(171e) Developing Fluorescent Viral Surrogate Particles As an Alternative to Live Virus in Viral Clearance Validation

When manufacturing protein-based biologic drugs in mammalian hosts, viral contaminations are of particular concern as they put patients at risk of contracting potentially fatal illnesses and cause manufacturing disruptions, pressing companies into costly decontamination and recall actions. Given these serious repercussions, regulatory agencies have issued guidelines requiring proof of sufficient viral clearance across the downstream process in case a contamination event should occur. This proof of clearance is currently accomplished by performing viral spiking studies. Viral spiking studies are executed by introducing high concentrations of live virus into drug-containing load materials and clearance capacity is evaluated across each downstream unit operation. These studies are costly in both time and money as they are typically performed externally with a certified contract research organization (CRO) with biosafety level (BSL)-2 capability; manufacturers will not knowingly introduce contaminating virus into their processes on-site owing to fears of recurring contamination. In this work, we address this technical gap by developing a series of fluorescent viral surrogate (FVS) particles, which are easily quantifiable through fluorescence-based measurements and have well-defined physicochemical characteristics aligning with enveloped and non-enveloped model viruses currently employed in viral clearance validation. Non-enveloped FVS’s were synthesized by covalently modifying Fluospheres™️ carboxylate-functionalized particles with fluorescent-labeled model proteins. Bare and coated Fluospheres™️ were characterized based on size, charge, and relative hydrophobicity, and these data were compared to those of live virus. To quantify FVS’s, a dead-end flow filtration unit operation was optimized for capture of FVS’s from solution to achieve limits of detection comparable to TCID₅₀ and plaque titer assays (used to quantify live virus). These optimized FVS’s will be characterized using clearance mechanisms across various separation unit operations, such as viral filtration (VF), affinity (AC), ion exchange (IEX), and hydrophobic interaction (HIC) chromatography. This will be done to formulate structure-function relationships and to assess the feasibility in employing FVS technology as an alternative to live virus in viral clearance validation.