Nelson et al.
`EJNMMI Radiopharmacy and Chemistry (2022) 7:27
`https://doi.org/10.1186/s41181-022-00180-1
`
`EJNMMI Radiopharmacy
` and Chemistry
`
`Open Access
`REVIEW
`Good practices for 68Ga radiopharmaceutical
`production
`
`Bryce J. B. Nelson1, Jan D. Andersson1,2, Frank Wuest1 and Sarah Spreckelmeyer3*
`
`
`
`*Correspondence:
`sarah.spreckelmeyer@charite.de
`1 Department of Oncology,
`University of Alberta, 11560
`University Avenue, Edmonton,
`AB T6G 1Z2, Canada
`2 Edmonton
`Radiopharmaceutical Center,
`Alberta Health Services, 11560
`University Ave, Edmonton, AB
`T6G 1Z2, Canada
`3 Department of Nuclear
`Medicine, Charité -
`Universitätsmedizin Berlin,
`Corporate Member of Freie
`Universität Berlin, Humboldt
`Universität Zu Berlin,
`and Berlin Institute of Health,
`Augustenburger Platz 1,
`13353 Berlin, Germany
`
`Abstract
`Background: The radiometal gallium-68 (68Ga) is increasingly used in diagnostic posi-
`tron emission tomography (PET), with 68Ga-labeled radiopharmaceuticals developed as
`potential higher-resolution imaging alternatives to traditional 99mTc agents. In precision
`medicine, PET applications of 68Ga are widespread, with 68Ga radiolabeled to a variety
`of radiotracers that evaluate perfusion and organ function, and target specific biomark-
`ers found on tumor lesions such as prostate-specific membrane antigen, somatostatin,
`fibroblast activation protein, bombesin, and melanocortin.
`Main body: These 68Ga radiopharmaceuticals include agents such as [68Ga]Ga-macro-
`aggregated albumin for myocardial perfusion evaluation, [68Ga]Ga-PLED for assessing
`renal function, [68Ga]Ga-t-butyl-HBED for assessing liver function, and [68Ga]Ga-PSMA
`for tumor imaging. The short half-life, favourable nuclear decay properties, ease of radi-
`olabeling, and convenient availability through germanium-68 (68Ge) generators and
`cyclotron production routes strongly positions 68Ga for continued growth in clinical
`deployment. This progress motivates the development of a set of common guidelines
`and standards for the 68Ga radiopharmaceutical community, and recommendations for
`centers interested in establishing 68Ga radiopharmaceutical production.
`Conclusion: This review outlines important aspects of 68Ga radiopharmacy, including
`68Ga production routes using a 68Ge/68Ga generator or medical cyclotron, standardized
`68Ga radiolabeling methods, quality control procedures for clinical 68Ga radiopharma-
`ceuticals, and suggested best practices for centers with established or upcoming 68Ga
`radiopharmaceutical production. Finally, an outlook on 68Ga radiopharmaceuticals is
`presented to highlight potential challenges and opportunities facing the community.
`Keywords: 68Ga-radiolabeling, Gallium-68, Automation, Cyclotron, Radiolabeling,
`68Ga-tracer, Radiopharmaceuticals
`
`Background
`The rise and increasingly widespread clinical use of positron emission tomography
`(PET) imaging with gallium-68 (68Ga) radiopharmaceuticals motivates providing guid-
`ance on aspects of 68Ga radiopharmaceutical production to aid the community in
`achieving consistent quality and reliable yields. Radiogallium isotopes have been exten-
`sively investigated, starting when gallium was first observed to accumulate at osteogenic
`activity in the late 1940s (Hayes 1978). Early clinical trials using reactor-produced 72Ga
` (t1/2 = 14.1  h) for therapy and diagnostic evaluation of malignant bone lesions were
`
`© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits
`use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original
`author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third
`party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate-
`rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or
`exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
`creat iveco mmons. org/ licen ses/ by/4. 0/.
`
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`ineffective, with investigation largely stopping by 1952 due to unsatisfactory patient ben-
`efits (Hayes 1978). A primary factor contributing to the negative diagnostic results was
`the poor detection equipment available at the time, while any further attempts exploit
`72Ga for therapy would have been limited by the high energy and intensity beta particle
`and gamma ray emissions depositing excess radiation dose in healthy tissue surround-
`ing the tumor sites. Subsequently, accelerator-produced 67Ga (t1/2 = 3.3 d) was inves-
`tigated for clinical use, and determined to be an effective tumor and abscess locating
`agent, with annual usage reaching nearly 250,000 patients by 1977 (Hayes 1978). In 1961,
`the first 68Ga generator system was developed, using decay of germanium-68 (68Ge)
`to provide a continuous supply of 68Ga for clinical studies (Gleason 1960). 68Ga was
`viewed as particularly attractive due to its short half-life permitting large activities to
`be administered for diagnostic imaging, with its rapid decay and clearance preventing
`excess patient radiation dose. Additionally, 68Ga nuclear decay exhibits a high positron
`branching ratio (88.9%) with minimal co-emitted gamma rays, positioning it favorably
`compared to other radiometals with respect to dose (https:// www. nndc. bnl. gov/ nudat2/
`reCen ter. jsp?z= 56&n= 77). Alongside advances in 67Ga, 68Ga was initially considered
`for potential use in PET imaging, however there was insufficient instrumentation at the
`time to achieve this application. The advent of 99mTc for single photon emission com-
`puted tomography (SPECT) imaging and 18F for PET imaging delayed the application of
`68Ga diagnostic imaging owing to widespread 99mTc generator commercial distribution,
`and the longer half-life of 18F compared to 68Ga providing ease of production and clini-
`cal application. Additionally, early 68Ge/68Ga generators precluded direct radiolabeling
`by providing 68Ga eluate complexed with EDTA, further slowing the development and
`utilization of 68Ga radiopharmaceuticals (Banerjee and Pomper 2013). With the recent
`emergence of more advanced PET cameras, and the next generation of GMP-grade com-
`mercially available 68Ge/68Ga generators that reliably provide 68Ga in chemically conven-
`ient dilute hydrochloric acid, 68Ga use for research and clinical application became more
`widespread. Development and production of many 68Ga radiopharmaceuticals ensued
`for various purposes including myocardial perfusion, renal and liver function, and tumor
`imaging. Somatostatin (DOTATOC/DOTATATE/DOTANOC) (Bauwens et  al. 2010;
`Decristoforo et al. 2007), prostate-specific membrane antigen (PSMA) (Fuscaldi et al.
`2021; Hennrich and Eder 2021), fibroblast activation protein (FAP) (Spreckelmeyer et al.
`2020; Loktev et al. 2018), bombesin (Schuhmacher et al. 2005; Richter et al. 2016) and
`melanocortin 1 (Froidevaux et al. 2004) targeting 68Ga radiotracers have been developed
`(Fig. 1), with their pharmacokinetics often well matched to the short physical half-life of
`68Ga (Banerjee and Pomper 2013).
`With an increasing number of centers using 68Ga on a regular basis for research and
`clinical application, several challenges have been maintaining consistency of reported
`parameters and providing sufficient process information for preclinical and produc-
`tion data of new 68Ga radiopharmaceuticals. This review will present a set of common
`guidelines and standards would be useful for the 68Ga community to report data in a
`uniform and reliable format. This review aims to outline key aspects of 68Ga radiophar-
`macy, including means of 68Ga production and purification via 68Ge/68Ga generators or
`medical cyclotrons, standard techniques for radiolabeling compounds with 68Ga, and
`established quality control procedures for clinical grade 68Ga radiopharmaceuticals. It
`
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`

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`Fig. 1 Structures of several 68Ga radiopharmaceuticals in clinical use (1) PSMA-11 (Fuscaldi et al.2021;
`Hennrich and Eder 2021) (2) PentixaFor (Sammartano et al. 2020; Spreckelmeyer et al. 2020) (3) FAPI-46
`(Spreckelmeyer et al. 2020) (4) R = H DOTA-TOC (Bauwens et al. 2010; Decristoforo et al. 2007); R = Carbonyl
`DOTA-TATE (5) Exendin peptide sequence = HGEGTFTSDL SKQ M EEEAVR LFIEWLKNGG PSSGAPPPS
`C = Exendin-4-Cys40(DOTA) (Velikyan et al. 2017) (6) Exendin peptide sequence = HGEGTFTSDL SKQ M
`EEEAVR LFIEWLKNGG PSSGAPPPS K = Exendin-4-Lys40(NODAGA) (Velikyan et al. 2017; Migliari et al. 2021)
`
`also suggests best practices for centers with existing or upcoming 68Ga radiopharma-
`ceutical production with respect to preparation of common 68Ga tracers, and reporting
`key production parameters to the community. To conclude, an outlook on the future of
`68Ga radiopharmaceuticals is presented to highlight some of the upcoming challenges
`and opportunities presenting the community.
`
`68Ga production routes: generators and cyclotrons
`68Ga generator production
`The most common method for obtaining 68Ga is via a 68Ge/68Ga generator. Genera-
`tors are convenient for many applications since the 270.93-day half-life of the par-
`ent nuclide, germanium-68 (68Ge), guarantees an ongoing supply of 68Ga sufficient
`for clinical use for up to a year. 68Ga/68Ge generators were first developed in the early
`1960s, however early generators utilizing liquid–liquid extraction and EDTA eluant
`to obtain 68Ga were not conducive to complex syntheses of 68Ga radiopharmaceuti-
`cals, and the advent of 99mTc and 18F radiopharmaceuticals slowed development of
`68Ga radiopharmaceuticals in the 1970s (Rösch 2013). Advances in radiochemistry led
`
`
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`to availability of new generators providing 68Ga3+ in hydrochloric acid eluate (Raz-
`bash et al. 2005). The eluted 68Ga, in the form of [68Ga]GaCl3, can be used for radi-
`olabeling and has led to significant advances in 68Ga chemistry and the development
`of targeted PET radiopharmaceuticals. Modern commercially available 68Ge/68Ga
`generators utilize TiO2, SiO2, CeO2, or SnO2 solid phase matrixes to provide [68Ga]
`GaCl3 by elution with dilute HCl while the mother 68Ge radionuclide remains on the
`matrix (Table 1). 68Ge content is less than 0.001% of 68Ga eluate throughout the life
`of the generator, with the eluate containing minimal metallic impurities (Rösch 2013;
`Chakravarty et  al. 2016; Romero et  al. 2020). A recent development is a 4.04  GBq
`68Ga/68Ge generator, capable of producing significantly higher 68Ga elution and drug
`product activities with a longer generator shelf-life compared to previous generators
`(Waterhouse et  al. 2020). The 68Ge generator parent radionuclide can be produced
`via several accelerator-based nuclear transformations, the most common being the
`69Ga(p,2n)68Ge reaction. The cross section for this reaction peaks just under 20 MeV,
`which is within the range of many medical cyclotrons, however, to achieve reasonable
`commercial scale yields (> 37 GBq) irradiations of 69Ga at 40–100 µA for several days
`are needed (IAEA PUB1436).
`68Ga can also be produced directly on the cyclotron via the 68Zn(p,n)68Ga nuclear reac-
`tion (Tieu et al. 2019; Alnahwi et al. 2020; Lin et al. 2018; Nelson et al. 2020; Thisgaard
`et al. 2021; Rodnick et al. 2020; Alves et al. 2017; Pandey et al. 2014, 2019; Riga et al.
`2018; Jensen and Clark 2011), with various production routes and yields presented in
`Table 2. Depending on the production technique, cyclotron 68Ga yields are typically one
`to several orders of magnitude greater than currently available 68Ga/68Ge generators.
`Significant development has been undertaken in the field of liquid targets for 68Ga pro-
`duction. Aqueous solutions of isotopically enriched zinc-68 (68Zn) were first subjected
`to proton bombardment in a regular niobium target mainly used for 18F production
`(Jensen and Clark 2011) and later upgraded to use a niobium foil as a beam degrader,
`producing 1800 MBq at end of bombardment (EOB) (Riga et al. 2018). Subsequently, a
`modified target design using an aluminum foil as beam degrader was developed (Pan-
`dey et  al. 2019) where zinc nitrate in nitric acid was irradiated at 20 µA, producing
`9.85 ± 2.09 GBq at EOB. Alternatively, solid targets using electroplated or pressed metal
`68Zn powder have been used, where 68Zn is electroplated or pressed onto metallic tar-
`get backings. Post-irradiation, the metallic 68Zn is dissolved for chemical separation and
`68Ga purification. An alternative target system combining irradiation and dissolution has
`recently been developed that aims to address the limitations of solid and liquid targetry.
`
`Table 1 Commercially available 68Ge/68Ga generators
`
`Manufacturer
`
`GMP
`
`Matrix
`
`Elution
`
`Size (GBq)
`
`IRE Elit
`ITG
`Eckert & Ziegler
`iThemba Labs
`Obninsk Cyclotron Co Ltd
`Pars Isotopes
`
`Yes
`Yes
`Yes
`No
`No
`No
`
`TiO2
`Octadecyl silica
`TiO2
`SnO2
`TiO2
`nano-SnO2
`
`0.1 M HCl
`0.05 M HCl
`0.1 M HCl
`0.6 M HCl
`0.1 M HCl
`1.0 M HCL
`
`1.85
`2/4.04 (Waterhouse et al. 2020)
`3.7
`1.85
`3.7
`2.59 (Romero et al. 2020)
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`Table 2 Liquid and solid target 68Ga cyclotron production routes
`
`Foil
`
`Beam
`
`Yield
`
`References
`
`Niobium
`
`15 MeV, 20 µA
`
`1800 MBq EOB
`
`Target
`
`[68Zn]ZnCl2
`
`[68Zn]Zn(NO3)2 (1.7 M)
`in HNO3 (0.2 N)
`[68Zn]Zn(NO3)2 (1.7 M)
`in HNO3 (0.2 N)
`1.4 M 68Zn(NO3)2 in
`1.2 N HNO3
`100 mg 68Zn(NO3)2
`1.0 M 68Zn(NO3)2 in
`0.3 N HNO3
`Pressed 68Zn
`
`Pressed 68Zn
`
`Electrodeposited 68Zn
`
`Pressed 68Zn
`Electrodeposited 68Zn
`
`Aluminum
`
`Niobium
`
`Aluminum
`
`Niobium
`Niobium/Havar
`
`Aluminum
`
`Aluminum
`
`14 MeV, 20 µA
`
`12 MeV, 20 µA
`
`192.5 ± 11.0 MBq/
`µA-hr EOB
`4.3 ± 0.3 GBq
`14 MeV, 40 µA, 60 min 9.85 ± 2.09 GBq EOB
`14 MeV, 45 µA, 50 min 6 GBq EOB
`14.3 MeV, 34 µA,
`4.6 ± 0.4 GBq
`60 min
`13 MeV, 80 µA,
`194 GBq EOB
`120 min
`12.5 MeV, 30 µA,
`73 min
`14.5 MeV, 30 µA,
`60 min
`13 MeV, 35 µA, 90 min 145 GBq
`14.5 MeV, 35 µA,
`6.30 GBq
`8.5 min
`
`37.5 GBq
`
`60.9 GBq
`
`Jensen and Clark
`(2011)
`Pandey et al. (2014)
`
`Riga et al. (2018)
`
`Pandey et al. (2019)
`
`Alves et al. (2017)
`Rodnick et al. (2020)
`
`Thisgaard et al. (2021)
`
`Nelson et al. (2020)
`
`Lin et al. (2018)
`
`Alnahwi et al. (2020)
`Tieu et al. (2019)
`
`To effectively establish 68Ga production, sites should select a liquid or solid target pro-
`duction route based upon their anticipated 68Ga demand, available infrastructure, and
`existing technical expertise. The following sections outline the advantages and disadvan-
`tages of liquid and solid 68Zn targetry and 68Zn/68Ga chemical separation techniques.
`
`68Ga solid and liquid cyclotron targetry
`Liquid 68Zn target solutions are prepared by dissolving isotopically enriched 68Zn metal
`or 68Zn oxide in nitric acid to produce [68Zn]Zn(NO3)2 (Rodnick et  al. 2020; Alves
`et al. 2017; Pandey et al. 2014, 2019; Riga et al. 2018). Alternatively, [68Zn]ZnCl2 can
`be employed (Jensen and Clark 2011), however [68Zn]Zn(NO3)2 is preferred, as it was
`found that irradiating ZnCl2 leads to a significant pressure buildup of hydrogen and oxy-
`gen resulting from beam-induced radiolysis of the target solution (Pandey et al. 2014).
`Target assemblies can utilize a combination of helium and water cooling to remove
`heat, with the target solution and cooling fluids separated by aluminum and niobium
`foils. Targets are typically irradiated at energies of 12–14 MeV up to 45 µA beam cur-
`rent (Rodnick et al. 2020; Alves et al. 2017; Pandey et al. 2014, 2019; Riga et al. 2018),
`with 68Ga yields dependent on the target pressure and concentration of 68Zn solution,
`yielding up to 9.85  GBq after a 60  min irradiation. While irradiating at higher beam
`energies increases 68Ga yield, it increases production of the 67Ga radionuclidic impu-
`rity, so irradiating at a lower energy of ~ 12 MeV improves radionuclidic purity through
`avoiding onset of the 68Zn(p,2n)67Ga reaction. However trace levels of undesired iso-
`topic impurities (0.1% 66Zn and 0.48% 67Zn) present in highly enriched 68Zn (99.3%) lead
`to unavoidable production of 66Ga and 67Ga from the 66Zn(p,n)66Ga and 67Zn(p,n)67Ga
`reactions, respectively (Nelson et al. 2020). To achieve higher beam currents on liquid
`targets, pressurized target assemblies are required due to cavitation of the target solu-
`tion. Advantages of liquid targets include ease of solution loading and removal from the
`
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`target assembly, use of existing cyclotron liquid target infrastructure and similarities
`to other liquid targetry (18F), and shorter 68Zn/68Ga chemical purification. Limitations
`of liquid targets include reduced yields resulting from beam energy degradation by the
`aqueous solution, potential increases in metallic impurities resulting from contact with
`the target assembly, and heat transfer constraints that limit beam current on-target.
`Solid 68Zn targets are prepared by either by electroplating or pressing 68Zn powder and
`irradiating at the same beam energies as liquid targets to achieve similar radioisotopic
`purity. Electroplated targets are produced using platinum, gold, or silver target backings,
`with the desired cyclotron beam-spot on the target backing immersed in a [68Zn]ZnCl2
`electroplating solution to deposit 40–250 mg 68Zn metal on a 7–10 mm target beam-
`spot (Tieu et al. 2019; Lin et al. 2018; Engle et al. 2012). Targets are then irradiated at
`currents up to 35 µA beam current at 14.5 MeV, yielding up to 60.9 GBq after a 60 min
`irradiation (Tieu et al. 2019; Lin et al. 2018). Pressed 68Zn targets are manufactured using
`68Zn metal powder that is compressed within a hardened stainless-steel die into a circu-
`lar pellet the diameter of the cyclotron beam-spot. 68Zn pellets are then sintered onto
`silver or aluminum target backings and irradiated at beam currents up to 80 µA, yield-
`ing up to 194 GBq after a 120 min irradiation (Nelson et al. 2020; Thisgaard et al. 2021;
`Zeisler et al. 2019). Similar to liquid targets, solid targets use helium and water cooling,
`with helium cooling provided across the front of the target assembly, while water cool-
`ing flows along the target backside. It is important to achieve an even cyclotron beam
`distribution across the 68Zn target material to avoid excessive heat loads concentrated
`on small areas of the target. For both electroplating and pressed powder targetry, an
`optimal 68Zn pellet thickness can be selected based upon production requirements, with
`thicker 68Zn targets yielding greater 68Ga for a given irradiation time at the cost of a
`greater material expense (Engle et al. 2012). Advantages of 68Zn solid targetry include
`much greater 68Ga yields compared to liquid targets, owing to the denser 68Zn target
`material and superior heat transfer to the target backing that enables greater cyclotron
`beam currents (Nelson et al. 2020). The much higher yields obtained with solid targetry
`mitigate the short half-life of 68Ga, enabling large-scale distribution of 68Ga to other PET
`centers surrounding a cyclotron facility. Limitations of solid targetry include additional
`dissolution and purification processing steps to separate 68Zn from 68Ga, the develop-
`ment of additional infrastructure for solid target retrieval and processing, and operator
`dose associated with processes involving manual retrieval of solid 68Zn targets.
`To address the limitations of solid and liquid targetry, a combined irradiation-disso-
`lution target system has been developed that combines advantages of liquid and solid
`targetry while avoiding their major limitations. Irradiation is performed on a remotely
`actuated multi-position target bar containing seven 20–40 mg solid 68Zn metal targets
`that permits multiple back-to-back irradiations, reducing operator exposure to radioac-
`tive dose. The irradiated target material is then dissolved within the target assembly, and
`the target solution is remotely transferred to a hot cell for 68Ga purification via a capil-
`lary line. This combined irradiation-dissolution assembly results in a system that com-
`bines the ease of use of liquid targets with the high yields of solid targets (https:// syniq.
`hu/ produ ct/ hybrid- target- system- for- 68ga- produ ction).
`To select a liquid or solid cyclotron target setup, all of the factors previously discussed
`should be considered with respect to cyclotron facilities’ infrastructure and technical
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`strengths. Sites may wish to employ liquid 68Zn targets if they possess existing liquid
`target infrastructure and expertise, have a suitable shielded tubing conduit for transfer-
`ring the irradiated 68Zn target solution to a hot cell for processing, and are primarily
`interested in routine production of smaller activities for use in local PET imaging cent-
`ers. Alternatively, sites may wish to employ solid 68Zn targets if they have existing solid
`target assemblies and production experience, and aim to produce large activities of 68Ga
`in single batches for many patients and distribute 68Ga to more remote PET imaging
`centers.
`
`68Zn target processing and 68Ga purification
`After irradiation, chemical separation must be performed to remove 68Zn target mate-
`rial and other metallic impurities that can interfere with subsequent radiolabeling.
`These separation procedures depend on using specific chemical concentrations and ion
`exchange column conditions to achieve reliable results, as small deviations in any of
`these parameters can often have a significant impact on the final 68Ga product yield and
`purity. Liquid and solid target solutions are downloaded to a hot cell via a capillary line
`containing an automated synthesis unit (such as a TRASIS All-in-One, NEPTIS Mosaic-
`LC, or GE FASTlab) employing ion exchange chromatography, or solvent extraction. A
`second ion exchange column is recommended if 68Zn target material contains significant
`levels of metallic impurities that can impact radiolabeling, or if the 68Ga elution solu-
`tion needs to be deacidified. The selection of this secondary column should be based on
`the chemical purity of the target material and the specific contaminants present in the
`clinic’s setup. Decay-corrected 68Ga yields have ranged from 74 to 96%, depending on
`the separation process (Nelson et al. 2020; Alves et al. 2017; Pandey et al. 2014).
`One liquid target purification involves passing [68Zn]Zn(NO3)2 solution over hydroxa-
`mate resin to trap 68Ga while eluting 68Zn, washing with 0.005 N HNO3 to remove resid-
`ual 68Zn, and elution of 68Ga using 5.5 N HCl to an AG-1X-8 column. The AG-1X-8
`column is subsequently eluted with 2 mL H2O to obtain concentrated 68GaCl3 for radi-
`olabeling (Pandey et al. 2014). Another technique utilizing [68Zn]ZnCl2 target solution
`employed a Waters C-18 Sep-Pak, where 68Ga sticks to the resin while [68Zn]ZnCl2 flows
`through. The Sep-Pak is washed with water, followed by 68Ga elution in 0.1 N HCl. The
` [68Zn]ZnCl2 is recovered by boiling up the eluate and water washes to the original solu-
`tion concentration (Jensen and Clark 2011). An additional separation method involved
`loading [68Zn]Zn(NO3)2 onto a 50W-X8 column, followed by elution with 3 N HCl onto
`a Biorad 1X8 column, which was then eluted with 0.1 N HCl (Alves et al. 2017).
`In contrast, solid 68Zn targetry requires an initial dissolution step prior to 68Ga separa-
`tion and purification. Several techniques have employed 10 N HCl to dissolve electro-
`plated or pressed 68Zn metal, prior to loading on a BioRad AG50W-X4 cation exchange
`resin. After washing with 10 N HCl to remove 68Zn and other metallic impurities, the
`68Ga is eluted in 4 N HCl and loaded onto a UTEVA resin column, where the 68Ga is
`eluted in several milliliters of 0.05–0.1 N HCl (Lin et al. 2018; Nelson et al. 2020). 68Zn
`metal can subsequently be recovered from the process solution using an electrolytic cell
`and manufactured into new targets, and has demonstrated comparable 68Ga yields upon
`irradiation to targets utilizing fresh 68Zn (Nelson et  al. 2020). An alternative process
`involves dissolving 68Zn in 7 N HNO3, followed by adjustment to pH 2 using NH4HCO2.
`
`
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`This solution is passed through a hydroxamate resin, followed by washing with 0.01 N
`HCl to remove 68Zn. 68Ga is eluted with 0.75  N HCl, and loaded onto a CUBCX123
`resin, washed with 0.01 N HCl, and the final 68Ga product is eluted with 5 M NaCl/5.5 N
`HCl (Alnahwi et al. 2020).
`Combined irradiation-dissolution targetry dissolves the 68Zn solid target within the
`target assembly in 7 N HCl, with the dissolution solution remotely transferred via a cap-
`illary line to a hot cell where it is loaded onto Zr resin, taking 15–20 min. The resin is
`washed with 10 N HCl, and eluted with 2 N HCl onto a TK200 resin, where it is washed
`with 2 N HCl and eluted in 0.05 N HCl. The 68Ga[Ga]Cl3 product was shown to comply
`with Ph. Eur. Specifications (https:// syniq. hu/ produ ct/ hybrid- target- system- for- 68ga-
`produ ction). An important consideration when utilizing any of the above methods is the
`use of concentrated corrosive acids, and particular caution should be taken in order to
`limit damage to the hot-cells used. Lining the hot cell with protective material, such as a
`chemically resistant plastic film, is a good option.
`The 68Zn/68Ga chemical separation and purification method should be selected based
`upon the cyclotron production method, purification time and product requirements. If
`a liquid target is used to produce 68Ga, the resins used in the initial steps of the down-
`stream purification method should be tailored to match the chemistry of the irradiated
`target solution. Similarly, solid target purification methods should utilize resins condu-
`cive to the initial 68Zn dissolution step. Owing to the short half-life of 68Ga, it is crucial
`to keep purification time to a minimum, provided it does not excessively sacrifice 68Ga
`product activity yield and radiochemical quality.
`
`68Ga radiopharmaceutical production techniques
`Radiolabeling with 68Ga is a single-step synthetic process that can be executed in three
`ways. 68Ga-radiopharmaceuticals can be produced either manually, by (semi)automated
`processes or by using a cold kit. Each of them will be described and discussed in this
`section.
`
`Manual production
`The first 68Ga-radiopharmaceuticals for human use were prepared manually, since
`68Ge/68Ga generators were not approved by authorities and therefore not available for
`the broader community. In addition, the demand for 68Ga was substantially lower than
`current use, as existing SPECT nuclear medicine infrastructure was not designed for
`PET imaging.
`Figure 2 depicts 68Ga-radiolabeling of a DOTA-precursor. First, the reaction mixture
`is prepared by adding 68Ga eluate to a mixture consisting of a suitable buffer, precursor,
`and additives if necessary. Second, the reaction mixture is incubated for a specific reac-
`tion time and temperature to achieve 68Ga chelation. Third, the reaction mixture can be
`purified using a solid phase extraction (SPE) method. The 68Ga-radiopharmaceutical is
`trapped on the column while free 68Ga, 68Ge impurity, and buffer pass through the col-
`umn and are discarded. Finally, the product is eluted and passed through a sterile filter
`as the fourth step (Meisenheimer et al. 2019).
`This process can be adapted to different non-DOTA-based precursors, while following
`the same four general production steps.
`
`Petitioner GE Healthcare – Ex. 1039, p. 8
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`Fig. 2 The four main steps of the 68Ga-radiolabeling procedure (Meisenheimer et al. 2019)
`
`A disadvantage of manual production is the manipulation of significant 68Ga activities
`near unshielded hands. The hands of the operator can be only protected from radioac-
`tive contamination by gloves, and from dose by extending the distance to the source of
`radioactivity with tweezers or tongs. Consequently, manual preparation results in high
`finger dose exposures for the operator (Bauwens et al. 2010). For example, the estimated
`absorbed body and hand dose for the manual synthesis of [68Ga]Ga-DOTA-TATE was
`2.0 and 27 µSv, respectively, while compared to an automated synthesis it was 1.3 and
`7.9 µSv, respectively (Decker and Turner 2012). Additional disadvantages are inconsist-
`ent radiochemical purities and/or radiochemical yields between productions and poten-
`tially non-GMP compliant processes, which is not acceptable for translation to a clinical
`setting.
`The key advantage of maintaining full manual control of the radiolabeling process is
`its usefulness for the early development of radiopharmaceuticals for research purposes.
`Studies regarding labeling kinetics and radiopharmaceutical stability can be performed,
`while keeping the hand dose low.
`Those labeling studies can be performed with lower radioactivity concentrations to
`minimize operator dose (< 100 MBq versus 1–2 GBq used for clinical application).
`
`(Semi)automated production
`Due to the increasing clinical demand for 68Ga-radiopharmaceuticals and the limitations
`of manual preparations discussed earlier, there is a need to perform automated.68Ga
`radiolabeling to increase synthesis yields, reliability, and reduce operator dose (Decris-
`toforo 2012). For this purpose, many different synthesis modules are now commercially
`available, with the majority cassette based. After each synthesis the used cassette is dis-
`posed, and a new cassette is installed for subsequent production runs (2019).
`Similar to the manual production process, automated production of 68Ga-radiophar-
`maceuticals can be grouped into four main steps, which are outlined in Fig. 3.
`First, the 68Ga-eluate starting material can be obtained either by a 68Ga/68Ge gen-
`erator or cyclotron production as described in Sect.  2. The specifications for the
`68Ga-eluate are defined in Ph. Eur. (2464) Depending on the source of 68Ga, different
`purification steps are needed before the radionuclide can be used for radiolabeling.
`In the case of the 68Ga/68Ge generator, it is important to distinguish if the genera-
`tor utilizes an organic or inorganic matrix (Velikyan 2015). The eluent for 68Ga must
`
`
`
`Petitioner GE Healthcare – Ex. 1039, p. 9
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`
`(1)
`Gallium-68 for
`radiolabeling
`
`(2)
`Post-elution
`purification
`
`(3)
`Reaction
`mixture
`
`•68Ge/68Ga generator
`•Cyclotron production
`
`•Fractionation
`•Anion-exchange-
`column
`•Cation-exchange-
`column
`Fig. 3 Schematic overview of automated radiolabeling process
`
`•Buffer
`•Ligand
`•Additives
`
`(4)
`Product
`purification
`
`•Column: C18, CM
`
`be selected based upon the matrix material, and is usually 0.05–2 N HCl, with elu-
`tion removing the 68Ga daughter while the 68Ge mother radionuclide remains on the
`column,. The molarity of the HCl is predefined by the supplier of the generator. A
`drawback is that the elution volume of the generator ranges between 5 and 10 mL,
`which is too large for radiolabeling significant activities in the small volumes required
`for application. Moreover, the eluate can contain metallic impurities resulting from
`either the matrix material of the column, impurities within the eluent, or the nuclear
`decay of 68Ga (discussed in detail in Sect. 5). In order to purify and concentrate the
`68Ga-radiolabeling solution to a small volume (~ 200–600 µL), post-elution processes
`are necessary (Decristoforo 2012).
`The resulting small volume and higher concentration of 68Ga allows for a reduced
`amount of ligand, and a faster radiolabeling reaction with