NO2 Sterilization as Part of a Batch Release Process for Clinical Trials Evan Goulet, Ph.D., Colleen A. Kase, and Erica G. Danko; Noxilizer, Inc., Baltimore, MD Abstract Process Definition Single Batch Release Process

The nitrogen dioxide (NO2) sterilization process is an ideal candidate ISO 14937:2009, Clause 8 Follows Guidance in AAMI TIR16 for the sterilization of the external, gas-accessible surfaces of prefilled Objective: Detailed Process Specification Objective: To provide a single batch of prefilled syringe product for clinical trials where sterility of external, gas accessible surfaces is required, without syringes. A process has been designed to accommodate temperature • Follows the “overkill approach” described in Annex D and pressure-sensitive products such as drugs or biologics in prefilled performing a complete validation. • Sublethal cycles to predict the half-cycle using exposure time as a variable • The guidance document is written for ethylene oxide, but the approach and syringes. The NO sterilization process is applied to the prefilled 2 • All other parameters were constant: NO Dose, %RH, Temperature, Aeration procedure are applicable to NO sterilization as well syringes (or other containers) with minimal vacuum at room 2 2 • Demonstrate a 6-log reduction in the BI at most-challenging location • Because the quality and safety of each batch is verified using USP <71> temperature. These attributes minimize the risks of sterilant ingress sampling guidelines, validation is not required • Prove that the BI is more resistant to the process than is the native bioburden via stopper movement and thermal degradation of the product. The • Each batch is essentially a mini validation • Sterilization process applied to exterior, gas-accessible surfaces of the syringe NO2 sterilization process rapidly inactivates microorganisms on Half-Cycle/Full-Cycle Approach: Product: 1-mL Long Syringe surfaces, and readily aerates from product, which results in relatively The syringe is shown in a • Half-cycle assures a 6-log reduction in BIs and short times at room temperature for product batches. • Glass barrel sterilization pouch with all negative results from Product Tests of Sterility Regulators have been requesting manufacturers to sterilize the • Pharma rubber/rigid plastic needle shield the BI in the diffusion • Sample sizes are based on USP <71>, and follow batch size -12 exterior surfaces of prefilled syringes that are intended to be used in • Pharma rubber plunger tip limited space within the • Full-cycle assures a sterility assurance level (SAL) of 10 glass barrel. • Product quality, packaging, and safety samples undergo 1.5 cycles the sterile field; particularly in ophthalmic applications. The NO single • Polypropylene plunger rod 2 • Each syringe was individually packaged in a Requirements for Release: Assume batch of 500 syringes, 0.2-mL fill volume batch release process allows manufacturers to expeditiously obtain Cycle Parameters: Tyvek-Mylar pouch NO Dwell Time BI Results PToS Results* Quality product for clinical trials without undergoing a full validation. The • Minimal vacuum is used for humidification Cycle 2 • Uncoated Tyvek 1073B (mg/L) (min) (Neg./Total) (Neg./Total) Tests process closely follows AAMI TIR16, which describes the batch release and NO2 dosing • 48 ga PET/2.0 mil PE (no tie layer) Half 10 30 10/10 20/20 process for ethylene oxide sterilization. The sampling rationales, along Parameter Set Pt. Tol. Pass Microbiological Challenges: Full 10 60 10/10 NA with the microbiological and process data will be discussed, as will the Vacuum 690 Torr +/- 10 • PToS in this case is on external surfaces of syringe and other gas accessible • Biological Indicator (BI): results of product sterility and NO2 ingress testing. surfaces. Still must test actual product for sterility per USP <71>. • 2.0 x 106 CFU Geobacillus NO2 Dose 5 mg/L +/- 1 stearothermophilus spores Dwell Time Variable +/- 10 s NO2 as a Sterilant • 7.2-mm stainless disc Aeration 90 min +/- 10 s NO Sterilant Ingress Test • Crimped to fit barrel 2 NO2 is a rapid and effective sterilant: • Dwell time employed as variable: o o • External Native Bioburden: Demonstrating Container-Closure Integrity • Can be applied over temperature range: 10 C to 30 C • Tested: 15, 30, and 60 minutes • For syringes manufactured in an aseptic • Saturated vapor pressure at 21°C is 1 atm Objective: To demonstrate that NO2 sterilant did not diffuse past the container- environment, one can expect <1 - • NO is normally a gas at room temperature Results: All requirements of Process Definition closure barrier via a colorimetric assay for nitrate (NO3 ) anions 2 CFU/syringe • Does not condense when compressed during processing were satisfied: • NO2 absorbed into water will form HONO and HNO3, both of which are • Product Test of Sterility (PToS) for external - • Vacuum is optional: Dwell Time BI PToS detectable via this assay as the NO3 anion surfaces via complete immersion of (min) (Neg./Total) (Neg./Total) • The European Pharmacopoeia places a limit of 0.2 ppm NO - on water for • NO2 will readily diffuse throughout the chamber 3 syringe in growth medium at 37°C (+/- 2) injection (WFI) • Fans must be used in the chamber to circulate the gas for 14 days 15 0/10 10/10 • • Syringes filled with 1-mL WFI were exposed to the sterilization process. Vacuum level can be varied to deal with complicated geometries Pressure Profile: 30* 10/10 10/10 like needle or other delivery devices • Ten exposed and ten control samples were tested: • Biological Indicator (BI) conforms to ISO 11138-1 60 10/10 10/10 Colorimetry Example: • Spores of Geobacillus stearothermophilus The pressure and *Minimum half-cycle defined Packaging Options: temperature profile • Sublethal cycles demonstrated the following: • Coated and uncoated Tyvek is shown for the • BI was more resistant than the native • Transitional or Legacy Full-Cycle Process. bioburden Standard Curve: • PETG trays • All negative PToS results • PET/PE films pouches • A 6-log reduction in BI population was achieved

• 5 mg/L NO2, 30-minute dwell time The pressure changes • A full-cycle of the process was specified in the cycle are with a safety factor: equivalent to driving • The NO2 dose and dwell time were Results: over the hills in doubled: 10 mg/L NO , 60-minute 2 • All exposed samples were 10X lower than the EP WFI limit Western Maryland. dwell • For regulatory completeness, manufacturers should still perform quality tests on the actual product per USP and/or design requirements.