During the production of protein biologics in mammalian hosts, viral contaminations pose a particular risk to patient safety and drug manufacturing operations. Thus, regulatory guidelines requiring viral spiking studies have been put in place to evaluate the extent of viral clearance across downstream processes. Viral spiking studies are executed by introducing high concentrations of representative live virus into drug-containing load materials and clearance capacity is evaluated across each downstream unit operation. In their current format, viral spiking studies are expensive and time consuming. The need to work with live virus normally requires collaboration with a contract research organization (CRO) with biosafety level (BSL)-2 capability, as manufacturers will not purposely introduce viruses on-site. To reduce time and resource cost of biopharmaceutical manufacturing, there is a clear need for more efficient viral clearance validation studies, that can be performed on-site. In our approach to this technical gap, we developed a series of fluorescent viral surrogate (FVS) particles which are easily quantifiable through fluorescence-based measurements and have well-defined physicochemical characteristics aligning with non-enveloped model viruses currently employed in viral clearance validation. Non-enveloped FVS’s were synthesized by covalently modifying Fluospheres™ carboxylate-functionalized nanoparticles with either fluorescent-labeled model proteins or recombinantly generated viral capsid proteins. After synthesis, FVS’s 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 TCID<sub>50</sub> and plaque titer assays, which are commonly used to quantify live virus. Here we focus on the feasibility of using normal flow-based capture to robustly quantify FVS’s using fluorescence and comparisons of FVS and live virus clearance mechanisms across platform downstream unit operations. Specifically, we evaluate clearance across representative viral filtration (VF), Protein A affinity (PrA), ion exchange (IEX), and hydrophobic interaction (HIC) chromatography operations to formulate structure-function relationships and assess feasibility in employing FVS’s as a potential alternative to live virus in viral clearance validation.

Fluorescent viral surrogates for simulation of non-enveloped viral clearance behavior across platform downstream unit operations

ACS Spring 2022

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