Radiolabeling of DOTA-like conjugated peptides with generator-produced 68Ga and using NaCl-based cationic elution method

Gallium-68 (68Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize 68Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system, 68Ga3+ of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3–4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of 68Ga radiopharmaceuticals (12–16 min).

The predominant advantage of 68 Ga radiopharmaceuticals is that the synthesis is based on generator-produced 68 Ga and that it can be performed on site (and on demand), without the need for a medical cyclotron. Nowadays, 68 Ge/ 68 Ga generator systems are widely available, and they consistently deliver high-purity 68 Ga for radiolabeling reactions. The parent radionuclide 68 Ge is accelerator-produced through the 69 Ga(p,2n) reaction, whereby Ga 2 O 3 is used as the target material 2 .
Several 68 Ge/ 68 Ga generators have been developed in which 68 Ge is adsorbed as a tetravalent compound onto an inorganic or organic matrix. The parent radionuclide decays with a half-life of 271 d to the trivalent 68 Ga 3+ cation. Because of distinct chemical differences, the daughter nuclide 68 Ga 3+ can be easily separated by elution from the generator with aqueous HCl. Depending on the carrier material (generator stationary phase), the concentration of eluent HCl varies from 0.05 to 1.0 M. The concentration of the parent nuclide 68 Ge in the eluent in currently available generators has been found to be acceptably low, and it has been thoroughly investigated 3 . These generators deliver 68 Ga in a high chemical purity with a low concentration of foreign ions. Heavy-metal ions such as iron and copper ions have a high impact on the labeling efficiency, as does zinc. Most important are iron impurities in the generator eluate or in the labeling reagents, which therefore must be avoided.
Numerous methods have been developed to synthesize 68 Ga-labeled peptides starting from 68 Ga elution from the generators. These methods can be classified into six different categories ( Table 1). All use the amphoteric behavior of 68 Ga to form cationic and anionic species such as cationic 68 Ga 3+ and anionic tetrachlorogallate [ 68 GaCl 4 ] − . Here we briefly describe the approach of each of these five classes of 68 Ga radiolabeling paradigms and we provide a detailed protocol for the use of a NaCl-based method (class 6) for radiolabeling of chelator-modified peptides and small molecules for molecular imaging applications.
The first method known from literature is based on enrichment of 68 Ga using a (tin(IV)oxide) SnO 2 -based generator. The fraction of the eluate with the highest concentration of activity (e.g., in 1-2 ml) is buffered for labeling, and a final purification step using a solidphase extraction (SPE; e.g., C18 cartridge) is required to trap the radiopeptide 4 . This purification step removes buffer compounds such as HEPES and unreacted 'free' 68 Ga, which are unwanted in the final product, and it also reduces the 68 Ge concentration resulting from small quantities of 68 Ge breakthrough from the generator. The final (highly pure) product is elutable from the SPE cartridge, usually with a small volume of ethanol, and the mixture can be diluted with 0.9% (wt/wt) NaCl solution.
In a second approach, the labeling begins by eluting the generator and converting the eluted 68 Ga 3+ into the anionic [ 68 GaCl 4 ] − species by the addition of concentrated HCl (final HCl concentration 5.5 M) and trapping anionic 68 Ga on an anion exchanger 5 . The excess of HCl is removed from the anion exchanger cartridge using a stream of inert gas. [ 68 GaCl 4 ] − can then be eluted with a small volume of water (0.2-0.4 ml) into the buffered precursorcontaining reaction vial. Basically, the pH for the labeling of DOTA-conjugated, NOTAconjugated or similarly conjugated peptides should be adjusted to between 3 and 4 (ref. 4). The labeling can be performed at 85-95 °C for DOTA-bearing peptides or at room temperature (21 °C) for, e.g., NOTA-conjugated compounds 6 . The use of concentrated HCl may be a disadvantage in the radiopharmaceutical practice.
A third approach to obtain 68 Ga-labeled tracers uses a cation exchanger to trap cationic 68 Ga 3+ directly from the generator eluate. The adsorbed 68 Ga can be eluted subsequently with mixtures of HCl-containing organic solvents (e.g., acetone/HCl) into the precursor peptide solution 7,8 . The organic solvent must be removed by heating the reaction mixture at 95 °C, and the labeled compound is separated using SPE cartridges. The resulting final product acetone concentrations are found to be well within regulatory limits for release. Labeling procedures using ethanol instead of acetone have also been published 9,10 . Because of the high concentration of ethanol in the radiopharmaceutical product, the determination of ethanol content is required in many countries. This method is patented and licensed for Eckert & Ziegler.
The fifth method for the utilization of 68 Ga combines the high efficiency of the cationic concentration with the anionic purification. In this method, concentrated HCl is substituted by a small volume of diluted HCl. Organic solvents are not required 11,12 . This procedure delivers 68 Ga in a high chemical and radiochemical purity, which allows the labeling of peptides at high specific activity. However, the requirement for two cartridges has limited the routine use of this method in practice.
All these methods are limited in their use for the routine production of radiopharmaceuticals, either because of the use of organic solvents or because of the use of semiconcentrated, corrosive-acting HCl.
In contrast, the procedure described in this protocol focuses on a 68 Ga radiolabeling procedure (method 6 in Table 1), which requires the use of only one single cation exchanger cartridge, and purification of 68 Ga for radiolabeling reactions is achieved just by manipulating cationic/anionic speciation of 68 Ga. The method, pioneered by Mueller et al., uses the behavior of 68 Ga to form anionic species present in concentrated NaCl solution 13 . In this method, 68 Ga 3+ of the generator eluate (usually in 0.1 M HCl or a lower concentration of HCl) is trapped on a cation exchange resin. Rinsing the column with acidified 5 M NaCl solution transforms the cationic 68 Ga 3+ instantly and in situ, which results in desorption and elution of 68 Ga as anionic [ 68 GaCl 4 ] − . This eluate is directed to a buffered solution that contains a chelator-modified DOTA-like peptide for labeling (see Fig.  1). The present protocol has been optimized for the use of DOTA-and NOTA-conjugated peptides [12][13][14][15][16] . The main advantages of this procedure are the high efficiency of desorption of the column and the absence of organic solvents.
Usually, the NaCl eluate of the cation-exchange resin is added to the reaction mixture, which contains 2-3 ml of water, and the NaCl concentration is therefore ∼1 M. Some proteins might be sensitive to high NaCl concentrations, and these usually precipitate by dehydration 17 . For many proteins and peptides, however, NaCl concentrations up to 1 M show stabilizing effects [18][19][20] . We recommend that preliminary experiments (i.e., appropriate biochemical assays) be performed to check that radiolabeling does not alter the biological activity of the peptides or proteins; if this labeling procedure is used to label compounds that are known to be sensitive, the binding affinity of the labeled products should be checked before routine production.
After labeling, neutralization and sterile filtration of the reaction mixture, quality control can be performed, and the 68 Ga-labeled radiopharmaceutical can be directly administered to patients. Appropriate quality control for this process is iTLC, HPLC and measurement of pH value. It is not necessary to determine the concentration of organic solvents, which reduces the overall synthesis-to-release time and simplifies the overall process for radiolabeling and quality control for 68 Ga-labeled radiopharmaceuticals. Furthermore, this procedure has been adapted easily to an automated synthesis module ( Supplementary Fig. 1), thus leading to a rapid preparation of 68 Ga-labeled radiopharmaceuticals (14 min; ref. 14).

Materials
Reagents ▲ CRITICAL Selection of ultra-high-purity reagents is crucial to the success of 68 Ga labeling procedures. Practitioners should carefully select reagents with the lowest possible metal content (e.g., trace metal grade) to avoid unwanted interference in the 68 Ga-DOTA labeling reaction. Examples of precise catalog numbers for trace metal grade reagents are given in this list of reagents. Reagents that are accompanied with a certificate of analysis stating metal concentrations at the parts per trillion level are preferred. • DOTA-like conjugated peptide (e.g., piChem, Bachem); DOTA-TOC, DOTA-NOC, DOTA-TATE, NODAGA-THERANOST and DOTA-SB3 are commonly used. Peptides carrying chelators that are different from DOTA or NOTA should be carefully checked for labeling efficiency when applying this protocol (e.g., for DATA-TOC, it was reported that this protocol led to incomplete labeling 10 ).

Equipment
▲ CRITICAL The equipment and apparatus presented here are for informational purposes.
For example, several manufacturers offer variants of the dose calibrator presented in this list that would be suitable for measurement of the final product radioactivity concentration. The cation-exchange and reverse-phase cartridges that are presented were available at the time of writing and were effective in our method development and evaluations presented here.
• Bond Elut SCX Cartridges, 100 mg, particle size 40 μm (Agilent, cat. no. 12102013)  Figure  2. Rinse the SCX cartridge with 1 ml of 5.5 M HCl and subsequently with 10 ml of water for injection.

Equipment Setup
Generators This procedure will work with a variety of different generators. We describe the specifications and elution conditions for three generators that we have experience with. ! CAUTION 68 Ga emits both positrons and gamma rays. It is imperative that employees using these procedures observe the guidelines set forth by their institution and the Nuclear Regulatory Commission, and that they observe ALARA (as low as reasonably achievable) protocols to minimize radiation exposure. When handling any radioactive material, proper protective equipment, shielding, body and ring dosimetry badges, and a survey meter are required. 4| Detach the SCX cartridge and dry it with a stream of air.

Eckert & Ziegler
5| Elute the 68 Ga, which is trapped onto the SCX, with 0.5 ml of 5 M NaCl/HCl solution into the reaction vial, which contains the reaction solution; the final pH in the reaction vial should be pH 3-4. Incubate the reaction mixture at 85-95 °C for 8-12 min.
6| (Optional) Determine the optimal reaction time by analyzing 1-to 2-μl aliquots of the reaction mixture by radio-iTLC or radio-HPLC, as described in Box 2.
▲ CRITICAL STEP We recommend that this initial experiment be done so as to achieve the required radiochemical purity (% incorporated 68 Ga) for final product release.
▲ CRITICAL STEP For radio-iTLC, a 1-to 2-μl aliquot of the reaction mixture can be directly applied to the iTLC plate.
7| Determine the efficiency of the reaction. To do this, remove a 1-to 2-μl aliquot of the reaction mixture and analyze it by radio-iTLC or radio-HPLC, as described in Box 2.
? TROUBLESHOOTING 8| At the end of the reaction period-after you are satisfied that the reaction has gone to completion or you have completed the steps in the Troubleshooting table-add 2 ml of sterile sodium phosphate buffer to the reaction mixture to adjust the pH of the solution to 6-7.
? TROUBLESHOOTING 9| Pass the reaction mixture through a sterile filter, as mentioned in the equipment list.
If the reaction mixture reaches a radiochemical purity (expressed as % of incorporation of activity in the DOTA-like peptide) that meets the stated release criterion (typically >95%) and the solution has been sterile filtered, the final product may be diluted further for dose administration to any desired volume for animal or patient studies, respectively.

■ PAUSE POINT
? TROUBLESHOOTING 10| The stability of the final product at room temperature should be investigated in a separate labeling experiment by radio-HPLC. If a radical scavenger is used, the labeled peptide can be usually applied within 4 h. For a clinical routine production of 68 Ga-labeled radiopharmaceuticals, it has been shown that a time distance of a minimum of 2 h after the first elution is sufficient for starting a second radiolabeling reaction. As described in the ANTICIPATED RESULTS, the decay of 68 Ge of the generator delivers enough new 68 Ga for the second synthesis.

? TROUBLESHOOTING
Troubleshooting advice can be found in table 2.

Anticipated Results
Our method for the labeling of DOTA-like conjugated peptides with 68 Ga has been published in earlier publications 13,14 . This labeling procedure was routinely used in >1,000 synthesis runs, for >3,000 patient scans and in several nuclear medicine facilities. If the labeling is carried out in the absence of heavy-metal contaminations (especially iron and copper), the probability of success using this protocol is high. Typically, the labeling efficiency of the 68 Ga-labeled DOTA-like conjugated peptides should be >95% within 12 min at 90 °C. By using the above-mentioned iTLC method, R f values of ∼0.8-1.0 are obtained for the labeled peptide, whereas any unchelated 68 Ga (colloidal) remains at the start point of the TLC plate or migrates with a retardation factor of ∼R f = 0-0.1 (Fig. 3). In the case of a radiopharmaceutical production of a 68 Ga-labeled peptide (e.g., for patient doses), the iTLC method may need to be changed to adhere to current local pharmaceutical regulations.
Depending on the hydrophobicity or hydrophilicity of the peptide, the HPLC column and the gradient applied, the retention times for different 68 Ga-labeled peptides will vary. A typical HPLC chromatogram of the 68 Ga-labeled peptide 68 Ga-DOTATOC is shown in Figure 4. By using the above-mentioned HPLC system and HPLC method, the 68 Ga-labeled peptide has an approximate retention time of 9 ± 3 min, and it can be determined by the radiodetector of the HPLC. Any nonchelated 68 Ga should be detected in the first 1-3 min of the run at a flow rate of 1.2 ml/min (void volume).

Elution behavior of the 68 Ge/ 68 Ga generator
After elution of the 68 Ge/ 68 Ga generator, new 68 Ga will be formed by decay of the parent radionuclide 68 Ge. The growth of 68 Ga subsequent to elution follows first-order kinetics described by well-known radioactive daughter growth equations. Thus, elution of 68 Ga after 68 min results in an approximate activity that is 50% of the total 68 Ge present in the column. It follows then that, after 136 min (two half-lifes of 68 Ga), the expected elution of 68 Ga is 75% of total 68 Ge present, which is a useful activity of 68 Ga to start a new synthesis run. After ∼10 h (10 half lives), >99% of the activity of 68 Ga will have grown into equilibrium and may be eluted (Fig. 5).
An example of the progress of 68 Ga activity after elution of the generator and start of an automated pharmaceutical synthesis run for the labeling of, e.g., DOTATOC, is shown in Figure 6. The synthesis is completed after 14 min, and an additional 6 min are required for the quality control (radio-iTLC, pH). Overall, the final radiopharmaceutical product is ready to be delivered to the clinics within 20 min from the start of the synthesis (including time for elution of the generator).
For routine use of 68 Ga-labeled peptides in clinical practice, a sequential production of 68 Ga-labeled radiopharmaceuticals is often required. Figure 7 shows the expected activity in sequentially performed synthesis runs. While the second synthesis starts 2 h after the first elution, the start activity is ∼25% lower compared with the first daily synthesis run (Fig. 7). Importantly, radioactive decay of 68 Ga produces measureable quantities of stable isotope 68 Zn. The concentration of zinc in the generator eluate increases as a function of time after elution 4 , as shown in Figure 8a. High concentrations of zinc ions may interfere with 68 Ga labeling reactions; therefore, the time between the last elution of the generator and the start of a radiolabeling experiment should not be longer than 48 h. Although the procedure that we describe here eliminates most cations (such as Zn), significant quantities of Zn indeed build continuously between generator elutions. Thus, it is prudent to minimize the potential for interference by frequent flushes of the generator to remove stable Zn and any other impurities that may be associated with the solid matrix generator materials. After 3 d post-prior elution of the 68 Ga/ 68 Ga generator, the calculated ratio of 68 Zn versus 68 Ga corresponds to 43 (Fig. 8b). The influence of zinc, iron, copper and other metal ions on labeling efficiency is described in detail in the literature 21,22 . The colorimetric test with thioacetic acid as standard procedure for the determination of critical concentrations of heavy metals, as described in the European Pharmacopoeia 23 , is a very useful tool for the routine quality control of the generator eluate. In some countries, the determination of the concentration of iron and zinc in the generator eluate is requested. The test kits, as mentioned in Reagents, are based on the formation of colored metal complexes with high extinction coefficients, and they allow the easy determination of iron and zinc in a range between 0.1 and 5 μg/ml.

Determination of iron • TIMING ∼6 min
Radiolabeling of tracers with 68 Ga requires that the reagents, tips, caps and reaction vessels used be as free as possible of trace metals, especially iron. If required, metal-free plastic needles must be used during the labeling reaction (Non-Vented Spike, e.g., Qosina, cat. no. 11757 or 11656). The determination of iron contaminations in reagents, generator eluate or other components using an atom absorption spectrometer is possible, but the use of the colorimetric test as mentioned above in Reagents is very helpful and easy to handle. The colorimetric test allows determination of iron concentrations between 0.1 and 5 μg/ml. The concentration of iron during the labeling should be <0.1 μg/ml.

ii.
Place a 1-to 2-μl spot of the sample of the final reaction mixture onto the start point of the iTLC strip (at the first centimeter of the iTLC strip above the level of the iTLC eluent in the chamber).

iii.
Place the iTLC strip upright in the iTLC chamber gently. Cover the chamber and allow the solvent to run up the support material.

iv.
When the solvent is ∼1 cm from the top of the strip, remove it from the chamber and dry the strip.

v.
Place the strip into a thin plastic sheet (e.g., Glad Clingwrap) and lay it onto the radio-iTLC scanner in the appropriate orientation for the scan.

B. Measurement of radiolabeling efficiency by Hplc
Take a sample (10-20 μl) of the final reaction solution and inject it on the RP-HPLC using the HPLC method as mentioned in this box.
Preliminary step. Usually, the sample can be injected directly without dilution using the HPLC radio detector, as mentioned in the Equipment Setup. Because of the pH of the reaction mixture (3μ4), a neutralization of the HPLC sample is not required. Care should be taken to optimize the aliquot volume and radioactivity concentration of the radio-HPLC aliquot to ensure that the radioactivity concentration does not overwhelm the radioactivity detector at the outflow of the radio-HPLC system. Preliminary experiments should be conducted to develop an understanding of detection efficiency and upper threshold of the detector before the preparation of a dose that will be used for important biological experiments or clinical work. Schematic drawing of the NaCl-based labeling procedure for the labeling of DOTA-or NOTA-conjugated peptides with 68 Ga. Preconditioning of the SCX cartridge, SCX cartridge with Luer-lock barb fitting.   Elution profile of the 68 Ge/ 68 Ga generator and formation of 68 Ga. 10 h after the former elution, there is an equilibrium of formation of 68 Ga and decay to 68 Zn, and the maximum of the elutable activity of 68 Ga is reached. By two half-lives after the former elution, 75% of the maximum 68 Ga activity is already elutable from the generator.  Progress of activity of sequentially performed synthesis runs for the routine pharmaceutical production of 68 Ga-DOTATOC-first start activity:   Table 1 Labeling methods.  Table 2 Troubleshooting table.

step(s) problem solution
Steps 7 and 8 Unchelated 68 Ga is discovered in the solution Take a sample and measure its pH. The final pH during the reaction should be between 3 and 4. If the pH is <3, add sodium acetate buffer (Reagent Setup) to the reaction mixture stepwise in 100-ml aliquots until the pH is between 3 and 4. If the pH is higher than 4, adjust the pH with diluted HCl or use a lower amount of sodium acetate buffer for the next labeling so that the pH of the reaction mixture is between 3 and 4 After re-adjusting the pH of the reaction mixture, incubate the reaction solution for an additional 7 min and analyze an aliquot by radio-iTLC and/or radio-HPLC.
Unchelated 68 Ga is still present in the solution after control of the pH of the reaction mixture Incubate the reaction solution for an additional 5 min and analyze another aliquot by radio-iTLC and/or radio-HPLC Unchelated 68 Ga is still present in the solution after the additional incubation Add 5-7 nmol DOTA-like conjugated peptide (if specific activity is not a main focus) and incubate for an additional 5 min. Analyze another aliquot by radio-iTLC and/or radio-HPLC Steps 7-9 Unchelated 68 Ga is still present in the solution after incubation with additional peptide Apply the reaction mixture to an activated SPE (e.g., Sep-Pak) cartridge a . Wash the cartridge with 5 ml of water (for injection) and elute dropwise the 68 Ga-labeled peptide with 0.5 ml of a 1:1 ethanol:saline solution Steps 7 and 8 Unchelated 68 Ga is present in the solution and you suspect that this is due to iron contamination of the reaction mixture Check all used reagents for iron contamination (including the generator eluate and other components (e.g., reaction vial)) using a colorimetric test (Box 1) or an atomic absorption spectrometer. Replace the contaminated reagent or component. If iron is detectable on the preconditioned SCX cartridge, rinse the SCX cartridges before the labeling with 1 ml of HCl (5.5 M), wait 2 min, and rinse again with 1 ml of HCl (5.5 M) and then with 10 ml of water. If iron is detectable in the reaction vial, rinse the vial before the labeling with 1 ml of HCl (5.5 M) and then with 10-20 ml of water Steps 7-10 Multiple radioactive peaks are observed from a sample that is known to be analytically pure and free of isomers Radiolysis may be occurring. Add ∼5 mg of a radical scavenger to the original reaction buffer and start the labeling procedure again. Possible radical scavengers include ascorbic acid, ethanol, gentisic acid and methionine a Sep-Pak purification of the labeled peptide: If a Sep-Pak C-18 cartridge is required to remove unreacted Ga 3+ , it should be activated by preconditioning with ethanol. For this purpose, rinse the cartridge with 5 ml of ethanol and then with 5 ml of water ('for injection' quality). Transfer the reaction mixture through the C-18 cartridge and rinse the cartridge again with 2 ml of water ('for injection' quality). After elution of the 68 Ga-labeled peptide with ethanol, it is essential to either remove the organic solvent from the resulting solution or to dilute the solution with isotonic saline solution before using the labeled compound as a radiopharmaceutical for animal studies or patient administration. The solvent can be evaporated by allowing a gentle stream of inert gas (helium, argon or nitrogen) to pass over the solution. To avoid adherence of the labeled peptide onto the inner surface of the reaction vial, the solution should not be concentrated to dryness.