Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

van der Veen, Elly L.; Antunes, Inês F.; Maarsingh, Petra; Hessels-Scheper, Janet; Zijlma, Rolf; Boersma, Hendrikus H.; Jorritsma-Smit, Annelies; Hospers, Geke A. P.; de Vries, Elisabeth G. E.; Lub-de Hooge, Marjolijn N.; de Vries, Erik F. J.

doi:10.1186/s41181-019-0062-7

EJNMMI Radiopharmacy and Chemistry

Table 1 Challenges encountered during translation from research grade to clinical grade [¹⁸F]FB-IL2 and adaptations made to the original preclinical protocol

From: Clinical-grade N-(4-[¹⁸F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

Preclinical production method	Challenge encountered	GMP-production method
Part 1: [¹⁸F]SFB synthesis
Purification by Oasis HLB cartridge.	Impurity in [¹⁸F]SFB product interfered with the conjugation of IL2.	Purification performed by HPLC, followed by formulation using an Oasis HLB cartridge.
[¹⁸F]SFB is produced in a non-classified hot cell with a Zymark robotic system. [¹⁸F]FB-IL2 produced manually.	GMP syntheses modules insufficient functionalities to accommodate the total labeling procedure, due to a change in purification method.	[¹⁸F]SFB produced as a starting material in a non-classified hot cell with a Zymark robotic system. Product collected via sterile filtration in sterile vial and transferred to a class C clean room. [¹⁸F]FB-IL2 produced with a PharmTracer Eckert & Ziegler synthesis module in a class C clean room and class C hot cell.
Part 2: [¹⁸F]FB-IL2 conjugation
Reconstituted IL2 stored at −80 °C. After defrosting added manually to the mixture immediately before conjugation.	IL2 labeling in automated synthesis module: IL2 added at start of synthesis. IL2 instable in warm hot cell during synthesis.	After production of [¹⁸F]SFB IL2 added, directly to cooled reaction vial. Temperature to 50 °C shortly before adding [¹⁸F]SFB.
	IL2 transported via disposable tubing to the reaction vial. Sticking of IL2 to tubing during transport to the reaction vial.
Conjugation reaction in borate buffer pH 8.3.	Low yields due to low labeling efficiencies. At pH > 8.7 hydrolysis of [¹⁸F]SFB.	Optimal pH 8.5 (±0.1).
HPLC purification after conjugation step.	Low recovery [¹⁸F]FB-IL2 from HPLC due to high lipophilicity and tendency to aggregate.	HPLC replaced by solid-phase extraction over a tC2 Sep-Pak cartridge.
No sterile filtration for animal experiments.	[¹⁸F]FB-IL2 tendency to stick to sterilization filter.	Filter size changed to smaller diameter.
End formulation in ~ 55% ethanol in water, diluted 1:10 with saline (ethanol < 10%) shortly before use.	Final formulation adapted for patient use: 9% ethanol, 4.5% glucose, 0.5% HSA, 0.1% SDS and 0.0001% phosphoric acid. An excipient in the final formulation occasionally precipitated.	Precipitation of HSA at low pH (< 4). Amount of phosphoric acid at washing/elution step adapted (100 times diluted).

Abbreviations: HLB hydrophilic-lipophilic balance, IL2 interleukin-2, HPLC high performance liquid chromatography, GMP good manufacturing practices, HSA human serum albumin, SDS sodium dodecyl sulfate

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