Microfluidic Preparation of 89Zr-Radiolabelled Proteins by Flow Photochemistry

Abstract

⁸⁹Zr-radiolabelled proteins functionalised with desferrioxamine B are a cornerstone of diagnostic positron emission tomography. In the clinical setting, ⁸⁹Zr-labelled proteins are produced manually. Here, we explore the potential of using a microfluidic photochemical flow reactor to prepare ⁸⁹Zr-radiolabelled proteins. The light-induced functionalisation and ⁸⁹Zr-radiolabelling of human serum albumin ([⁸⁹Zr]ZrDFO-PEG₃-Et-azepin-HSA) was achieved by flow photochemistry with a decay-corrected radiochemical yield (RCY) of 31.2 ± 1.3% (n = 3) and radiochemical purity >90%. In comparison, a manual batch photoreactor synthesis produced the same radiotracer in a decay-corrected RCY of 59.6 ± 3.6% (n = 3) with an equivalent RCP > 90%. The results indicate that photoradiolabelling in flow is a feasible platform for the automated production of protein-based ⁸⁹Zr-radiotracers, but further refinement of the apparatus and optimisation of the method are required before the flow process is competitive with manual reactions.

Keywords: flow chemistry; photochemistry; protein conjugation; radiochemistry.

Figure 1

Illustration depicting the general concept of the light-activated preparation of ⁸⁹Zr-radiolabelled protein conjugates in a microfluidic photochemistry device.

Scheme 1

Synthesis of DFO-PEG₃-Et-ArN₃ (1). Reagents and conditions: (a) imidazole-1-sulphonyl azide HCl, CuSO₄⋅5H₂O, K₂CO₃, MeOH, rt, 18 h; (b) N-Boc-4,7,10-trioxa-1,13-tridecanediamine (3), HATU, DIPEA, anhyd. DMF, rt, 24 h; (c) TFA, CH₂Cl₂, rt, 1 h; (d) succinic anhydride, anhyd. DMF, rt, 48 h; (e) DFO-mesylate, HATU, DIPEA, anhyd. DMF, rt, 48 h.

Figure 2

(A) FutureChemistry FlowStart B-222 photochemistry module. (B) LedEngin, Inc twin LEDs connected in series. (C) Emission spectra for the twin LED light source (combined plots). (D) Microfluidic chip (top), oriented in the direction of flow (left to right); and microfluidic chip holder with standard ¼ inch-28 screw-connectors for input and output.

Scheme 2

Preparation of the [⁸⁹Zr]ZrDFO-PEG₃-Et-azepin-HSA protein conjugate via sequential radiolabelling and flow photoconjugation.

Figure 3

Radioactive chromatography: (A) Radio-ITLC chromatograms of [⁸⁹Zr]ZrDFO-PEG₃-Et-ArN₃ before (blue) and after (black) photolysis at 395 nm. The elution profile of [⁸⁹Zr][Zr(DTPA)]⁻ (red) is shown as a control. (B) HPLC chromatograms show the elution profiles of compound 1 (black); the non-radioactive [^natZr]Zr-complexes before (green) and after (red) photolysis; and the [⁸⁹Zr]ZrDFO-PEG₃-Et-ArN₃ complex (blue).

Figure 4

Radioactive chromatography depicting: (A) radio-ITLC chromatograms of purified (black) [⁸⁹Zr]ZrDFO-PEG₃-Et-azepin-HSA (RCP > 95%) and control (blue) [⁸⁹Zr][Zr(DTPA)]⁻. (B) Analytical SEC (PD-10) elution profiles displaying crude (blue) and purified (black) samples of [⁸⁹Zr]ZrDFO-PEG₃-Et-azepin-HSA. (C) Radioactive (black) and electronic absorption (blue) SEC gel-filtration HPLC chromatograms of the purified [⁸⁹Zr]ZrDFO-PEG₃-Et-azepin-HSA (RCP > 90%). Note that (*) designates aggregated protein.