Millipore - The Effect of Vaporous Hydrogen Peroxide on Steritest Sterility Testing Devices

Millipore Technical Publications

		TN- TB1002EN00
The Effect of Vaporous Hydrogen Peroxide on Steritest Sterility Testing Devices
Lit Number:	TB1002EN00
Year:	2001

I. Introduction

Closed, disposable Steritest sterility test devices reduce the risk of false positives during the sterility testing of pharmaceuticals. To further reduce the risk of false positives, some manufacturers use the devices inside an isolator. Typically, the filtration devices are placed in the isolator while the isolator is decontaminated using sterilization gases.

In this study, pre-sterilized Steritest devices were exposed to two 90-minute vaporous hydrogen peroxide (VHP) cycles. This design simulates one particular sterilization scenario in which filtration devices are placed inside an isolator prior to a sterility test and are treated with VHP. The devices are not used during the sterility testing and remain in the isolator until the next test day. Prior to the second sterility test, the isolator (and the Steritest devices) is treated with VHP again.

The purpose of this study is to answer the following questions:

Does exposure to VHP affect device performance?
If there is an effect, are different membrane filter materials (PVDF and MCE) affected differently?
Is device performance affected over time?

Different tests were selected and executed on Steritest units before and after contact with VHP to answer the questions posed in this study:

Membrane bubble point test was conducted to ensure that neither the membrane nor device was damaged.
A panel of five microorganisms was chosen for bacteriostasis/fungistasis testing in FTG and TSB media. Two strains of Staphyloccoccus were chosen due to their particularly high sensitivity to VHP. Bacteriostasis/ fungistasis tests were performed on Steritest units after two rinsing steps with 100 mL of Fluid A per canister. Negative media controls were also included.
Gravimetric extractables tests (non-volatile residues, FTIR) were conducted after extraction in water (24-hour soak). NVR was used for quantitative analysis and FTIR for qualitative identification of extractables.

The results are only valid for the conditions set for this study. Since isolators, isolator configurations, isolator contents, and sterilization protocols may vary, isolator users should validate their test independently.

II. VHP Sterilization Cycle Parameters

The tested Steritest units were exposed to two consecutive 90-minutes VHP cycles. The VHP treatments were performed on-site with the client's isolator. The isolator characteristics and the details of these cycles are shown below.

Isolator volume: 22 ft²(0.6 m²)

Dehumidify air set: 22 scfm (623 L/min)

Dehumidify to: 2.3 mg/L

Dehumidify time: 5 min

Preheater temperature set: 90 ^oC (194 ^oF)

Vaporizer temperature set: 100 ^oC (212 ^oF)

Condition air set: 16 scfm (453 L/min)

Condition injection rate: 4 g/min

Sterilize air set: 16 scfm (453 L/min)

Sterilize injection rate: 2.5 g/min

Sterilize time: 90 min

Aerate air set: 22 scfm (623 L/min)

Aerate time: 4 hr

Blower set to run after
aerate time out:
12 hr

Isolator volume:	22 ft²(0.6 m²)
Dehumidify air set:	22 scfm (623 L/min)
Dehumidify to:	2.3 mg/L
Dehumidify time:	5 min
Preheater temperature set:	90 ^oC (194 ^oF)
Vaporizer temperature set:	100 ^oC (212 ^oF)
Condition air set:	16 scfm (453 L/min)
Condition injection rate:	4 g/min
Sterilize air set:	16 scfm (453 L/min)
Sterilize injection rate:	2.5 g/min
Sterilize time:	90 min
Aerate air set:	22 scfm (623 L/min)
Aerate time:	4 hr
Blower set to run after aerate time out:	12 hr

III. Experimental Design

The study included two phases. In Phase 1, a full factorial, two factor (two level), balanced block experiment was performed to evaluate the bubble point of the membranes in the Steritest canisters. The bubble point test is the most sensitive way to measure membrane performance.

Based on the results of Phase 1, a test design was established for Phase 2, which was comprised of bacteriostasis/fungistasis testing and gravimetric extractables evaluation.

PHASE 1 (Figure 1)

Factor 1: two membrane polymers (two lot numbers for each Steritest type)

MCE (Mixed Cellulose Esters membrane used in Steritest HA)
PVDF (Polyvinylidene Fluoride membrane used in Steritest HV)

Factor 2: two time points

Initial study
7 months later

Trials done on:

Steritest pre-VHP sterilization cycles.
Steritest post-VHP sterilization cycles
- Two different VHP runs A and B (we assumed there was variability between VHP loads, thus all factors and levels were accommodated within the test block).
- There was a one-to-one comparison with the control (untreated) samples.

Note: the experiment included a total of 32 canisters or 16 Steritest canister sets (8 PVDF sets and 8 MCE sets).

Figure 1. PHASE 1. Bubble point test. The tree diagram displays the full factorial, two factor, two level design of the study. The treatment was two 90-minute cycles of VHP.

PHASE 2 (Figure 2)

Factor:1 two membrane polymers (four lot numbers for each Steritest type)

MCE (Mixed Cellulose Esters membrane used in Steritest HA)
PVDF (Polyvinylidene Fluoride membrane used in Steritest HV)

Factor 2: two time points

Initial study
7 months later

Trials done:

Steritest pre-VHP sterilization cycles.
Steritest postÐVHP sterilization cycles
3 replicates.
4 different VHP runs (we assumed there was variability between VHP loads.
There was a one-to-one comparison with the control (untreated) samples.

Note: Phase 2 required 48 canister sets with MCE membrane from four different lot numbers and 48 canisters sets with PVDF membrane from four different lot numbers.

To ensure that Steritest canisters from the three replicates were not exposed to the same VHP cycle, the three bacteriostasis/fungistasis replicates were divided into four VHP runs (Table 1).

Table 1. Sterilization Plan

Each grouping (for example, Replicate 1, MCE, VHP Run 1) included five Steritest canister sets plus one negative control canister set.

Extractables study

Materials used for this study:

1 control with water.
1 Steritest unit TTHALA210 (MCE membrane) pre-VHP sterilization.
1 Steritest unit TTHALA210 (MCE membrane) post-VHP sterilization.
1 Steritest unit TLHVSL210 (PVDF membrane) pre-VHP sterilization.
1 Steritest unit TLHVSL210 (PVDFmembrane) post-VHP sterilization.

Figure 2. PHASE 2. Bacteriostasis/fungistasis test design. The tree diagram displays the full factorial, two factor, two level design of the study. The treatment was two 90-minute cycles of VHP.

IV. Material and Methods

PHASE 1

Steritest HA,
Millipore cat. no. TTHA LA2 10
Lot 1 R5JM60636
Lot 2 R5MM60824

Steritest for Solvents,
Millipore cat. no. TLHV SL2 10
Lot 3 F5HM81611
Lot 4 F5KM81550

Bubble Point Test (Figure 3)
Purpose: to measure the integrity of Steritest sterility test devices using "bubble point" as the measurement criterion. Equipment: Automatic Integrity Tester, IntegritestâII (Millipore cat. no. XEIT110 11)

Figure 3. Steritest Bubble Point Test Using an IntegritestâII Automatic Integrity Tester

PHASE 2

Bacteriostasis/Fungistasis Test
Purpose: to evaluate the presence or absence of inhibitory residuals due to VHP treatment.

In this test, each canister was rinsed twice with 100 mL of Fluid A before the filtration of the last 100 mL Fluid A with the test microorganism. Then, the growth medium was added.

Steritest units used for this study:
Steritest HA,
Millipore # TTHA LA 210
Lot R5JM60626
Lot R5MM60824
Lot R6CM68235
Lot R6EM13545

Steritest for Solvents,
Millipore # TLHV SL 210
Lot F6AM81551
Lot F5NM81788
Lot F5HM81611
Lot F5KM81550

Test microorganisms:
Bacillus subtilis (ATCC^â6633)
Candida albicans(ATCC 10231)
Clostridium sporogenes(ATCC 11437)
Staphylococcus epidermidis(ATCC 12228)
Staphylococcus aureus(ATCC 6538)

Acceptance Criteria:

Negative controls shall demonstrate no growth as determined visually after incubation for 14 days.
Inoculum concentration of each test microorganism delivered to the specified Steritest canister shall be between 10 and 100 cfu (cf. Plate count results).
Positive controls shall demonstrate growth of the specified test microorganism (USP24: growth within 5 days for bacteria, yeast, and molds).
Inoculated test canisters shall demonstrate growth that is visually comparable to the positive control.

Gravimetric Extractables
Purpose: the weight of extractables and the HPLC and FTIR profiles for Steritest sterility test devices have been determined after soaking at ambient temperature (20 oC) in ASTM type1reagent-grade water for 24 hours.

Steritest units used for this study:
Steritest HA,
Millipore # TTHA LA 210
Lot R5JM60626 x 2

Steritest for Solvents,
Millipore # TLHV SL 210
Lot F5HM81611 x 2
Equipment: Drying oven capable of 80 ^oC ± 5 ^oC

V. Results

Bubble Point Test
All of the 32 canisters met the acceptance criteria for bubble point. The Initial bubble point range was:

PVDF: 24.1 to 26.3
MCE: 31.1 to 35.3

There is no statistical difference between VHP-treated versus non-VHP-treated devices for bubble point at T_initial or T_7Months.

Bacteriostasis/Fungistasis Test
Growth of all test microorganisms were as expected. No static or cidal effects were observed. All canisters met the acceptance criteria for the tests being conducted.

A) Bacteriostasis/Fungistasis Tests at TInitial

Table II. Time _Initial Plate Count Results

Table III. Time Initial Results for Staphylococcus epidermidis

* Result was negative on a Friday and positive on a Monday. The units were not examined over the weekend.

Table IV. Time Initial Results for Staphylococcus aureus

* Result was negative on a Friday and positive on a Monday. The units were not examined over the weekend.

Table V. Time Initial Results for Candida albicans

* Test was not performed. The tubing was accidentally damaged prior to the test.

Table VI. Time Initial Results for Bacillus subtilis

* Result was negative on a Friday and positive on a Monday. The units were not examined over the weekend.

Table VII. Time Initial Results for Clostridium sporogenes

* Because Clostridium sporogenesis an anaerobe, the test was performed in Fluid Thioglycollate Medium only.

Table VIII. Time Initial Negative Controls

*NG denotes no growth was observed after 14 days of incubation at the appropriate temperature for the
medium being tested.

B) Bacteriostasis/Fungistasis Tests at T7Months

Table IX. Time 7 Months Plate Count Results

Table X. Time 7 Months Results for Staphylococcus epidermidis

* Result was negative on a Friday and positive on a Monday. The units were not examined over the weekend.

Table XI. Time 7 Months Results for Staphylococcus aureus

Table XII. Time 7 Months Results forCandida albicans

Table XIII. Time 7 Months Results for Bacillus subtilis

Table XIV. Time 7 Months Results for Clostridium sporogenes

* Because Clostridium sporogenesis an anaerobe, the test was performed in Fluid Thioglycollate Medium only.

Table XV. Time 7 Months Negative Controls

*NG denotes no growth was observed after 14 days of incubation at the appropriate temperature for the medium being tested.

Gravimetric Extractable Analysis

The water extraction study showed a significant difference in the amount of non-volatile residue for the VHP-treated Steritest for Solvents Canisters with PVDF membrane as compared to the other values. The IR spectra of all the extractions suggest the presence of inorganic materials such as salts. However, nothing unusual couldbe identified.
Table XVI. Non-Volatile Residue Gravimetric Extractables Results

Figure 4
Figure 5

Figure 6
Figure 7

Figures 4-7. FTIR Spectra of Untreated and VHP-Treated Steritest PVDF and MCE Devices.

VI. Conclusions

It appears that the primary function of Steritest canisters is not impaired by two 90-minute VHP cycles.
Bubble point results suggest no membrane structural and performance changes over time due to VHP treatment.
Bacteriostasis/fungistasis tests done with standard microorganisms described in USP 24 and with Staphyloccoccus epidermidis (ATCC12228) sensitive to VHP show no evidence of inhibitory residuals over time after rinsing the membrane twice with 100 mL Fluid A per canister.
Qualitative extractable analysis did not identify unusual chemical components. No correlation was possible between the amount of non-volatile residues and contact of the Steritest with VHP.
These conclusions are only valid for the conditions evaluated in this study. Each isolator must be validated independently because each isolator's configuration, contents, and sterilization cycle protocols vary. In the "normal" conditions used in this study, repeated exposure to isolator sterilization cycles did not adversely affect the characteristics of the Steritest devices that are pertinent for a sterility test.