochemical yield is not determined by taking the
starting [11C]CO2 or [11C]CH4 as the references, as their activities are difficult to
quantitate with the radiosynthesis module, due to the extremely variable geometry of
the above gases when they are trapped by the respective columns.
As already stated above, the [11C]choline radiosynthesis, purification and
formulation is an automated, continuous process, carried out in a closed system (the
“synthesis module”). For this reasons, the active principle [11C]choline, as well as
other intermediates, are, as a rule, not isolated.
The synthesis module may be considered as a closed system because the
starting materials (reagents, solvents, precursor), the intermediates and the final
product, including the purification subset, are never exposed to the external
atmosphere in any step of the whole process.
2.2.1.S.2.3 Control of Materials
For most of the starting materials, controls are limited to a visual inspection, to verify
the integrity of the packaging and products, and a check on the analysis certificate
and expiry date.
The most critical starting material is certainly the DMAE precursor. Moreover,
it has to be underlined that in case of “gas phase” method, several starting materials
might be re-used several times, and they are not replaced at the end of every
synthesis. Examples are represented by I2, the nickel catalyst, the solid support used
to trap volatile by-products and/or reagents, such as Carbosphere, Porapak, ascarite
or P2O5. They are indeed replaced every 10 synthesis or more, depending on their
stability and the rate of contamination.
In Table 3 a list of the starting materials used in the preparation of
11
[ C]choline via “gas phase” method using a GE Tracerlab FxC-Pro, together with the
proposed verification test, is reported.
4
Materials
Dimethylaminoethanol
(DMAE)
[11C]CO2
P2O5
N-14 + 0.1-1% O2
Test and acceptance criteria
HPLC; purity should conform with the indication of the
supplier, based on analysis certificate
C-11 has T1/2 = 20.3 min; this starting material cannot
be isolated and tested during the radiosynthetic
process
See the attached analysis certificate
See the attached analysis certificate; additionally, an
irradiation test is performed every time the gas cylinder
containing the gaseous mixture N2 + O2 is replaced
See the attached analysis certificate
See the attached analysis certificate
See the attached analysis certificate
See the attached analysis certificate
Water for injection
EtOH
Iodine
NaCl 0,9% injectable
solution
CH3CN
See the attached analysis certificate
Ni catalyst
See the attached analysis certificate
H2
See the attached analysis certificate
Carbosphere
See the attached analysis certificate
Porapak
See the attached analysis certificate
Accel CM cartridge
See the attached analysis certificate
(SepPak)
Acrodisc 0,2 µm filter,
See the attached analysis certificate
Supor membrane
Sterile vials
See the attached analysis certificate
Vented Millex GS 0,22 µ
See the attached analysis certificate
filter
Table 3 – List of the starting materials used in the preparation of [11C]choline via “gas
phase” method using a GE Tracerlab FxC-Pro.
2.2.1.S.2.4 Control of Critical Steps and Intermediates
The “in-process” controls are limited to the monitoring of the critical parameters (e.g.
activity, reaction temperatures and pressures, inert gas flow rate) through the
graphical control software interface. Printouts of representative preparation process
diagrams are usually provided. A full quality control program is set for the final
product (see section # 2.2.1.P.4.2.)
2.2.1.S.2.5 Process Validation and/or Evaluation
Please refer to the section 2.2.1.P.3.5
2.2.1.S.2.6. Manufacturing Process Development
Please refer to the section 2.2.1.P.2.3
2.1.2.S.3 Characterisation:
2.1.2.S.3.1 Elucidation of Structure and other Characteristics
[12C]choline is provided with a suitable CoA, which states that NMR and MS spectra
have been performed to confirm the choline structure. No further structure elucidating
analyses are requested. With the aim to confirm the purity, an analytical HPLC
control of the reference standard is performed. The experimental conditions are given
in the section # 2.2.1.P.4.2.
5
2.1.2.S.3.2 Impurities
Radionuclidic purity:
Several European Pharmacopoeia Monographs of 11C labelled radiopharmaceuticals
have been published to date. Acceptance criteria for radionuclidic purity for
[11C]methionine, [11C]raclopride, [11C]flumazenil, [11C]acetate is usually set to > 99%
of 11C, with maximum admitted amount of other radioisotopic contaminants being no
more than 1%. As the radionuclidic impurities are generated during the cyclotron
irradiation process, and considering that there are no significant differences between
the [11C]choline preparation procedure and the above cited 11C radiolabelled
compound, the same acceptance criteria has been adopted for the interested
radiopharmaceutical.
As for the possible radioisotopic contaminants, in addition to the main nuclear
reaction 14N(p,)11C, there are other nuclear reactions that may take place at the
cyclotron target level. The most prominent are 14N(p,n)14O and 16O(p,)13N.
Other impurities arising from the irradiation of target foils (vacuum foil and
target foil) are usually not of concern in case of gaseous target products such as
[11C]CO2, as they do not transfer from the solid foils to the gaseous phase, and they
are thus not swept out by the target during the unloading process.
13
N and 14O are both positron emitting radionuclides, and their presence
cannot thus be detected through the gamma emission spectra analysis. On the other
hand, 14O may be ruled out as a significant contaminant, considering its ultra-short
half-life (T1/2 = 70.6 sec), combined with an average elapsed time between the end of
bombardment (EOB) and the end of the synthesis (EOS) of 15-30 min, which means
a number of decays in the range 12-25, more than enough to remove any trace of the
above impurity. The only radionuclidic contaminant potentially present in the final
product at End of Synthesis (EOS) is thus 13N, in the form of gaseous 13N labelled
oxides (e.g. [13N]NO, [13N]NO2, etc.). Decay profile analysis may be conducted (see
also section # 2.2.1.P.5.1 and Ph. Eur. monograph N. 0125, “Radiopharmaceuticals”)
by measuring the activity of a radiopharmaceutical sample at three time points (T0, T1
and T2), using a suitable, calibrated instrument (e.g. dose calibrator, gamma
spectrometry).
Specification for 11C half-life, as reported in the Ph. Eur. chapt. N. 50700
(“Table of physical characteristics of radionuclides”) is 20.385 min. The above cited
Ph. Eur. monographs for 11C labelled radiopharmaceuticals usually report an
acceptance criteria in the range 19.9-20.9 min (i.e. ±2.5%), which has been adopted
in the present document.
Radiochemical purity
The acceptance criteria for radiochemical purity might be, as already seen for
radionuclidic purity, defined on the basis of the relevant published Ph. Eur.
monographs for other 11C labelled radiopharmaceuticals, and set to > 95% of
[11C]choline. Analytical method for radiochemical purity determination is analytical
HPLC with integrated radiochemical detector. More details are given in section
2.2.1.P.5.2.
Chemical purity
The more plausible chemical impurities in [11C]choline preparations, are: i) the DMAE
precursor, and ii) residual solvents such as acetonitrile, which is typically the reaction
solvent, and ethanol/acetone, which are used for radiosynthesis module cleaning
purposes. As neither dedicated Ph. Eur. monograph is available, nor the other 11C
labelled radiopharmaceutical relevant monographs are of help in this case, the
acceptance limit for the precursor DMAE has been established on the scientific
literature and toxicity data basis (see for instance the paper “Doses as high as 1200
mg daily produce no serious side effects; and single 2500 mg dose taken in suicide
attempt had no adverse effects”. [Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N.
6
Gleason. Clinical Toxicology of Commercial Products. 4th ed. Baltimore: Williams
and Wilkins, 1976., p. II-240]), which indicate how the amount of precursor that could
be likely present in the final preparation, even considering the worst case where all
the DMAE used in the labelling reaction is detected in the finished product vial, is not
of concern from the toxicity point of view, and negligible risks are thus associated.
As for the residual solvents, their limits are defined in the document “EMEA
note for guidance on impurities: residual solvents” (CPMP/ICH/283/95), and in
chapter 5.4 of Ph. Eur. The analysis is usually performed using gas-chromatography.
More details are given in the section 2.2.1.P.5.2.
2.2.1.S.4 Control of the Drug Substance:
Details on methods for [11C]choline analysis, their validation, the batch analysis, and
the justification of specifications will be provided in the appropriate 2.1.P sub-sections
2.2.1.S.5 Reference Standards or Materials:
The list of reference standards is provided in Table 4. There are two distinct kind of
reference standard: i) chemical standard, ii) radionuclide calibrated sources. As for
the chemical standard, it has to be noted that a choline bitartrate standard has been
added to the list. Indeed, the high hygroscopicity of the true standard choline
chloride, make it difficult to handle and accurately weigh, thus decreasing the
reliability of repeatability and linearity test.
The chemical reference standards are commercially available, chemical
grade products. The specifications for purity have been set by the supplier, and
accepted by the Applicant. The specifications, and most of the analytical test (e.g.
1
H-NMR and/or 13C-NMR, mass spectrometry) are performed by the supplier, and
described in the attached Certificate of Analysis. The chemical reference standards
are re-tested for chemical purity by the applicant using HPLC, as detailed in the
section 2.2.1.P.5.
The radioactive reference standards are used to verify the calibration status
of the gamma spectrometer and the dose calibrator, respectively. Their composition
and the activity(ies) of the radionuclide(s) at reference time and date are described in
the attached Certificate of Analysis. The calibrated source are metrologically
referable to recognized standards, and a verification of their identity or purity is in this
case non applicable
Reference
standard
Choline bitartrate
Choline chloride
Multinuclide
source
Mononuclide
source
Aim
Choline identity
Choline identity
Gamma spectrometer
calibration
Test
HPLC
HPLC
Not
applicabile
Not
Dose calibrator
applicabile
Table 4 - List of reference standards
Acceptance
criteria
Purity > 95%
Purity > 95%
Not applicabile
Not applicabile
2.2.1.S.6 Container Closure System
For the reasons already stated above, container closure system will be described for
the drug product, in the appropriate sections.
2.2.1.S.7 Stability
For the reasons already stated above, stability will be described for the drug product,
in the appropriate sections.
7
2.2.1.P INVESTIGATIONAL MEDICINAL PRODUCT UNDER TEST
2.2.1.P.1 Description and Composition of the Investigational Medicinal Product:
Each vial contains 15 mL of a saline physiological solution of [11C]choline, with
radioactive concentration in the range 250-3700 GBq/mL, at reference time.
Reference time is taken as the time when the activity contained in the finished drug
product is measured with the dose calibrator. The [11C]choline solution is contained in
20 mL borosilicate glass vials (Ph. Eur. type I), sealed with bromobutyl rubber
stopper (Ph. Eur. compliant), which is in turn secured with an aluminum flip-off cap.
Composition
The composition of the finished drug product is given in Table 5:
Component
Quantity
Function
Reference
[ C]choline
370-3700 MBq/mL Active substance N.A.
NaCl 0,9% solution 14-16 mL
Excipient
Ph. Eur.
11
Table 5 – Composition of the injectable solution of [ C]choline
11
2.2.1.P.2 Pharmaceutical Development
[11C]choline is prepared using an automated synthesis module, by means of which
the desired product is simply purified, at the end of the labelling reaction, via the well
known “solid phase extraction” technique, using cation exchange cartridge, which
selectively retains [11C]choline. After washing the cartridge with water, [11C]choline is
then eluted with saline physiological solution. The final formulation is thus very simple
and NaCl 0.9% is the only excipient included in the product vial. The active
substance is never isolated in the course of the preparation process, and it is
available for quality controls only at the end of formulation step. The finished product
has to be considered as a multi-dose preparation, and the activity at reference time
may vary, depending on the clinical trial need (e.g. the higher the number of daily
subjects, the higher the number of individual radiopharmaceutical doses that should
be prepared).
Stability studies have been performed, demonstrating that the composition of
the [11C]choline solution does not undergo significant changes to chemical and
radiochemical purity during the considered time (2 hours). For more information
about stability, see section 2.2.1.P.8.
2.2.1.P.2.3 Manufacturing Process Development
Not applicable
2.2.1.P.3 Manufacture:
2.2.1.P.3.1 Manufacturer(s)
Institution Name
Address
Person responsible for the
small scale preparation of
radiopharmaceuticals
e-mail address
Phone
Fax
2.2.1.P.3.2 Batch Formula
A batch of [11C]choline is usually represented by a single, multi-dose vial. The
materials used in the preparation of a typical batch via the “gas phase” method and
8
using a GE Tracerlab FxC-Pro radiosynthesis module are listed in Table 6. The
starting materials marked by an asterisk are not single use, and they are replaced
with a frequency established during the automated device validation procedures.
Starting materials
Amounts
DMAE
0,025 mL
*P2O5
2g
Water for injections (WFI)
10 mL
*Ascarite
15 g
*Iodine
3,5 g
NaCl 0,9% physiological solution 15 mL
CH3CN
0,475 mL
*Ni Catalyst
200 mg
H2
50 mL/min
*Carbosphere
450 mg
*Molecular sieves
300 mg
SepPak Accell cartridge
1
Table 6 – List of starting materials used in a [11C]choline
batch production via “gas-phase”
Batches of [11C]choline to be used in clinical trials are typically made of a single vial.
2.2.1.P.3.3 Description of Manufacturing Process and Process Controls
The [11C]choline preparation process has already been described in section
2.2.1.S.2.2 of the present document. However, a flow chart is added below:
9
Fig. 3: flowchart of the preparation process of [methyl-11C]choline
2.2.1.P.3.4 Controls of Critical Steps and Intermediates
Description of in-process controls and intermediated has already been given in
section 2.2.1.S.2.4
2.2.1.P.3.5 Process Validation and/or Evaluation
Process validation has been performed by preparing and controlling three
consecutive batches of [11C]choline. Validation runs have been performed in the
same operating conditions (e.g. starting [11C]CO2 activity, amount of precursor,
physicochemical reaction parameters) characteristic of the process; i.e. using the
same instrumentation and starting materials in the stated quantities normally set for
typical runs.
The parameters evaluated and their acceptance criteria are found in Table 7:
10
Parameters
Acceptance criteria
Radioactive concentration
0.37 - 3.7 GBq/mL
Final volume
15  1 mL
Table 7 – Production parameters evaluated during the process validation
The experimental data are reported in the following table:
[11C]choline activity
Radioactive
concentration
Volume
Conform
BATCH 1
BATCH 2
BATCH 3
(Prod.
(Prod.
(Prod.
29/09/10)
07/10/10)
08/10/10)
10,7 GBq
8,4 GBq
10,22 GBq
0,713 GBq/mL
0,56 GBq/mL
0,681 GBq/mL
15 mL
15 mL
15 mL
□ yes
□ No
□ yes
□ No
□ yes
□ No
Table 8 – Experimental production data for process validation
Each batch has been analyzed following the complete quality control program. The
results are summarized in Table 9. Specifications are described in section
2.2.1.P.5.1.
11
QUALITY CONTROL
Batch 1
Acceptance
Test
Batch 2
Batch 3
Confor
results
Conform
results
Conform
results
4.5-8.5
6.5
□ Yes □ No
6.22
□ Yes □ No
6.47
Clear,
colourless
≤ 4.1
mg/Vmax*
≤ 50
mg/Vmax*
0.1 mg/Vmax*
Clear,
colourless
4.37
g/mL
414
g/mL
0
□ Yes □ No
□ Yes □ No
Clear,
colourless
□ Yes □ No
0 mg
□ Yes □ No
570 g/mL
□ Yes □ No
Clear,
colourless
1.38
g/mL
287
g/mL
0
□ Yes □ No
0
100%
□ Yes □ No
100%
□ Yes □ No
100%
506 KeV
□ Yes □ No
505 KeV
□ Yes □ No
505 KeV
 99.9%
 99.9%
□ Yes □ No
 99.9%
□ Yes □ No
 99.9%
Half-life
19.9-20.9
min
20.35 min
□ Yes □ No
20.6 min
□ Yes □ No
20.2 min
Bubble point
≥ 46 psig
48 psig
□ Yes □ No
55 psig
□ Yes □ No
48 psig
□ Yes □
No
Sterile
□ Yes □ No
Sterile
□ Yes □ No
Sterile
□ Yes □
No
<5
EU/mL
□ Yes □ No
<5
EU/mL
□ Yes □ No
< 5 EU/mL
□ Yes □
No
criteria
pH
Appearance
CH3CN
EtOH
DMAE
Radiochemical
purity
Identification:
gamma spect.
Radionuclidic
purity
Sterility
Bacterial
endotoxins
≥ 95% of
[11C]choline
511  10
KeV
Ph. Eur.
Conform
Ph. Eur.
Conform
(<175/Vmax* in
IU/mL)
□ Yes □ No
□ Yes □ No
Table 9 – Experimental quality control data for process validation
*= max. recommended dose (mL)
2.2.1.P.4 Control of Excipients:
2.2.1.P.4.1 Specifications
The excipients used in the preparation process of the finished investigational
radiopharmaceutical should meet the specifications reported in Table 10.
Excipient
Specification
0.9% NaCl
Ph. Eur.
Table 10 – List of excipients
The 0.9% NaCl solution is approved following a check for packaging integrity, expiry
and Certificate of Analysis. The appearance should also be verified. For the above
reasons, for excipient control there is no need to describe analytical procedures, as
well as their validation and justification of specification(s). No novel excipients have
been used.
2.2.1.P.5 Control of the Investigational Medicinal Product:
2.2.1.P.5.1 Specifications
Each batch of [11C]choline is submitted to quality control, with the aim to evaluate
chemical, radiochemical, radionuclidic and biological purity of the finished product.
The QC tests are summarized in Table 11.
12
m
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
□ Yes □
No
Parameters
Appearance
Test
Visual
inspection
Identification
HPLC
Identification
Gamma
Spectrometry
Radiochemical purity
DMAE
Chemical Purity
EtOH*
CH3CN*
Radionuclidic purity*
Half-life
pH
Bacterial endotoxins*
Filter integrity
Sterility*
HPLC
HPLC
GC
GC
Gamma
spectrometry
Gamma
spectrometry/
Dose
calibrator
pH meter
Specification
Clear, colourless solution
The principal peak in the
radiochromatogram obtained with the test
solution of [11C]choline has approximately
the same retention time as the principal
peak in the chromatogram obtained with a
reference solution of “cold” choline
chloride (or choline bitartrate)
Gamma photons have an energy of 511
KeV and, depending on the measurement
geometry, a sum peak of 1022 KeV may
be observed
[11C]choline ≥95%
≤0.1 mg/max. recommended dose (mL)
≤50 mg/max. recommended dose (mL)
≤4.1 mg/max. recommended dose (mL)
11C
≥ 99 %
19.9-20.9 min
4,5-8,5
Ph. Eur.
<175 EU/ max. recommended dose (mL)
Bubble point
Following specification of the supplier
Ph. Eur.
Sterile
Table 11 – Specifications and acceptance criteria for [11C]choline
*The tests labelled with an asterisk are performed after the release of the
radiopharmaceutical, due to both the test duration not compatible with radionuclide
half-life (e.g. sterility test or radionuclidic purity), and/or for radiation protection
reasons (e.g. bacterial endotoxins test).
2.2.1.P.5.2 Analytical Procedures
Analytical procedures are described in detail in relevant Standard Operating
Procedures, which are available on request.
2.2.1.P.5.2.1 Identification and determination of the chemical (DMAE) and
radiochemical purity using HPLC
[11C]choline analysis: specifications
- Identification: the main radiochemical peak should have the same
retention time evidenced by the SST test for standard choline chloride (or
bitartrate) ±0.2 min
- Radiochemical purity: peak area for [11C]choline has to be > 95% of the
total peak areas
- Chemical purity (DMAE): the peak area for DMAE should not be more
than the corresponding area of the peak obtained with reference SST
solution
13
2.2.1.P.5.2.2 Determination of residual solvents using gas-chromatography
Instrumentation:
Gas-chromatograph
 Head space injection system
 FID-Detector (Flame Ionization Detector)
 Capillary column: 30m, 0.32 mm, 1 µm,
 Acquisition data system
 Carrier Gas: helium
Operative conditions:



Temperature ramp: 0-80°C at 10°C/min, etc.
Flow: 1mL/min
Acquisition time: 10min
[11C]choline analysis: specification
-
Acetonitrile: maximum admitted concentration = 4.1 mg/V, where V is the
maximum recommended dose (mL).
-
Ethanol: maximum admitted concentration = 50 mg/V, where V is the
maximum recommended dose (mL).
2.2.1.P.5.2.3 Gamma spectrometry
Instrumentation:
 Gamma Spectrometer
 NaI detector
 Acquisition data software
Identification
Method: the activity of the [11C]choline sample should be in a suitable range, such
that the instrument dead time is < 5%. Analyze the sample for a suitable time (e.g. 10
min), and energy spectrum (e.g. 0-1800 KeV).
Specification: the only gamma photons have an energy of 0.511 ± 10% MeV and,
depending on the measurement geometry, a sum peak of 1.022 MeV may be
observed.
Radionuclidic Purity
Method: a sample of [11C]choline with suitable volume and geometry is left to decay
for a time sufficient to allow the complete decay of the main radionuclide (11C).
Usually this decay time should be estimated such as the remaining 11C activity
should be negligible (e.g. Tdecay >20 half-lives). Analyze the sample for a suitable time
(e.g. 60 min), and energy spectrum (e.g. 0-1800 KeV).
Specifications: the sum of the areas due to the possible contaminants, recalculated
to the activity at shelf-life of the radiopharmaceutical preparation, and related to the
total radioactivity of the preparation itself, should be <1% of the total peak areas.
2.2.1.P.5.2.4 Determination of the half-life
Instrumentation:
 Gamma Spectrometry
 NaI detector
 Acquisition data software
14
Or

Dose calibrator
Method:
Decay analysis should be performed by measuring the sample at least two times (T 0
and T1), with a suitable interval between T0 and T1 (e.g. 10 min).
[11C]choline: specifications
Half-life of [11C]choline should be in the range 19.9-20.9 min. Calculations may be
done using the decay equation:
where:
-
t = time between the two activity measurements
N0 = activity at T0
N = activity at T1
2.2.1.P.5.2.5 Determination of pH
Instrumentation: pH meter
- Before to proceed with the analysis, a calibration test using suitable reference
buffer solutions has to be performed.
-
Insert the pHmeter electrode into the sample solution, and record the value
[11C]choline analysis: specification
pH of the test solution should be in the range 4.5-8.5
2.2.1.P.5.2.6 Endotoxins
Instrumentation:
 Endosafe PTS Reader
[11C]choline analysis: specifications
Bacterial endotoxins level should be <175 EU/V, where V is the maximum
recommended dose (mL).
2.2.1.P.5.2.7 Filter Integrity Test
This test is required if the finished product is sterilized using an 0.22 m filter
membrane.
Instrumentation:
- Remotely operated device specifically designed for filter integrity test
Method
- Connect a suitable gas source upstream to the filter membrane
- Gradually increase the pressure
- Record the pressure value at which bubble formation is observed
[11C]choline: specifications
Bubble point depends on the filter characteristics, and the specifications are given by
the manufacturer.
2.2.1.P.5.3 Validation of Analytical Procedures
The reference guidelines are as follows:
15

ICH Harmonised Tripartite Guideline “Text on validation of analytical
Procedures” Step 4 of the ICH Process, November 2005

ICH Harmonised Tripartite Guideline “Validation of Analytical Procedures:
Text and Methodology” Step 4 of the ICH Process, November 2005
The above guidelines may not always apply to validation of radioactive compounds,
due to their peculiar nature. Exceptions will be discussed.
2.2.1.P.5.3.1 Method validation for the determination of chemical purity using
HPLC
Validation of analytical method for the determination of chemical purity of injectable
solution of [11C]choline is here presented.
In Table 12 the validation parameters and their acceptance criteria are summarized:
Acceptance criteria
Test
≥ 2,5
Specificity
CV %  2%
Repeatability
Fcalc≤ value of Ftab
Intermediate precision
R2 ≥ 0,99
Linearity
Quantification Limit (LOQ)
Limit of detection (LOD)
CV %  2% for standard
choline
CV %  5% for impurity
--Fcalc≤ value of Ftab
Robustness
Table 12 – Test and acceptance criteria for the validation of the method for the
determination of chemical purity using HPLC
Specificity
Specificity determination is performed analyzing mixture containing critical
components that might be present in the finished product [11C]choline solution, and
demonstrating that the method is capable to distinguish the various components
present at the limit concentration for the considered standards. The [11C]choline
preparation method development did not prompt for chemical impurities, except for
the DMAE precursor. Thus, analyses were performed using a series of solution
containing the precursor itself, together with the choline chloride (or bitartrate)
standard.
Peak resolution may be calculated using the following equation:
Rs 
1,18  Trb  Tra 
Wa  Wb
where:
Trb= retention time of the compound b
Tra = retention time of the compound a
Wb= full width at half-height of the compound b
Wa= full width at half-height of the compound a
16
Linearity
The statistical function used in these cases is linear regression with least squares.
The curve equation, the correlation coefficient and the determination coefficient (r 2)
are then calculated.
Equation:
y = ax + b
where: a = slope
b = intercept
y = peak area
x = analyte concentration
r2 should be  0,99, within the concentration operating range.
The above evaluation requires the preparation of at least 5 standard solutions with
different concentrations for each of the interested analytes (DMAE and standard
choline). Such solutions are usually prepared by a series of dilution starting from a
“mother” solution, with the highest concentration.
Precision
Precision may be considered at different levels, as a measure of repeatability or
intermediate precision.
a. Repeatability:
Repeatability may be calculated based on the content of standard DMAE and
choline. The statistical parameter of concern is the variation coefficient (CV%) (or
Relative Standard Deviation. RSD), which is determined using the following equation:
CV % 
s
 100
m
where:
s = standard deviation of the peak areas
m = average of the peak areas
CV% should be < 2% for the standard choline, and < 5% for the DMAE precursor.
The necessary experimental data may be obtained by injecting 6 times a sample of
the desired analyte, whose concentration should fall within the range established
during linearity test.
b. Intermediate precision
Intermediate precision may be determined through the variance calculation
(ANOVA), which allows, in turn, for Fisher value calculation.
Limit of quantitation (LOQ)
Experimentally, LOQ may be determined by analyzing a series of diluted solutions of
DMAE and standard choline, until a concentration level quantified with a precision
>95% is reached.
The experimental value determined as above described need to be confirmed
through a precision analysis, using a sample at the concentration corresponding to
the found LOQ. Acceptance criteria is CV% <5%.
Limit of detection (LOD)
The LOD may be determined experimentally by successive dilutions, until the above
minimum concentration is found.
Robustness
In case of HPLC analysis, a critical parameter might be the mobile phase flow. Once
the critical parameter has been selected, three consecutive analyses of the desired
analyte have to be performed, following a deliberate modification of the pump flow.
17
Data may be evaluated through the variance analysis (ANOVA). In the
present document, the parameters whose variation will be analyzed are retention
time and peak areas.
2.2.1.P.5.3.2 Method validation for the determination of radiochemical purity
using HPLC
Validation of the analytical method for the determination of the radiochemical purity is
here presented. In Table 13, the validation parameters and their acceptance criteria
are summarized:
Test
Specificity
Repeatability
Intermediate precision
Linearity
Acceptance criteria
Not applicable
CV %  2%
Not applicable
R2 ≥ 0,99
Limit of quantitation (LOQ)
Not applicable
Limit of detection (LOD)
Not applicable
Robustness
Not applicable
Table 13 – Test and acceptance criteria for the validation of the method for the
determination of radiochemical purity using HPLC
In case of validation of methods of radioactive compounds, some of the ICH
guidelines validation parameters may not be of concern and do not apply. In
particular:
-
Specificity would require the analysis of at least two radioactive analytes with
comparable activity, and this is usually not applicable in case of very short
half-life radionuclides labelled compounds, for experimental reasons.
-
Flow cell radiochemical detectors typically used in the analysis of
radiolabelled compounds are inherently not very suitable for quantitation
purposes; moreover, they are typically used to determine relative ratios
between the activity of the various labelled compounds that might be present
in the sample, rather than for true activity quantitation purposes, and the final
required outcome is a percentage areas calculation. Thus, both LOQ and
LOD analyses have been considered of no concern.
-
Similar considerations may be done for intermediate precision analysis, which
is strictly related to the possibility of quantitation of the desired analytes.
-
As for the robustness, the results obtained during the tests for validation of
chemical purity, using “cold” analytes (DMAE and standard choline), provide
response and data that may apply to the radioactive analytes, as well. A
18
repetition of the test would in this case represent a useless radiation burden
for the operators.
-
Last but not least, the ALARA (“as low as reasonably achievable”)” radiation
protection concept should indeed always be kept in mind, while designing the
necessary validation tests.
Linearity
Considering the radioactive nature and the very short half-life of 11C, the typical
experimental approach, based on the preparation of a series of solution with different
concentrations does not apply. On the contrary, in this case one sample solution
only, with a suitable radioactive concentration, is analyzed 5 times, at defined time
intervals. Indeed, the radioactivity being the physical parameter of concern for
radiochemical detectors, the radionuclide decay itself provide the necessary linear
series of values.
r2 may thus be extrapolated from the calibration curve by analyzing 5 different
radioactive concentration of [11C]choline.
Repeatability
Here also apply the same considerations described for linearity; that’s, the decay of
the radionuclide 11C inevitably lead to a decrease in time of the radioactivity.
However, repeatability may be evaluated analyzing a series of HPLC runs obtained
with repetitive injections of a single [11C]choline sample, and recalculating the
obtained peak area values with the decay equation:
lnA0= ln A + λt
λ= 0,693/t1/2
where:
A0= corrected peak area
A= measured peak area
t= time interval between the considered injection and the first one
t1/2= half-life (11C = 20,3 min)
The peak area values, normalized for decay, may then be compared and yield a
consistent statistical analysis. Average, standard deviation and (CV%) are then
calculated.
Repeatability has to be determined in three different days, to verify the
instrument outcome during the time course.
2.2.1.P.5.3.3 Validation of the analytical method for the determination of
residual solvent using gas-chromatography
Gas-chromatography is used to evaluate the amount of residual solvents in the
finished product solution of [11C]choline.
The validation parameters are practically the same already described for the
validation of the method for the determination of chemical purity (see Table 12,
section 2.2.1.P.5.3.1). For this reason, discussion will not be repeat in this context.
2.2.1.P.5.3.4 Validation of the analytical method for the determination of
radionuclidic purity and identification test using gamma spectrometry
Validation parameters are described in Table 14:
19
Test
Specificity
Repeatability
Intermediate precision
Acceptance criteria
Rs ≥ 1
CV %  2%
Not applicable
Linearity
R2 ≥ 0,99
Accuracy
90-110%
Limito of quantitation (LOQ)
Limito of detection (LOD)
Robustness
Not applicable
Not applicable; MDA*
Not applicable
Table 14 – Test and acceptance criteria for the validation of the method for the
determination of radionuclidic purity and identification test using gamma spectrometry
*LOD may be suitably replaced by minimum detectable activity (MDA) values, which
are determined by the instrument every time a sample is measured. MDA is a
parameter depending by several factors such as geometry, activity, background,
counting time, etc. It is assumed that all the intended measurements are performed
keeping into account the above factors, so as to obtain consistent data.
As already discussed in section 2.2.1.P.5.3.2, validation parameters stated by
ICH guidelines are not always applicable when dealing with radioactive samples,
essentially due to inherent decay phenomena. Thus, intermediate precision and
robustness may not be evaluated with sufficient reliability, and the remaining
validation parameters need to be adapted to the peculiar nature of the radioactive
samples.
LOD may be considered, provided that the instrument is periodically
calibrated for efficiency.
In the case of robustness, it is not easy to apply deliberate and controlled
variations to the normal operating detector parameters, and for this reason this part
of the validation program is not considered.
Specificity
Specificity may be determined using a suitable, preferably multi-nuclide, calibrated
source, with an overall emission energy spectrum capable to include as much as
possible the instrument operating range (usually 0-2000 KeV). For this reason,
[11C]choline samples are not useful in this case, as their emission energy is likely to
be attributable to the expected 511 KeV only, and they usually do not contain
significant and detectable amounts of radioisotopic impurities. Thus, the specificity
analysis may just provide a general validation of the inherent physical characteristic
of the instrument, more than a true evaluation of the [11C]choline injectable solutions.
Acceptance criteria for specificity (Rs) should take account of the above
considerations and, in particular, of the differences in resolution between NaI and
HPGe (or other similar high resolution detectors). Indeed, the former have a lower
resolution and Rs values should necessarily be lower, as well. For calculation
purposes, the following formula has been used:
20
Rs 
1,18  Erb  Era 
FWHM a  FWHM b
where:
Erb= Energy at the centroid of the radionuclide b peak
Era = Energy at the centroid of the radionuclide a peak
Wb = full width at half-height of radionuclide b peak
Wa = full width at half-height of radionuclide a peak
Linearity
As the desired analyte is [11C]choline, whose activity decrease with time, the same
consideration already discussed in section 2.2.1.P.5.3.2 apply.
Repeatability
The same set of data obtained following the linearity tests may be used to determine
repeatability, as already described in section 2.2.1.P.5.3.2.
Accuracy
Accuracy has been evaluated using a multinuclide calibrated source containing 60Co,
which has been selected, among the others, due to its long half-life (T1/2 = 5.27 y)
and its widespread availability. Accuracy has been determined by comparing
calculated activity of 60Co with the activity quantified by the instrument.
2.2.1.P.5.3.5 Validation of analytical method for the determination of pH
As the pH meter and related methods are very simple, validation following the ICH
guidelines is not necessary. The only suggested test is to check the repeatability of
the analytical method. CV% may be determined by analyzing 6 times a series of
suitable buffer solutions, whose pH brackets a significant portion of the pH working
range (e.g. buffer solutions with pH of 4.7 and 10, respectively). Anyhow, the
instrument calibration is checked every time it has to be used, using the same buffer
solutions above depicted.
2.2.1.P.5.4 Batch Analyses
Data related to the [11C]choline batches are included in the respective certificates of
analysis. They report information related to the number and batch size, as well as
information on the production site, methods of production, quality control and
acceptance criteria.
2.2.1.P.5.5 Characterization of Impurities
The discussion about the possible impurities in injectable solution of the finished
product [11C]choline has already been described in section 2.2.1.S.3.2, related to the
active substance.
2.2.1.P.5.6 Justification of Specification(s)
As already mentioned in the appropriate sections, the analytical test, the methods
and acceptance criteria have been derived, where applicable, from the general Ph.
Eur. monograph “Radiopharmaceuticals” and from the Ph. Eur. monographs
specifically dedicated to 11C labelled radiopharmaceuticals above cited.
It is noteworthy that the radiopharmaceutical [11C]choline is a very well
established radiopharmaceutical, for which a comprehensive scientific and clinical
literature is available, that cover all the aspects related to the preparation and quality
control of the radiopharmaceutical and, most noticeably, to its clinical outcome. Thus,
the specifications adopted in the present document may be considered as a suitable
summary of numerous experiences and propose a preparation process already
validated, in practice, by several production sites.
21
2.2.1.P.6 Reference Standards or Materials
Reference standards and materials have already been discussed in section
2.2.1.S.5.
2.2.1.P.7 Container Closure System
The radiopharmaceutical product is contained in a glass vial, Ph. Eur. type I, sterile
and pyrogen free, covered with a bromo (or chloro) butyl rubber stopper (Ph. Eur.
conform), sealed with a flip-off aluminum cap. Attached are the certificate of analyses
of the vials and stopper manufacturer. No further test other than visual inspection is
applied to these materials.
2.2.1.P.8 Stability
The radiopharmaceutical [11C]choline, due to the short half-life of 11C, is typically of
immediate use, and its administration to patients immediately follows the preparation
and quality control operations. Generally speaking, stability does not need to be
determined in case of very-short half-life radionuclides such as 11C. However, a
stability study has been performed, with the aim to provide evidence on how the
quality of [11C]choline may vary with time and to establish a shelf life for the finished
product, under recommended storage conditions. During the whole shelf-life, the
radiopharmaceutical characteristics of purity have to meet quality criteria discussed
and established in the previous sections.
The goal is thus to define expiry time for the radiopharmaceutical product.
For stability study purposes, three consecutive batches of [11C]choline have to be
prepared by using the maximum possible starting activity of the radionuclide 11C, in
order to obtain batches with the highest radioactive concentration, that allow to
evaluate the effect of radiolysis of the active substance in “worst case” conditions.
For each batch of radiopharmaceuticals, the following information should be
provided:
- batch number
- preparation date and time
- calibration time
- activity at calibration time
- radioactive concentration at calibration time
Specifications and acceptance criteria for each batch are those defined in section
2.2.1.P.5.1, Table 11. The test program follows the matrix listed below:
22
Test
Appearance
Identification: HPLC
Identification: gamma
spectrometry
Radiochemical purity
DMAE
Chemical
Purity
EtOH
CH3CN
Radionuclidic purity*
Half-life
pH
Bacterial endotoxins
Filter integrity
Sterility**
Specification
Clear, colourless solution
The principal peak in the
radiochromatogram
obtained with the test
solution of [11C]choline
has approximately the
same retention time as
the principal peak in the
chromatogram obtained
with a reference solution
of “cold” choline chloride
(or choline bitartrate)
Gamma photons have an
energy of 511 KeV and,
depending
on
the
measurement geometry,
a sum peak of 1022 KeV
may be observed
[11C]choline ≥95%
≤0.1 mg/max.
recommended dose (mL)
≤50 mg/ max.
recommended dose (mL)
≤4.1 mg/ max.
recommended dose (mL)
11C
≥ 99,9 %
T0
X
T1
X
T2
X
X
/
/
X
/
/
X
X
X
/
X
/
X
/
/
X
/
/
X
/
/
X
/
X
X
X
X
/
X
/
X
/
/
/
/
/
19.9-20.9 min
4,5-8,5
<175 EU/ max.
recommended dose (mL)
Following specification of
the supplier
Sterile
Table 16 – Matrix of the test to be performed during stability study (“X” means: to be
tested; “/” means: not to be tested)
*The test has to be performed at least 12 h after the batch preparation, to allow the
complete decay of the main radionuclide 11C
**Sterility test is performed after adequate decay of the radioactivity contained in the
finished product
For example, in case the proposed shelf-life is 2 h, the analyses will have to be
repeated three times, at the following time intervals: T 0 (EOS), T1 (T0 + 60 min) and
T2 (T0 + 120 min).).
Considering the very short half-life of 11C, and the typical amounts of the
above radionuclides that may reasonably be produced using commercially available
cyclotrons and target systems, it seems to be reasonable to propose a shelf-life no
longer than 2 h.
23