ALISI – Automated Light induced Synthesis of 89Zr-Radiolabeled Antibodies for ImmunoPET
The standard production of 89Zr-radiolabeled antibodies (89Zr-mAbs) relies on the pre-conjugation of desferrioxamine B (DFO) to the purified protein, followed by isolation and characterization of the functionalized intermediate, and then radiosynthesis on demand. Although highly successful, this route entails several technological and economic hurdles that limit the more wide-spread development of 89Zr-mAbs at smaller research and clinical facilities. Here, we adapted our one-pot photoradiosynthesis approach for automation and designed ALISI – a synthesis box that can produce and isolate 89Zr-mAbs starting from [89Zr(C2O4)4]4– (89Zr-oxalate), our photoactivatable DFO-PEG3-ArN3 chelate, and clinical-grade Herceptin without pre-purification of trastuzumab.
Methods: We constructed our prototype radiosynthesizer unit by using a combination of open-source microcontrollers (Arduino) and computer-aided design (CAD), coupled with additive manufacturing (3D-printing) techniques. Liquid handling components and fluidic pathways employ commercially available, single-use tubing and three-way switching valves operated by digital servo-motors. Liquid transfer is driven pneumatically with a syringe pump. For the in-line automated purification, we designed custom-made SK-10 that flow under gravity and allow for increased resolution between the small-molecule byproducts and the higher molecular weight 89Zr-mAbs. Results: ALISI can produce 89Zr-radiolabeled proteins formulated in sterile solution in 89Zr-oxalate stock, chelate radiolabeling, and light-induced protein conjugation, followed by 89Zr-mAb purification, formulation and sterile filtration. Conclusion: Full automation of the simultaneous radiolabeling and photochemical bioconjugation process was demonstrated on a custom-made radiosynthesis unit. The ALISI box performs all of the essential steps required to make 89Zr-mAbs in high radiochemical purity at the touch of a button.
A.J. Poot,K.W.A. Adamzek,A.D.Windhorst,M J.W.D.Vosjan,S.Kropf,H.J.Wester,G.A.M.S.Van Dongen,D.J.Vugts, J.Nucl.Med. 2019, 60, 691–695.
M. Patra, S.Klingler, L.S.Eichenberger, J. P. Holland, iScience 2019, 13, 416–431.
S.Klingler,R.Fay,J.P.Holland, J. Nucl. Med. 2020, 61, 1072–1078.
A.Guillou,D.F.Earley,M.Patra,J.P.Holland, Nat. Protoc. 2020, 15, 3579–3594.
A.Guillou,D.F.Earley,J.P.Holland, Chem.–A Eur.J. 2020, 26, 7185–7189.