field notes #2 validation of a quality control method for ... · 7/1/2017 · field notes #2...

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

Disclosure

No relevant financial relationships with commercial interests.

Highlights

• Release Criteria for [11C]Methionine – routine clinical tracer at our institution

• Details of QC Testing for [11C]MET

• Issues with QC of [11C]MET along the way...and how we overcame them

• Best Practices in QC of MET – pointers and helpful tools

• Issues with QC of [18F]Fluorodopamine

L-[methyl-11C]Methionine Synthesis

• Trapping of [11C]CH3I bubbled through of chilled acetone (< 0°C).

• L-homocysteine thiolactone HCl dissolved in 32 mM NaOH (0.1 mg @ 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 17-25 minutes.

HSOH

NH2

OS

OHNH2

O

H311C

[11C]CH3I

100°C

Quality Control – 20 minutes Test Acceptance Criteria

Appearance and Color Clear and Colorless Radiochemical purity &

identity ≥ 90% C-11 Methionine

RT within 15% of standard Chemical Purity

(annually) ≤ 4% D-methionine

Radionuclidic Purity (annually) ≥ 95%

Volatile Organic Impurities

Acetone < 0.5% Tetrahydrofuran < 0.072%

Ethanol < 0.5% Diacetone Alcohol < 0.04%

pH 4.5 – 7.5 Filter Integrity ≥ 50 psi breaking pressure

Radionuclidic Identity 18.36 to 22.44 min (20.4 ± 10%)

Bacterial endotoxin (LAL) ≤ 5 EU/mL

Sterility No colony growth out to 14 days

Quality Control Details

• Visual inspection for clear and colorless with no evidence of particulate matter.

Appearance and Color

• HPLC of [11C]MET by coinjection and comparison to standards (2 before, 1 after – system suitability)

• Confirm [11C]MET represents at least 90% of total radioactivity and < 15% RT difference between RAD and UV

Radiochemical Purity & Identity

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

QC (2)

• [11C]MET is counted in a MCA using 4 - 4 min analyses over 20 min from 40-2500 keV.

• ≥ 95% of the observed gamma emissions should correspond to the 511 keV, 1022 keV, or Compton scatter peaks of 11C.

Radionuclidic Purity (annually)

Volatile Organic 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.

QC (3)

• Confirm in range of 4.5 – 7.5 by spotting on pH paper pH

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.

• Activity measurement taken in dose calibrator every 5 min over a period of 20 min. Calculated t½ must be 20.4 min ± 10%.

Radionuclidic Identity

QC (4)

• 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.

Bacterial Endotoxin

• Approximately 2 mL [11C]MET reserved for sterility testing with 1 mL each innoculated (within 24 hours) into two media types for incubation – thioglycollate and trypticase.

• Over a 14 day period, samples are monitored for bacterial growth (cloudiness).

Sterility

Radiochemical Purity

• HPLC analysis of a co-injection of L-methionine reference standard (100 µg/mL) with [11C]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

-1

1

3

5

7

9

11

0 1 2 3 4 5 6 7

Abs

orba

nce

(mA

U)

Time (minutes)

UVRadioactivity

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.

99.76% pure

98.44% pure

SAME PRODUCT, DIFFERENT

INJECTED ACTIVITY

HPLC - Injected Activity

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16 18 20

MET

Pea

k Ar

ea (m

AU)

Approximate Radioactivity Injected (µci)

10 uL5 uL3 uL2 uL1 uL

Volatile Organic Impurities

• Testing for organic solvents used in synthesis OR for cleaning/sterilization.

• Used to be called RESIDUAL SOLVENTS….this is misleading and doesn’t encompass all possible volatile organic impurities

• Keep in mind other molecules are volatile – not only typical solvents.

User Experience - GC

• While validating the QC method for [11C]Methionine…..

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.

Acetone

Ethanol

THF

STANDARD

???

FINAL PRODUCT

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

Troubleshooting

Homocysteine + NaOH + Acetone Homocysteine + NaOH + Acetone – 100° NaOH + Acetone – 100°

Possible Byproducts

O

H2C

O

O

OH

O

CH2

O

O

H2C

O

OHH

OO

- H2O

Self-Aldol Condensation

base

Michael addition

- H2O

Acetone MW = 58.08

Diacetone alcohol MW = 116.6

Mesityl oxide MW = 98.14

Isophorone (MW = 138.21)

Robinson Annulation?

Mass Spec

• Confirmation by mass spec and also GC standard.

Diacetone alcohol – H2O

Diacetone alcohol + H+

Diacetone alcohol + Na+

New Standard and Patient GC

SolventAcetone 0.086% AcetoneTHF 0.000% THFEthanol 0.009% EthanolDAA 0.002% Diacetone Alcohol

Release Criteria≤ 0.5%

≤ 0.04%

RESULTS

≤ 0.072%≤ 0.5%

Residual Hexane (needle rinse)

Acetone

THF

Ethanol

DAA

Ramp temperature after 2 min to shorten run to 4 min

STANDARD

FINAL PRODUCT

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 LD50 = 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 LD50 = 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

Aberrant Peak


Acetone

THF

Ethanol

DAA

STANDARD

FINAL PRODUCT

Comparison of Solvents

0

50

100

150

200

2501.

171.

241.

311.

391.

461.

541.

611.

681.

761.

831.

911.

982.

062.

132.

202.

282.

352.

432.

502.

572.

652.

722.

802.

872.

953.

023.

093.

173.

243.

323.

393.

463.

543.

613.

693.

763.

843.

913.

984.

064.

134.

214.

284.

354.

434.

504.

584.

654.

734.

804.

874.

95

pA

Time (minutes)

ETHA

NO

L -1

.73

ACET

ON

ITRI

LE-1

.99

TOLU

ENE

-2.1

6

AMM

ON

IUM

ACE

TATE

-3.

99

DMSO

-4.8

9

MET

HAN

OL

-1.6

2

DMF

-3.4

4

BUTY

L AC

ETAT

E -2

.25

DIG

LYM

E -3

.36

NIT

ROM

ETHA

NE

-2.6

0

TRIF

LUO

ROTO

LUEN

E -1

.87

ETHY

L BE

NZE

NE

-2.4

7

THF

-1.5

5

ANIS

OLE

-3.

41

ETHY

L AC

ETAT

E -1

.59

IPA

-1.7

0

HEXA

NE

• 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.

GC Calculations Volatile Organic Impurity Analysis by GC

Tracer: METBatch #:

Performed by: ERB

CALCULATIONS(patient samples)


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 DATAEnter 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 1.42 101.42296 101.96733 88.52475 87.13136 102.45029THF 1.53 12.22383 12.25668 0.00000 0.00000 12.45126Ethanol 1.7 101.51244 103.22704 9.58524 9.43543 103.31979DAA 3.51 9.42761 9.55293 1.66103 1.68544 9.93029

20170530METa

SYSTEM SUITABILITY PASSED


≤ 0.04%

RESULTS

≤ 0.072%≤ 0.5%

Volatile Organic Impurity Analysis by GCTracer: MET

Batch #:Performed by: ERB




Solvent Value


Acetone < 10% 10.188% THF 0.01%

THF < 10% 0.999% Ethanol 0.10%

Ethanol < 10% 0.991% DAA 0.01%

DAA < 10% 2.715%




20170530METa

SYSTEM SUITABILITY FAILED


≤ 0.04%

RESULTS

≤ 0.072%≤ 0.5%






Solvent Value


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%




20170530METa



≤ 0.04%

RESULTS

≤ 0.072%≤ 0.5%






Solvent Value


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%




20170530METa



≤ 0.04%

RESULTS

≤ 0.072%≤ 0.5%

Excel spreadsheet with conditional

formatting provides double check

Enantiomeric Purity

• Ishiwata and colleagues* have shown that by limiting the concentration of NaOH to less than 250 mM, less than 4% D-methionine is formed.

-10

0

10

20

30

40

50

0 2 4 6 8 10 12UV

Abs

orba

nce (

mA

U)

TIme (minutes)

UV @ 254 nm

Radioactivity

L D

Phenomenex Chirex-33126 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) 39:311-314.

Chiral Purity

• 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?

5.4% D-MET!!

Chirality of Precursor

New Lot (new vendor)

Current Lot

Old Lot (original vendor)

Results with Pure Precursor

• We have added routine testing of any new lot of precursor prior to use.

• We confirmed that the vendor DOES NOT test chiral purity. They assume all is L because their precursor is L. Purity listed is purity of homocysteine, not chiral purity.

6-[18F]Fluorodopamine - Synthera

Use commercially available synthesis modules and kits to make fluorinated compounds quickly and easily.

F-DA Synthesis – 2 Syntheras

K18F, Kryptofix 2.2.2 Azeotrope evap: 90 sec @ 110°

12 mg, 600 µL CH3CN Evap @ 50°C

HI, 600 µL 4 min @160°

NBoc

Boc

IO

O

OOTf

NBoc

Boc

IO

O

O

18F

NH2

18FHO

HO

Toluene, 900 µL 4 min @155°

NBoc

Boc

18FO

O

O

I+

NBoc

Boc

18FO

O

SYNTHERA 1

SYNTHERA 2

25% uncorrected, HPLC-purified yield

in 60 min

Release Criteria

Acid-Labile Precursor = 1 Synthera

• Residual levels of Kryptofix were higher than the release limit established by the USP (< 50 µg/mL) even after HPLC purification.

• One-pot synthesis removed intermediate purification that eliminated Kryptofix

K18F, Kryptofix 2.2.2 Azeotrope evap: 2 min @ 110°

15 mg 10/90 MeCN/Toluene

Decomposition 8 min @ 140°

4 M HCl 20 min @ 95°

NBoc

Boc

IO

O

OOTf

O

O

NBoc

Boc

IO

O

O

18F

O

O

NBoc

Boc

18FO

OO

O

O

I+

NH2

18FHO

HO

Replacement?

• Any attempt to remove K2.2.2 also removed F-DA. It is believed they coelute on the HPLC purification.

• Tetraethylammonium bicarbonate (TEA HCO3) and tetrabutylammonium bicarbonate (TBA HCO3) 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 Org Chem (2014), 28:6145-6149; Reed et al. J Fluor Chem (2012) 143:231-237.

Excellent Yield

(n = 19) (n = 9) (n = 8) (n = 7)

22.9% 23.0% 22.5% 26.0%

0%

5%

10%

15%

20%

25%

30%[18

F]F-

DA Y

ield

(unc

orre

cted

)

Kryptofix/K2CO3 2.5 mg TEA HCO3

3.4 mg TEA HCO3

2-2.5 mg TBA HCO3

Residual TBA HCO3

• TBA HCO3 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 HCO3 concentration.

• < 100 µg/mL residual TBA HCO3, and possibly < 50 µg/mL, when visually compared to reference standards (n = 8)

0 50 100 150 200

TBAB (µg/mL)

Patient Sample

Kuntzsch, M. et al., Pharmaceuticals (2014) 7: 621-633

Justification

• USP does not provide a recommended release limit for TBA HCO3, however, the European Pharmacopoeia has an established release limit of 2.6 mg/injected volume.*

• Synthesis requires only 2.0 mg of TBA HCO3 - even if all TBA HCO3 were carried through to the final product, the release limit would not be reached.

• Majority of the TBA HCO3 will be purified out of the final product via HPLC during the final purification.

• Residual TBA HCO3 in several [18F]F-DA preparations were consistently lower than 0.1 mg/mL.

* The European Pharmacopoeia, EDQM (2014) Strassbourg, France.8.0..

Volatile Excipient?

F-DA FINAL PRODUCT


Ethanol

Ammonium Acetate

STANDARD

Acetonitrile

Toluene

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 Organic 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, 2nd Ed. 2009. Informa Healthcare USA, New York, NY.

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

field notes #2 validation of a quality control method for ... · 7/1/2017 · field notes #2...

Documents