Viruses can be lipid-coated or non- enveloped. Virus inactivation works by one of the following two mechanisms:  By attacking the viral envelope or capsid and destroying its ability to infect or interact with cells.  By disrupting the viral DNA or RNA and preventing replication. Virus inactivation involves dismantling a virus’s ability to infect cells without actually eliminating the virus. FDA specifies that a clearance of at least 3 logs must be achieved for mAb production.

Solvent/Detergent (S/D) Inactivation Effective with lipid-coated viruses Disrupts the interactions between molecules in the lipid coat, rendering the coat dysfunctional and impeding replication Commonly used and has a reliable safety record Chemicals need to be removed downstream Pasteurization/Heat Effective with non-lipid and lipid-coated viruses Typically in a liquid at 60 o C for 10 hours Protein product must have a higher thermal resistance than the virus Requires the addition of stabilizers, which will need to be removed downstream Low pH Treatment Most effective with lipid-coated viruses Acidic conditions deactivate virus Protein product must have a higher resistance to low pH conditions than that of the viral particles No additional clearance steps are needed downstream

Virus Inactivation Tank Ion Exchange Chromatography Storage Tank and Diafiltration

StreamMass Flow Rate (kg/batch) ComponentMass Composition (%) Connection S Acetic acid Impurities mAb WFI From storage tank S Polysorbate 80 WFI Chemical inlet S Nitrogen Oxygen Vent to atmosphere S Acetic acid Impurities mAb Polysorbate 80 WFI To polishing filter

 There was no way to measure concentrations of active and inactive viruses  Manipulating temperature, holding time, and flow rates had very little effect in our simulated unit  Theoretically, a longer holding time and a higher S/D concentration should results in higher levels of inactivation  The volume increased minimally as the detergent concentration increased

SuperPro® Specification for Polysorbate80 at 0.01% wt Value Volume L Max Allowable Working Volume 90% Height/Diameter Ratio 3 Height 2.138m Diameter 0.713m Design Pressure1.52bar

Supplier UsedNew Range of Cost For Tank with agitator Cost for Mixing Unit Cost for Tank without agitator Total Cost of Tank and Agitator Machinery and Equipment Company, Inc. $12, $25, $100, Aaron Equipment Company$8, $10, Slimline Manufacturing $1,000.00$127, $128, Pope Scientific Inc. $11, Apache Stainless Equipment Corporation $15,000.00$20,000.00$35,000.00

Operating CostsCost Steam (per batch)$0.18 Polysorbate 80 (per batch)$2.32 Usage Rate (per equipment-hr)$2, Labour Hours (labour hrs/hr)8 TOTAL $2, Labour Hours Operating conditions  Steam: normal pressure, 42.5 kg/batch  Polysorbate 80: kg/batch Simulation Unit Operation: In Place Steaming  Transfer In  Pull In  Holding  Transfer Out  In Place Cleaning

 Develop a way to simulate active and inactive virus concentrations › Optimize for temperature, holding time, and S/D concentration  Polysorbate 80 concentration of wt%  Purchase a used blending tank from Aaron Equipment Company for $8000