Decontamination of Aseptic Pharmaceutical Isolators Using Vaporized and Aerosolized Hydrogen Peroxide

Pharmaceutical Aseptic Process Isolator

Case Study: A Comparison of Methods and Benefits for the Industry

Isolation of Aseptic Processes in the Pharmaceutical Industry

Pharmaceutical and laboratory isolators are glovebox-type containment chambers designed to:

• Protect operators from substances associated with high occupational exposure risks (OEB 4-7) or from microorganisms being tested or processed inside the isolator

• Protect sensitive products from external environmental conditions such as oxygen, humidity, or biological contamination

  
  

They are used both in large-scale manufacturing and in research and development of new products.

Aseptic isolators provide microbiological safety by preventing contamination from bacteria, viruses, and other microorganisms. To effectively fulfill this function, aseptic chambers must be capable of decontamination, that is, the reduction or elimination of biological agents within the chamber.

Decontamination of Aseptic Pharmaceutical Isolators

Decontamination, or sterilization, involves introducing a biocidal agent into the isolator chamber. Today, in most cases, the agent used is hydrogen peroxide (H₂O₂), a powerful oxidizing compound.

Hydrogen peroxide is typically introduced in one of two forms:

• Vaporized Hydrogen Peroxide (VHP)

• Aerosolized Hydrogen Peroxide (AHP), which represents the latest generation of technology adopted by global leaders in aseptic process isolation

  
  

The average particle size of the aerosol introduced during AHP decontamination is typically between 4 and 8 micrometers, often referred to as a "dry fog". The small droplet size facilitates rapid evaporation, resulting in the simultaneous presence of both hydrogen peroxide vapor and aerosol within the chamber.

The maximum concentration of hydrogen peroxide in the gas phase during aerosol decontamination can reach 500-700 ppm, which is comparable to concentrations typically used in purely gaseous VHP systems.

The aerosol droplets that do not fully evaporate provide an additional biocidal effect. As a result, AHP surface decontamination benefits from both:

1. Hydrogen peroxide in vapor form

2. A thin liquid film formed on decontaminated surfaces

   
   

This dual-action mechanism contributes significantly to the high effectiveness of the process.

Decontamination Cycle

The decontamination cycle for both VHP and AHP systems is very similar and can be divided into the following stages:

1. Pressure Test

Verification of chamber integrity and leak-tightness

2. Drying

Reduction of humidity within the chamber

3. Conditioning

Introduction of hydrogen peroxide in the form of vapor or aerosol

4. Decontamination

Maintenance of the target H₂O₂ concentration for a defined exposure period

5. Aeration

Removal of hydrogen peroxide until a safe concentration level is reached, typically below 1 ppm

Pharmaceutical Industry Expectations for Isolators

A comparison of gas-phase hydrogen peroxide decontamination (VHP) and aerosolized hydrogen peroxide decontamination (AHP) was conducted using two comparable systems in terms of size and chamber volume:

• Isolator 1: VHP system

• Isolator 2: AHP system

Both systems were tested under unloaded conditions (empty chambers) to ensure equivalent testing conditions.

Table 1 compares the parameters of two successful decontamination cycles performed using VHP and AHP, respectively. Both methods utilized 35% hydrogen peroxide solution as the decontamination agent.

Geobacillus stearothermophilus ATCC 12980 spores were used as biological indicators (BIs) to evaluate and validate cycle efficacy.

	VHP	AHP
Isolator chamber volume [m³]	1	0.83
Biological Indicator (BI) Result	Negative (no growth) in 15/15 BIs	Negative (no growth) in 11/11 BIs
Aqueous H₂O₂ Consumption per Cycle [g]	67.5	10
Maximum H₂O₂ Concentration Reached in the Chamber [ppm]	750	650
Total Cycle Time [min]*	70	15

Table 1. Comparison of decontamination cycle parameters for two successful cycles: VHP (Isolator 1) and AHP (Isolator 2).

* Cycle time is measured from the start of hydrogen peroxide injection until a concentration of 1 ppm is reached after aeration.

Results

A successful VHP decontamination cycle (all biological indicators showing negative growth) required 70 minutes, whereas successful AHP decontamination was completed in only 15 minutes.

A dose of 67.5 g/m³ of hydrogen peroxide solution was required to achieve effective VHP decontamination, while only 12 g/m³ was needed for AHP decontamination.

Lower peroxide doses were insufficient for complete spore inactivation. For example, introducing 48 g/m³ of hydrogen peroxide during a VHP cycle resulted in growth of some biological indicators.

Figure 2 compares hydrogen peroxide concentration profiles inside empty chambers of:

• Isolator 1 (VHP) – left side

• Isolator 2 (AHP) – right side

  
  

Total hydrogen peroxide doses applied:

• 48 g/m³ (VHP)

• 12 g/m³ (AHP)

  
  

The blue arrows indicate the period during which biological indicators remained exposed to hydrogen peroxide concentrations exceeding 100 ppm, defined as the contact time.

Contact times were:

• 26 minutes for VHP

• 7 minutes for AHP

  
  

Despite a contact time more than three times longer in the VHP cycle, some biological indicators survived and demonstrated positive growth results.

This suggests that the effectiveness of aerosolized hydrogen peroxide is likely due to a dual interaction mechanism:

1. Exposure to hydrogen peroxide vapor.

2. Direct exposure of biological contaminants to a thin liquid film of hydrogen peroxide deposited on surfaces.

Figure 2

To illustrate performance at lower doses, Figure 2 presents a separate experiment using 48 g/m³ (VHP) and 12 g/m³ (AHP).

Aerosolized Hydrogen Peroxide Decontamination: Benefits for the Industry

The greatest advantage of aerosolized hydrogen peroxide decontamination (AHP) is its exceptionally high efficacy combined with a significantly shorter cycle time, providing a substantial advantage over conventional vapor-phase decontamination.

In addition, the use of catalytic aeration systems in AHP-equipped isolators further reduces aeration time, resulting in a considerable reduction in overall cycle duration.

Because hydrogen peroxide is generated in a concentrated form directly at the point of use, inside the isolator chamber, it can be utilized with minimal losses. This contributes not only to high decontamination performance but also to low biocide consumption.

Furthermore, aerosol generators are compact and energy-efficient because they do not require heating of the liquid hydrogen peroxide solution. This enables:

• Miniaturization of the dosing system

• Reduced energy consumption

• Lower operating costs

• Simplified system integration

As a result, AHP technology offers pharmaceutical manufacturers an effective, economical, and operationally efficient alternative to traditional VHP decontamination systems.

Authors: Dr Kamila Gawel, Dr Tomasz Regula, Dec Poland