Published on IVT Network (http://www.ivtnetwork.

com)

Microbiological Assessment of Compressed Gases in Pharmaceutical Facilities

By Tim Sandle Aug 17, 2015 11:00 pm PDT

Peer Reviewed: Microbiology

ABSTRACT

Compressed gasses are used at different stages of the pharmaceutical manufacturing process for numerous applications.
Compressed gas sampling for microorganisms is an important part of contamination control assessment. This paper
addresses topics related to microbial assessment of compressed gases. These include gas purity including moisture
considerations, sterile filtration, compressed gas standards, microbial contamination, microbial survival, sampling and testing
requirements, instrumentation, and various sampling considerations including growth medium choice and frequency, and
reporting requirements. The topics addressed in this paper should be built into the site biocontamination control program.

INTRODUCTION

Compressed gasses are used at different stages of the pharmaceutical manufacturing process. Applications include weighing
of vials on process line or the removal of fallen vials. Compressed gases such as air, nitrogen, and carbon dioxide are
deployed in operations involving purging or overlaying.

Compressed gas sampling for microorganisms is an important part of contamination control assessment (1). While sampling
is important, the method of sampling can hindered by the design of the gas system where sampling is not easily conducted in
an aseptic manner, or by the design of the air-sampling instrument. This paper reviews the important aspects of compressed
air sampling for microbiological assessment and looks at possible sources of contamination if microorganisms are recovered.

COMPRESSED GAS

Compressed gas is a general term for gas stored or held under pressure that is greater than atmospheric pressure. Gas is
compressed via a compressor -- the greater the quantity of the gas, the higher the pressure (degree of compression). The
compressor takes gas or air, which occupies a given space, and reduces it to a smaller space. The greater mass of air or gas
within the container produces a greater pressure. For instance, compressed air operating at 100 pound-force per square inch
will have been compressed down to 1/8th of its original volume. 100 pounds per square inch equals 7 bar; or to use SI units,
one pound per square inch equals approximately 6894 Pascals.
Types of compressed gas include air, colorless and odorless, with composition of 78% nitrogen, 21% oxygen, and remainder
trace elements. Other common gases are oxygen, carbon dioxide, and nitrogen. Aside from air, nitrogen is the most
commonly used gas in the pharmaceutical sector. Nitrogen has inert characteristics that lead to its ideal use as a pressurizing
agent. Tanks, pipelines, hoses, vessels, and other process equipment can be tested for leaks with nitrogen gas. Nitrogen gas
can also be used to dispense or transfer most fluids from storage tanks or reservoirs. Nitrogen gas should be composed of a
minimum of 99% nitrogen gas, with trace impurities such as carbon monoxide, carbon dioxide, and oxygen permitted at
<0.001% (2).

Purity Including Moisture

Purity is a factor that needs to be maintained with compressed gas; hence the gas should be supplied oil free. Purity overall is
achieved through a combination of filtration, purification, and separation. The process of creating the compressed gas can
additionally introduce water vapor. A process must therefore be in place to remove water vapor before the gas is expelled into
a critical zone such as a pharmaceutical cleanroom. Compressed gas is typically discharged from the compressor hot and it
will contain water vapor. Temperature is reduced by using a post-compressor cooler and, as the gas condenses, the water
vapor and other impurities can be removed. The risk of water vapor is particularly high with compressed air, which is drawn
into a compressor via the atmosphere. Atmospheric air contains a high proportion of water vapor (water in a gaseous form).
Water removal is achieved through a combination of filtration and dehumidification.

Sterile Compressed Air

Where moisture is drawn in from the outside, the process of drawing in also introduces microorganisms, which require filtering
out. The level of filtering depends upon whether sterile air is required (absence of viable microorganisms) or air with a low
bioburden.

Compressed gas can be supplied at source either sterile or non-sterile. Sterility is achieved through the use of a bacterial
retentive membrane filter (0.2 m pore size). A sterile-filtered gas is required where a gas contacts a sterilized component or
material. It is important that the sterilizing grade filter is maintained dry for condensate in a gas filter will most probably cause
blockage or lead to microbial contamination. Risks of condensate are controlled by heating and use of hydrophobic filters (to
prevent moisture residues in a gas supply system). Filters should also be changed periodically. As part of on-going quality
control, filters must be integrity-tested at installation and at end of use.

Compressed gas standards

Although national standards bodies have guidance documents for compressed air sampling and reference is made within FDA
and EU GMPs, the general approach and requirements for compressed gasses are set out in a ISO 8573. This standard
consists of the following parts (3):

Part 1: Contaminants and purity classes

Part 2: Test methods for aerosol oil content
Part 3: Test methods for measurement of humidity
Part 4: Test methods for solid particle content
Part 5: Test methods for oil vapor and organic solvent content
Part 6: Test methods for gaseous contaminant content
Part 7: Test method for viable microbiological contaminant content
Part 8: Test methods for solid particle content by mass concentration
Part 9: Test methods for liquid water content.

Part 1 outlines the required purity classes based on the concentration of particles and level of impurities. The potential impure
contaminants for compressed air, that can affect whether a required purity class is met include:

Particles (such as dirt, rust, pipe scale), with particles assessed by size. For example, as result of the mechanical
compression process, additional impurities may be introduced into the air system. Generated contaminants include
compressor lubricant, wear particles, and vaporized lubricant. Fittings and accessories can also contribute to particles.

Water (in both vapor and liquid forms). Water is typically assessed by vapor pressure dew-point. Temperature at which
the air can no longer "hold" all of the water vapor with which it is mixed.
Oil (including aerosol, vapor, and liquid forms).
Microbiological Contamination

Microbial content itself does not influence the purity class assigned, although the standard recommends that microbial levels
are assessed. Acceptable microbial numbers are subject to a separate assessment; with such an assessment is based on an
interpretation of GMP. For example, the 2004 FDA Aseptic Filling Guidance document states (4):

A compressed gas should be of appropriate purity (e.g., free from oil) and its microbiological and particle quality after filtration
should be equal to or better than that of the air in the environment into which the gas is introduced.

With purity, many parts of the pharmaceutical industry will use class 1 compressed gas based on the maximum number of
permitted particulates. The particle limits are:

ISO 8573 class Particle size limits per m3

0.1 - 0.5 m 0.5 - 1.0 m 1.0 - 5.0 m
1 ? 20,000 ? 400 ? 10
Outside of the ISO 8573 standard, supporting information is contained within the ISPE Good Practice Guide - Process Gases
(2011) (5). Here Table 7.1 of the guide indicates that particle counts (both viable and inert) should be "Typically equal to the at
rest condition of the area served." That is, for EU GMP Grade A / ISO 14644 class 5 areas, the microbial count should be <1
CFU/m3 and the particle levels conform to the area at rest ? 3,520 particles per m3.

A separate standard exists for the production of compressed air. This is ISO 12500, a four part standard:

ISO 12500-1:2007 - Filters for compressed air -- Test methods -- Part 1: Oil aerosols
ISO 12500-2:2007 - Filters for compressed air -- Test methods -- Part 2: Oil vapors
ISO 12500-3:2009 - Filters for compressed air -- Test methods -- Part 3: Particulates
ISO 12500-4:2009 - Filters for compressed air -- Methods of test -- Part 4: Water

ISO 12500 has no specific microbial testing requirements.

MICROBIAL SURVIVAL

Based on the above, the maximum level of microorganisms will be applicable to the cleanroom class. This places a tight limit
on ISO 14644 class 5 / EU GMP Grade A areas. Although compressed gas and air systems are relatively harsh
environments, they can aid microbial survival if there are available nutrients. The availability of nutrients is dependent upon
the purity of the gas and airline. Nutrients suitable for metabolizing by microorganisms include water and oil droplets. Another
factor that can affect survival is temperature, especially where temperatures are warmer (6).

In addition to vegetative cells, bacterial spores are well equipped to survive the harsh environmental conditions. Spores are
resistant to the types of temperature ranges and moisture levels found within compress gas lines. Another risk exists with
biofilm, where microbial communities can potentially form and develop through attachment to air lines and tubing.

Although these risk factors exist, typically no microorganisms would be expected to be recovered from compressed gas lines.
Research has shown that many microorganisms can survive and multiply in pressurized systems up to 10 bar and some are at
least able to recover after being pressurized. However, at 160 bar pressure and upwards, survival rates are very low. Where
low level counts are recovered, these require investigation. More often the source is adventitious contamination, although a
fault with the compressed air line cannot be ruled out.

Although microbial contamination of compressed air or gas is a rare event, incidents can occur. Sources of contamination
include:

Source of the air of gas. Intake air from surroundings which can contain oil, dirt/dust and moisture/ water vapor, and
microorganisms.
Piping distribution systems. Piping distribution and air storage tanks, more prevalent in older systems, will have
contaminant in the form of rust, pipe scale, and mineral deposits in addition to bacteria.
Bacterial retentive filter. The filter may become blocked, lose its integrity, or become wet.
Compressor failure. The compressor itself can create a contaminated environment. For example; the compressor's
 pre-filters can become overloaded with dust and lint, causing the filter to cease functioning properly.
Sample valve. The point-of-use sample valve may not be designed correctly or become faulty.

SAMPLING AND TESTING REQUIREMENTS

As indicated above, compressed gas requires assessment against a number of parameters, including particles and viable
microorganisms. The part of the standard used for making assessments is ISO 8573 Compressed air Part 7: Test method
for viable microbiological contaminant content (7).

When sampling compressed air for microorganisms, it is important that the air is depressurized and that the flow rate is
controlled. Control of the flow rate is important to ensure that a cubic meter of air is sampled within the required sampling time
(time is instrument-dependent). With this, compressed gas is typically at 160 pounds per square inch or greater. An external
regulator will be needed to bring this down to the sampling rate of instrument. If the air sampler takes 36 minutes to capture a
cubic meter of air, then it will be sampling at one cubic foot per minute. The regulator will need to reduce the air down to allow
for this. This is assessed using a flow meter. Pressure reduction to atmospheric conditions is of great importance. Knowing
the flow allows the agar exposure time to be assessed so that one cubic meter of air is sampled.

It is also important that isokinetic sampling of the air occurs and that air velocity is reduced until it is within the range of the
sampler as identified by the manufacturer. This is not only necessary for obtaining the correct sample size; it also impacts on
the possibility of microbial survival. The level of impact stress has been shown to affect microbial recovery on agar and be
dependent upon the impaction velocity of the cells into the agar as well as the design and operating parameters. Due to the
fact that any microorganisms present are transported under pressure and then suddenly released into atmospheric conditions,
they may be damaged by the immediate expansion of the gas and the resulting shearing forces.

The head of the instrument and any attachments must be sterile before use to avoid contamination. The culture medium used
with the instrument should be sterile (normally by irradiation) and a representative item should have been tested for growth
promotion. With most samplers the head will be autoclavable. Some users disinfect the tubes and hoses used to connect the
sampler with a disinfectant such as 70% isopropyl alcohol. This is mentioned as an option in the ISO standard, although this
is erroneously described as "sterilization." Where a disinfectant is used it is important to run the air through the sampler
without any agar plate in place; this is necessary to evaporate the disinfectant and to remove any residues. The presence of
disinfectant could potentially lead to a false negative.

With sampling, the sample inlet is connected to the compressed gas line and air is directed over an agar plate or strip. The
method works by compressed gas under reduced pressure (called partial flow) is forced over the surface of an agar plate.
Any microorganisms are impinged onto the surface of the agar.

The sampling time should be sufficient in order to sample one cubic meter of the agar. After sampling, the agar plate or strip
is removed and incubated within a microbiology laboratory. At the end of incubation, the agar is examined for colony forming
units. If colony forming units are recovered, these should be assessed against the appropriate limit. It is good practice to
identify the contaminants recovered; the identification may provide important information to help determine the origin of the
bacteria.

INSTRUMENTATION

The type of instrument recommended in the ISO standard is a slit-sampler, a type of impaction air tester, although alternative
samplers can be used if justified. With a standard impactor sampler, air is drawn through a sampling head via a pump or fan
and accelerated usually through a perforated plate (sieve samplers) or through a narrow slit (slit samplers). This process
creates a laminar flow through the sampler head. The air sampler should be fitted with a diffuser capable of maintaining
laminar flow conditions. This is necessary so that particles pass through the sample head in a controlled flow.

The velocity of the air is determined by the diameter of the holes in sieve samplers and the width of the slit in slit samplers.
When the air strikes the collection surface on the agar plate, it makes a tangential change of direction. This causes any
suspended particles to be thrown out by inertia, impacting onto the collection surface. When the correct volume of air has
been passed through the sampling head, the agar plate can be removed and incubated (8).
When selecting a suitable sampler, three parameters should be checked and evaluated. These are (9):

Physical efficiency of the sampler. This is the relative efficiency of the sampler in collecting particles over a range of
sizes. Physical efficiency is measured against membrane filtration sampling and the d50 value assessed. The d50 is
the aerodynamic diameter, above which the collection efficiency of the impactor approaches 100%. Knowing the d50
value gives an indication of the sizes of particles likely to be collected by the sampler for the d50 is equivalent to
particle size at which 50% of the particles are collected, and 50% pass through the sampler because that are too small
to impact (10).
Biological efficiency. This is the relative efficiency of the sampler in collection of microorganisms on a surface so
that they remain viable and can be counted post-incubation. Biological efficiency is compared with an established
reference sampler such as the Casella slit sampler. Assessment of air samplers involves the use of a controlled
microbial population passed into a nebulizing chamber.
Flow rate of the sampler. With all sample sizes, the flow rate of air through the sampling head is critical to the
accuracy of the result.

In terms of culture media, sampler models available either collect air samples onto contact plates (55mm or 84mm diameter),
standard petri dishes (90mm diameter), or onto agar strips.

Outside of these requirements, the ideal device should be portable to permit sampling throughout a series of cleanrooms.
Devices should also be cleanable and resistant to common cleanroom disinfectants. The ideal material of construction is
stainless steel.

SAMPLING CONCERNS

Compressed air sampling should form part of an environmental monitoring program, along with cleanroom assessments. The
program should take into account air points to be tested. This could be every point, points considered to be of greater risk
such as product contact, or representative points along a loop. The frequency of testing must also be considered based on risk
assessment.

An appropriate agar must be selected. An example is tryptone soya agar, which is a generally nutritious medium designed to
recover a range of bacteria and fungi. A key factor to take into account is whether the process of sampling leads to undue
desiccation of the agar rendering any recovered microorganisms unable to grow on the gar due to depletion of growth
nutrients. This will be affected by the flow rate, type of compressed gas, and the model of air-sampler, together with the type
of culture medium. A risk will remain that microbial cells will become damaged by mechanical stress during the sampling
process and lose viability. These factors should be evaluated through an experimental study (11). In addition, the agar
medium should be removed from the sampler as quickly as is practicable and transferred to the required incubator. This is to
avoid the culture medium from drying out or deteriorating.

With the incubation conditions selected, the time and temperature should be suitable for the recovery of the a general range of
microorganisms, particularly Gram-positive organisms given that such bacteria are better equipped to survive in dry
environments (12). The typical requirement is to look for mesophilic bacteria and fungi (those that would grow across the
temperature range 20-30oC). Some users would elect to use one representative temperature whereas others would elect to
use a two-step incubation regime, such as (13):

20-25oC for 3-5 days, followed by

30-35oC for 3-5 days.

The selected incubation time should be based on growth promotion studies. If certain microorganisms are considered a
problem, alternate incubation times or culture media can be considered.

Sampling frequencies

The user will need to determine whether each compressed gas line requires testing and the frequency of testing. Certainly all
product contact compressed gases should be assessed. A sampling plan should also consider and adapt to the following:

Increased or reduced production schedules

Seasonal changes
 Equipment changes and modifications
Replacement of hardware or filters and dryers
Inactivity of system.

REPORTING REQUIREMENTS

When reporting the results from a compressed air sampling session regarding microbial counts, the following information is
advised in the ISO 8573 standard:

1. Whether the compressed airline was sterile or non-sterile

2. The date of sampling
3. The date of measurements
4. The location of the sample.

The result expressed as colony forming units per cubic meter of air (CFU/m3) should also be added to the report.

BACTERIAL ENDOTOXIN

The ISO 8573 standard has the option of sampling compressed air for bacterial endotoxin. Such testing remains relatively
uncommon and it is only necessary should the compressed gas have a direct product contact and where there is a concern
with Gram-negative bacteria. In most cases there should be no likelihood of endotoxin being present.

The sampling method for bacterial endotoxin is tricky and inexact. Either colonies are examined for Gram-negative bacteria
and assessment is made about endotoxin risk; or the compressed is passed through pyrogen-free water.

SUMMARY

The assessment of compressed gas is an important part of quality control within pharmaceutical organizations whether that
assessment is for chemical impurities, particulates, or microorganisms. This paper has assessed the appropriate standards
for compressed air and focused on microbiological sampling. In doing so, the important features of air sampling have been
raised together with the factors that can lead to microbial contamination occurring. These aspects should be built into a
biocontamination control program.

REFERENCES

1. Sandle, T. (2013). Contamination Control Risk Assessment in Masden, R.E. and Moldenhauer, J. (Eds.)
Contamination Control in Healthcare Product Manufacturing, Volume 1, DHI Publishing, River Grove: USA, pp423-474
2. Baseman, H. (2010) Sterile product manufacture using form fill seal technologies. In Agalloco, J. and Akers, J. (Eds.)
Advanced Aseptic Processing Technology, Informa Healthcare, New York, USA, pp164-165
3. ISO 8573-1:2010 Compressed Air Contaminants and Purity Classes, International Standards Organization, Geneva,
Switzerland
4. FDA (2004) Guidance for Industry Sterile Drug Products Produced by Aseptic Processing Current Good
Manufacturing Practice, U.S. Department of Health and Human Services Food and Drug Administration, Rockville, MD,
USA
5. IPSE Good Practice Guide: Process Gases, 2011, IPSE, USA, pp81-86
6. Stewart, S. L., S. A. Grinshpun, K. Willeke, S. Terzieva, V. Ulevicius, and J. Donnelly (1995) Effect of Impact stress on
microbial recovery on an agar surface. Appl. Environ. Microbiol. 61:1232-1239.
7. ISO 8573-7:2003 Test method for viable microbiological contaminant content, International Standards Organization,
Geneva, Switzerland
8. Sandle, T. (2011): Key points for active air samplers, Clean Air and Containment Review, Issue 5, pp8-10
9. Sandle, T. (2010) Selection of active air samplers, European Journal of Parenteral and Pharmaceutical Sciences, 15
(4): 119-124
 10. Hinds, W. C. (1982) Aerosol technology, John Wiley & Sons, New York, p. 104126
11. Morring, K. L., W. G. Worenson, and M. D. Attafield (1983) Sampling for airborne fungi: a statistical comparison of
media. Am. Ind. Hyg. Assoc. J. 44: 662-664
12. Moissl-Eichinger C, Rettberg P, Pukall R. (2012) The first collection of spacecraft-associated microorganisms: a public
source for extremotolerant microorganisms from spacecraft assembly clean rooms, Astrobiology 12(11):1024-34
13. Sandle, T. (2014) Examination of the Order of Incubation for the Recovery of Bacteria and Fungi from Pharmaceutical
Cleanrooms, International Journal of Pharmaceutical Compounding, 18 (3): 242 247

Source URL: http://www.ivtnetwork.com/article/microbiological-assessment-compressed-gases-

pharmaceutical-facilities