Tangential flow filtration as a novel approach for fluorescent tracer purification and production up-scaling: cetuximab-800CW and ustekinumab-800CW as a proof-of-concept.

Fluorescence molecular imaging using fluorescently labeled antibodies enables identification of target cells and provides pharmacological insight into local drug distribution. The current GMP manufacturing method of tracers using PD-10 columns is subject to methodological constraints when handling low antibody concentrations, large quantities, or incompatible antibodies. This study presents Tangential Flow Filtration (TFF) as a novel and scalable approach to manufacture clinical-grade fluorescent tracers and demonstrates its applicability using cetuximab-800CW and ustekinumab-800CW. TFF system settings were optimized for purification of cetuximab-800CW and ustekinumab-800CW with labeling parameters and quality control similar to tracer production with PD-10 columns. Cetuximab-800CW production was successfully transitioned from PD-10 to TFF. QC-data after manufacturing with TFF (n = 7) or PD-10 (n = 21) were compared at release and up to 24 months of storage at 2-8°C. This revealed TFF purification to be more efficient and robust, with label efficiencies of 93.5 ± 2.3% with TFF compared to 68.3 ± 6.9% with PD-10, and 0.0 ± 0.1% unconjugated dye with TFF compared to 0.5 ± 0.3% with PD-10. Stability data were comparable for both methods. TFF manufacturing yielded clinical-grade cetuximab-800CW batches stable for at least 24 months. Concentrations ranged from 0.99-1.08 mg/mL and target binding affinity from 65-98%. Detected free dye and aggregates remained well below 3.0%. Moreover, ustekinumab-800CW was successfully manufactured using TFF after PD-10 manufacturing failed due to particle formation. Ustekinumab-800CW was stable up to 24 months at 2-8°C. Together, this demonstrates the feasibility of using TFF for clinical-grade fluorescent tracer manufacturing under GMP conditions.