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. 11C decays to the stable isotope 11B, through the emission of β+ (99,8%), with a physical half-life of 20,38 min.
Parent Nuclide / T1/2 / Decay mode / b+ Emax / Relativeintensity / γKeV / Daughter nuclide
11
6 / C
/ 20.38 min / β+ / 960.2 KeV / 99.759% / 511 /
11
5 / B
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 chlorideAppearance / 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
11C is generated “in-target” in the form of [11C]CO2 by means of an accelerated cyclotron proton beam via the nuclear reaction:
14N(p,a)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 mA, 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.
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 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 25-35%, 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 [11C]choline via “gas phase” method using a GE Tracerlab FxC-Pro, together with the proposed verification test, is reported.
Materials / Test and acceptance criteriaDimethylaminoethanol (DMAE) / HPLC; purity should conform with the indication of the supplier, based on analysis certificate
[11C]CO2 / C-11 has T1/2 = 20.3 min; this starting material cannot be isolated and tested during the radiosynthetic process
P2O5 / See the attached analysis certificate
N-14 + 0.1-1% O2 / See the attached analysis certificate; additionally, an irradiation test is performed every time the gas cylinder containing the gaseous mixture N2 + O2 is replaced
Water for injection / See the attached analysis certificate
EtOH / See the attached analysis certificate
Iodine / See the attached analysis certificate
NaCl 0,9% injectable solution / See the attached analysis certificate
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 (SepPak) / See the attached analysis certificate
Acrodisc 0,2 µm filter, Supor membrane / See the attached analysis certificate
Sterile vials / See the attached analysis certificate
Vented Millex GS 0,22 µ filter / See the attached analysis certificate
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.
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,a)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,a)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.
13N 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).