Field Notes #2 Validation of a Quality Control Method For a Clinical PET Tracer

Transcription

1 Field Notes #2 Validation of a Quality Control Method For a Clinical PET Tracer Amy L. Vāvere, PhD Head Radiochemist Department of Diagnostic Imaging St. Jude Children s Research Hospital Memphis, TN

2 No relevant financial relationships with commercial interests. Disclosure

3 Highlights Release Criteria for [ 11 C]Methionine routine clinical tracer at our institution Details of QC Testing for [ 11 C]MET Issues with QC of [ 11 C]MET along the way...and how we overcame them Best Practices in QC of MET pointers and helpful tools Issues with QC of [ 18 F]Fluorodopamine

4 L-[methyl- 11 C]Methionine Synthesis HS NH 2 H [ 11 C]CH 3 I 100 C H 3 11 C S NH 2 H Trapping of [ 11 C]CH 3 I bubbled through of chilled acetone (< 0 C). L-homocysteine thiolactone HCl dissolved in 32 mm NaH (0.1 2 mg/ml) is added to the reactor vial, sealed and heated to 100 C. Acetone is evaporated with heat and argon flow and solution is neutralized with 10 mm acetic acid and passed through a C18 Sep- Pak Plus followed by sterile water. Average yield is 25-30% (uncorrected) in minutes.

5 Quality Control 20 minutes Test Appearance and Color Radiochemical purity & identity Chemical Purity (annually) Radionuclidic Purity (annually) Volatile rganic Impurities Acceptance Criteria Clear and Colorless 90% C-11 Methionine RT within 15% of standard 4% D-methionine 95% Acetone < 0.5% Tetrahydrofuran < 0.072% Ethanol < 0.5% Diacetone Alcohol < 0.04% ph Filter Integrity Radionuclidic Identity Bacterial endotoxin (LAL) Sterility 50 psi breaking pressure to min (20.4 ± 10%) 5 EU/mL No colony growth out to 14 days

6 Quality Control Details Appearance and Color Visual inspection for clear and colorless with no evidence of particulate matter. Radiochemical Purity & Identity HPLC of [ 11 C]MET by coinjection and comparison to standards (2 before, 1 after system suitability) Confirm [ 11 C]MET represents at least 90% of total radioactivity and < 15% RT difference between RAD and UV Chemical Purity (annually) Chiral HPLC by comparison to standards of L- and D-MET (2 before, 1 after system suitability) Confirm D-MET is 4% of total D- + L-MET

7 QC (2) Radionuclidic Purity (annually) [ 11 C]MET is counted in a MCA using 4-4 min analyses over 20 min from kev. 95% of the observed gamma emissions should correspond to the 511 kev, 1022 kev, or Compton scatter peaks of 11 C. Volatile rganic Impurities GC on Carbowax column with comparison to standards (2 before, 1 after system suitability). Confirm presence of solvents are below limits for release more details to follow.

8 QC (3) ph Confirm in range of by spotting on ph paper Filter Integrity Sterilizing filter is placed on a compressed air line with a pressure gauge and the outlet of the filter is placed under water. The gas pressure is increased slowly until a steady stream of bubbles is observed. The pressure when the bubble stream begins must be 50 psi to pass. Radionuclidic Identity Activity measurement taken in dose calibrator every 5 min over a period of 20 min. Calculated t ½ must be 20.4 min ± 10%.

9 QC (4) Bacterial Endotoxin Endosafe -PTS unit utilizing cartridges (pre-filled with LAL) reagents as controls) to spectrophotometrically determine the endotoxin concentration. Final product will contain < 5 EU/mL which, assuming injection of the total 10 ml of final product, would remain well within the USP bacterial endotoxin limit for radiopharmaceuticals of < 175 EU/V. Sterility Approximately 2 ml [ 11 C]MET reserved for sterility testing with 1 ml each innoculated (within 24 hours) into two media types for incubation thioglycollate and trypticase. ver a 14 day period, samples are monitored for bacterial growth (cloudiness).

10 Radiochemical Purity 11 Absorbance (mau) UV Radioactivity Time (minutes) HPLC analysis of a co-injection of L-methionine reference standard (100 µg/ml) with [ 11 C]MET. Luna 10 μm SCX 100Å column (4.6 x 250 mm) run at 2 ml/min with 0.10 M potassium phosphate, ph 2.6

11 Radiochemical Purity - Sensitivity If multiple HPLC systems are in use, confirm similar sensitivity settings and results between systems. Establish the accuracy threshold for injected activity and your radioactive detector % pure 99.76% pure SAME PRDUCT, DIFFERENT INJECTED ACTIVITY

12 HPLC - Injected Activity MET Peak Area (mau) ul 5 ul 3 ul 2 ul 1 ul Approximate Radioactivity Injected (µci)

13 Volatile rganic Impurities Testing for organic solvents used in synthesis R for cleaning/sterilization. Used to be called RESIDUAL SLVENTS.this is misleading and doesn t encompass all possible volatile organic impurities Keep in mind other molecules are volatile not only typical solvents.

14 User Experience - GC While validating the QC method for [ 11 C]Methionine.. Acetone STANDARD Ethanol THF FINAL PRDUCT??? Carbowax column with oven temp of 60 C for 1 min then ramped to 140 C at 40 C/min for total of 4 min. Injection volume is 0.5 ul and the front inlet heater is at 250 C. Split ratio is 15:1, hydrogen flow is 40 ml/min and the air flow is 400 ml/min.

15 Mystery Peak Very well defined peak it s real and must be identified. Tested rinses of multiple components looking for something volatile, but not extremely so possibly residue from manufacturing? Considered side products that might be forming amongst the relatively few chemicals involved in synthesis and cleaning, that is also a volatile compound. Possible reactants: Homocysteine thiolactone Water Sodium hydroxide Ethanol Acetone

16 Troubleshooting Homocysteine + NaH + Acetone 100

17 Possible Byproducts Acetone MW = Diacetone alcohol MW = Mesityl oxide MW = base - H 2 Self-Aldol Condensation H H 2 C Michael addition H 2 C - H 2 H H CH 2 Isophorone (MW = ) Robinson Annulation?

18 Mass Spec Diacetone alcohol H 2 Diacetone alcohol + H + Diacetone alcohol + Na + Confirmation by mass spec and also GC standard.

19 New Standard and Patient GC STANDARD Acetone Ethanol Ramp temperature after 2 min to shorten run to 4 min Residual Hexane (needle rinse) THF DAA FINAL PRDUCT RESULTS Solvent Acetone Release Criteria 0.5% 0.086% Acetone THF 0.072% 0.000% THF Ethanol 0.5% 0.009% Ethanol DAA 0.04% 0.002% Diacetone Alcohol

20 Setting Release Limit Wording added to our CMC section of the IND: There is no dose limit listed by the USP for diacetone alcohol, however it has an HMIS rating of 2 and is WHMIS class D-2B, similar to acetone, acetonitrile and THF. Based on the available toxicity data (rat oral LD 50 = 2520 mg/kg) diacetone alcohol it seems closest in hazard to acetonitrile (rat oral LD50 = 2460 mg/kg), but less toxic than THF (rat oral LD 50 = 1650 mg/kg). To be conservative, we have set the release criterion for diacetone alcohol at 0.04%, equal to the USP limit for acetonitrile. Accepted by the FDA

21 Aberrant Peak Acetone STANDARD Ethanol Residual Hexane (needle rinse) THF DAA FINAL PRDUCT

22 Comparison of Solvents pa HEXANE THF ETHYL ACETATE METHANL IPA ETHANL TRIFLURTLUENE ACETNITRILE TLUENE BUTYL ACETATE ETHYL BENZENE NITRMETHANE ANISLE DIGLYME DMF Isopropyl alcohol elutes 0.03 min before ethanol. IPA can enter the FPV if sterile needles are introduced before the IPA-swabbed septum is dry Also possible if the QC dose needle punctures the FPV before the septum is dry. AMMNIUM ACETATE DMS Time (minutes)

23 Volatile rganic Impurity Analysis by GC Tracer: MET Batch #: METa Performed by: ERB CALCULATINS (patient samples) SYSTEM SUITABILITY PASSED FAILED RESULTS Solvent Acetone Release Criteria 0.5% 0.081% 0.086% 0.575% Acetone THF 0.072% 0.000% THF Ethanol 0.5% 0.009% Ethanol DAA 0.04% 0.002% Diacetone Alcohol GC Calculations Excel spreadsheet with conditional formatting provides double check SYSTEM SUITABILITY STANDARD VALUES (Relative standard deviation of standards) Concentration in MET GC Standards Solvent Value Solvent Release Criteria Rel Std Dev Acetone 0.10% Acetone < 10% % 0.504% THF 0.01% THF < 10% 0.999% Ethanol 0.10% Ethanol < 10% 0.991% DAA 0.01% DAA < 10% 2.715% RAW DATA Enter peak areas from the Area Percent Report PEAK AREA (pa*s) Solvent RT(min) Standard 1 Standard 2 Patient 1 Patient 2 Standard 3 Acetone THF Ethanol DAA

24 Enantiomeric Purity Ishiwata and colleagues* have shown that by limiting the concentration of NaH to less than 250 mm, less than 4% D- methionine is formed. L D UV Absorbance (mau) nm Radioactivity TIme (minutes) Phenomenex Chirex D-penicillamine column (4.6 x 50 mm) with a mobile phase of 1 mm copper sulfate : 2-propanol, 95:5. The flow rate is 0.5 ml/min * Ishiwata et al. Int J Rad Appl Instrum A (1988) 39:

25 Chiral Purity 5.4% D-MET!! Routine annual validation revealed increase in D-MET in product. Performed synthesis on two different radiosynthesizers with similar results. Synthesis is successful, but what about the chiral purity of the precursor L-homocysteine?

26 Chirality of Precursor Current Lot New Lot (new vendor) ld Lot (original vendor)

27 Results with Pure Precursor We have added routine testing of any new lot of precursor prior to use. We confirmed that the vendor DES NT test chiral purity. They assume all is L because their precursor is L. Purity listed is purity of homocysteine, not chiral purity.

28 6-[ 18 F]Fluorodopamine - Synthera Use commercially available synthesis modules and kits to make fluorinated compounds quickly and easily.

29 F-DA Synthesis 2 Syntheras Tf SYNTHERA 1 I Boc N Boc K 18 F, Kryptofix Azeotrope evap: mg, 600 µl CH 3 CN 50 C 18 F I Boc N Boc Toluene, 900 µl 4 I + 18 F Boc N Boc 25% uncorrected, HPLC-purified yield in 60 min H H 18 F NH 2 HI, 600 µl 4 18 F Boc N Boc SYNTHERA 2

30 Release Criteria

31 Acid-Labile Precursor = 1 Synthera Tf I Boc N Boc K 18 F, Kryptofix Azeotrope evap: mg 10/90 MeCN/Toluene 18 F I Boc N Boc Decomposition I + 18 F Boc N Boc Residual levels of Kryptofix were higher than the release limit established by the USP (< 50 µg/ml) even after HPLC purification. 4 M HCl H NH 2 ne-pot synthesis removed intermediate purification that eliminated Kryptofix H 18 F

32 Replacement? Any attempt to remove K2.2.2 also removed F-DA. It is believed they coelute on the HPLC purification. Tetraethylammonium bicarbonate (TEA HC 3 ) and tetrabutylammonium bicarbonate (TBA HC 3 ) were selected as possible replacements based on previous successes.* Fluorination testing demonstrated incorporation by either tetralkylammonium salt comparable to Kryptofix. * Brichard et al., Eur J rg Chem (2014), 28: ; Reed et al. J Fluor Chem (2012) 143:

33 Excellent Yield 30% [ 18 F]F-DA Yield (uncorrected) 25% 20% 15% 10% 5% 0% 22.9% 23.0% 22.5% 26.0% Kryptofix/K 2 C mg 3.4 mg mg TEA HC 3 TEA HC 3 TBA HC 3 (n = 19) (n = 9) (n = 8) (n = 7)

34 Residual TBA HC Patient Sample TBAB (µg/ml) TBA HC 3 standards and F-DA samples were spotted on silica and overspotted with a methanol/25% ammonium hydroxide solution (9:1). Development in an iodine chamber for one minute allowed visual comparison for interpolation of TBA HC 3 concentration. < 100 µg/ml residual TBA HC 3, and possibly < 50 µg/ml, when visually compared to reference standards (n = 8) Kuntzsch, M. et al., Pharmaceuticals (2014) 7:

35 Justification USP does not provide a recommended release limit for TBA HC 3, however, the European Pharmacopoeia has an established release limit of 2.6 mg/injected volume.* Synthesis requires only 2.0 mg of TBA HC 3 - even if all TBA HC 3 were carried through to the final product, the release limit would not be reached. Majority of the TBA HC 3 will be purified out of the final product via HPLC during the final purification. Residual TBA HC 3 in several [ 18 F]F-DA preparations were consistently lower than 0.1 mg/ml. * The European Pharmacopoeia, EDQM (2014) Strassbourg, France.8.0..

36 Volatile Excipient? F-DA FINAL PRDUCT STANDARD Ethanol Residual Hexane (needle rinse) Acetonitrile Toluene Ammonium Acetate

37 Not an Impurity Ammonium acetate is added as a neutralization buffer to the F-DA product with a final concentration of 0.15%. This is below the approved level of 0.4% for intravenous injection of ammonium acetate as an excipient for sterile dosages in the formulation of pharmaceuticals.* Not included in Volatile rganic Impurity calculations because the percentage is known and the addition is intentional, not an impurity. * Niazi, SK. Chapter 13: Approved Excipients in Sterile Dosage Forms. Handbook of Pharmaceutical Manufacturing Formulations: Sterile Products. Volume Six, 2 nd Ed Informa Healthcare USA, New York, NY.

38 Acknowledgements SJCRH Clinical PET Team Scott Snyder, PhD - Director Elizabeth Butch, PhD Victor Amador Cody Thompson Jitendra Mishra, PhD Past members: Christopher Surdock PharmD, PhD; Zapporah Young, PhD; Tracy McGraw; Tino Goronga; Joe Cook, MD