Cycle Parameters:

• Minimal vacuum is used for humidification and NO2 dosing

• Dwell time employed as variable:

• Tested: 15, 30, and 60 minutes

Product: 1-mL Long Syringe

• Glass barrel

• Pharma rubber/rigid plastic needle shield

• Pharma rubber plunger tip

• Polypropylene plunger rod

• Each syringe was individually packaged in a Tyvek-Mylar pouch

• Uncoated Tyvek 1073B

• 48 ga PET/2.0 mil PE (no tie layer)

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

Process Definition ISO 14937:2009, Clause 8

AbstractThe nitrogen dioxide (NO2) sterilization process is an ideal candidate

for the sterilization of the external, gas-accessible surfaces of prefilled

syringes. A process has been designed to accommodate temperature

and pressure-sensitive products such as drugs or biologics in prefilled

syringes. The NO2 sterilization process is applied to the prefilled

syringes (or other containers) with minimal vacuum at room

temperature. These attributes minimize the risks of sterilant ingress

via stopper movement and thermal degradation of the product. The

NO2 sterilization process rapidly inactivates microorganisms on

surfaces, and readily aerates from product, which results in relatively

short times at room temperature for product batches.

Regulators have been requesting manufacturers to sterilize the

exterior surfaces of prefilled syringes that are intended to be used in

the sterile field; particularly in ophthalmic applications. The NO2 single

batch release process allows manufacturers to expeditiously obtain

product for clinical trials without undergoing a full validation. The

process closely follows AAMI TIR16, which describes the batch release

process for ethylene oxide sterilization. The sampling rationales, along

with the microbiological and process data will be discussed, as will the

results of product sterility and NO2 ingress testing.

NO2 as a SterilantNO2 is a rapid and effective sterilant:• Can be applied over temperature range: 10oC to 30oC• Saturated vapor pressure at 21°C is 1 atm

• NO2 is normally a gas at room temperature• Does not condense when compressed during processing

• Vacuum is optional:• NO2 will readily diffuse throughout the chamber

• Fans must be used in the chamber to circulate the gas• Vacuum level can be varied to deal with complicated geometries

like needle or other delivery devices• Biological Indicator (BI) conforms to ISO 11138-1• Spores of Geobacillus stearothermophilus

Packaging Options:• Coated and uncoated Tyvek

• Transitional or Legacy• PETG trays• PET/PE films pouches

Parameter Set Pt. Tol.

Vacuum 690 Torr +/- 10

NO2 Dose 5 mg/L +/- 1

Dwell Time Variable +/- 10 s

Aeration 90 min +/- 10 s

Objective: Detailed Process Specification

• Follows the “overkill approach” described in Annex D

• Sublethal cycles to predict the half-cycle using exposure time as a variable

• All other parameters were constant: NO2 Dose, %RH, Temperature, Aeration

• Demonstrate a 6-log reduction in the BI at most-challenging location

• Prove that the BI is more resistant to the process than is the native bioburden

• Sterilization process applied to exterior, gas-accessible surfaces of the syringe

The syringe is shown in a sterilization pouch with the BI in the diffusion limited space within the glass barrel.

Microbiological Challenges:

• Biological Indicator (BI):

• 2.0 x 106 CFU Geobacillus stearothermophilus spores

• 7.2-mm stainless disc

• Crimped to fit barrel

• External Native Bioburden:

• For syringes manufactured in an aseptic environment, one can expect <1 CFU/syringe

• Product Test of Sterility (PToS) for external surfaces via complete immersion of syringe in growth medium at 37°C (+/- 2) for 14 days

Pressure Profile:

Results: All requirements of Process Definition were satisfied:

*Minimum half-cycle defined

• Sublethal cycles demonstrated the following:

• BI was more resistant than the native bioburden

• All negative PToS results

• A 6-log reduction in BI population was achieved

• 5 mg/L NO2, 30-minute dwell time

• A full-cycle of the process was specified with a safety factor:

• The NO2 dose and dwell time were doubled: 10 mg/L NO2, 60-minute dwell

Dwell Time(min)

BI(Neg./Total)

PToS(Neg./Total)

15 0/10 10/10

30* 10/10 10/10

60 10/10 10/10

Single Batch Release ProcessFollows Guidance in AAMI TIR16

Objective: To provide a single batch of prefilled syringe product for clinical trials where sterility of external, gas accessible surfaces is required, without performing a complete validation.

• The guidance document is written for ethylene oxide, but the approach and procedure are applicable to NO2 sterilization as well

• Because the quality and safety of each batch is verified using USP <71> sampling guidelines, validation is not required

• Each batch is essentially a mini validation

Half-Cycle/Full-Cycle Approach:

• Half-cycle assures a 6-log reduction in BIs and all negative results from Product Tests of Sterility

• Sample sizes are based on USP <71>, and follow batch size

• Full-cycle assures a sterility assurance level (SAL) of 10-12

• Product quality, packaging, and safety samples undergo 1.5 cycles

Requirements for Release: Assume batch of 500 syringes, 0.2-mL fill volume

• PToS in this case is on external surfaces of syringe and other gas accessible surfaces. Still must test actual product for sterility per USP <71>.

CycleNO2

(mg/L)Dwell Time

(min)BI Results

(Neg./Total)PToS Results*(Neg./Total)

Quality Tests

Half 10 30 10/10 20/20Pass

Full 10 60 10/10 NA

NO2 Sterilant Ingress TestDemonstrating Container-Closure Integrity

Objective: To demonstrate that NO2 sterilant did not diffuse past the container-closure barrier via a colorimetric assay for nitrate (NO3

-) anions

• NO2 absorbed into water will form HONO and HNO3, both of which are detectable via this assay as the NO3

- anion

• The European Pharmacopoeia places a limit of 0.2 ppm NO3- on water for

injection (WFI)

• Syringes filled with 1-mL WFI were exposed to the sterilization process.

• Ten exposed and ten control samples were tested:

Colorimetry Example:

Standard Curve:

Results:

• All exposed samples were 10X lower than the EP WFI limit

• For regulatory completeness, manufacturers should still perform quality tests on the actual product per USP and/or design requirements.

The pressure andtemperature profileis shown for the Full-Cycle Process.

The pressure changesin the cycle are equivalent to drivingover the hills in Western Maryland.