Main feature
A history of isolator and containment technology
Part 1: Early containment leading to flexible
film isolators
Doug Thorogood
Abstract
This is the first of five papers that will
describe the history and development
of containment technology in the
field of research, medicine and
pharmaceutical applications.
The paper commences with the
development of containment for various
purposes but essentially for experiments
to determine if the raising of germ free
animals was possible and practical.
The paper goes on to describe how the
experimental work in the late 1920s
led to the development of metal and
then flexible film isolators for use in
veterinary research, human medicine
and surgery. The notable contributions
of Philip Trexler, known to everyone as
Trex, from the 1930s right through to
the 1960s, especially in patient care and
treatment, are recorded.
Introduction
The concept of isolation from the
surrounding environment is not new.
In the late 1800s botanists used simple
bell jars to cover growing plants in
assessing their various development
and physiological properties.
In World War I a number of limb
isolator designs were tried. These
consisted of a leak-proof rubber device
fitted with inlet and outlet tubes to
enclose a limb. The surfaces of the limb
so enclosed could be continuously
irrigated with various agents to treat
burns and the effects of mustard gas.
However, historically, a starting
point of isolators design and use is the
research and development of germ-free
animals. Pasteur in 1885 (1) mused on
the effect of rearing germ-free species
and that they would not survive.
Schottelius (2,3,4) who was a supporter
of Pasteur took up the challenge and
published a number of papers on a
method of raising germ-free chickens.
The idea was that germ-free animals
could be used to determine the effect of
various gut bacteria on growth related
to nutrition and later to evaluate
14 Clean Air and Containment Review | Issue 18 | April 2014
probiotic agents. Still later single organism
animals were used to observe the effects
of various drugs under investigation
against specific pathogens. The term
gnotobiotic is used to cover both germ-
free and single organism animals
although strictly speaking gnotobiotic
should apply to animals that have one
known gut bacteria.
Further development took place
including the work of Nuttal and
Thierfelder on the raising of germ-free
guinea pigs in an isolator system based
upon the use of modified bell jars. (5,6,7)
The animals were delivered by caesarean
section but they survived for only two
weeks. The feeding was thought to have
been inadequate and difficult.
Kuster (10) avoided problems of feeding
by using kid’s milk, carefully heated in an
entry port of a sterile glove box.
Metchinkoff reported looking at the
bacteriology of the human gut and
theorised about germ-free people. (8,9)
Further experiments were conducted
but it was found that there were problems
in maintaining a germ-free environment
mainly because of inadequate methods
of supplying food and water into the
enclosures which were modified glove
boxes. Disinfection methods were also
being evaluated.
In 1928 within the University of
Notre Dame, Indiana, USA a small
nucleus of scientists led by J.A.Reyniers
started to look at methods for raising of
germ-free animals. Using the facilities
at the university they developed new
ideas about containment and isolators.
Figure 1: Steam sterilizable metal isolator
(Notre Dame University)
Reyniers (11) reported on the use of
germ-free guinea pigs in bacteriology.
He had developed a steam sterilizable
metal isolator (see Figure 1). Using this
type of device they were able to maintain
small colonies of germ-free animals and
debated the technology to maintain
much larger colonies of germ-free animals.
Philip C. Trexler, recently graduated
in microbiology, was appointed as
Reyniers ‘biological apprentice’. His first
four years were spent in engineering
work on the metal isolators, manufactured
at Reynier’s father’s company in Chicago.
In 1939 a symposium was held
at the Notre Dame University covering
micrurgical (microsurgical) and
germ-free methods. The metal isolator
depicted in Figure 1 was described in
detail as well as its use in rearing
germ-free guinea pigs, rats, rabbits,
chickens and a monkey.
From 1941 onwards the apparatus
and facilities were devoted to research
related to the war. It had been
demonstrated that there was the capability
to maintain a sterile workspace for a
long period without contamination.
Trexler was so confident that the
containment was 100% that he and a
co-worker spray dried hazardous toxins
and pathogens inside with little risk of
contamination to themselves. The
isolator had to be leak tested, subjected
to internal steam sterilization followed
by a vacuum to allow for cooling and
drying. After the spray drying process
the isolator was steam sterilized again
or subjected to a chemical sterilant. It is
of note that a slight negative pressure in
relation to ambient was used during the
spray drying process.
To expand upon various studies the
University formed the LOBUND Institute
(Laboratories of Bacteriology at the
University of Notre Dame) with Reyniers
as the Director. Figure 3 shows Reyniers
with Trexler at this time. It was found
that the cost of routinely producing
animals from the Reyniers Isolator
System was much too high. To lower
www.cleanairandcontainment.com
 Main feature

costs a room was erected that could be

steam sterilized. This was a steel tank
2.4 m in diameter and 4.6 m long.
Personnel entered the tank in a sealed
ventilated garment made of PVC via a
disinfectant shower area and a dunk
tank. Only a couple of hundred germ-
free rats were produced.
By 1957 PVC (polyvinyl chloride)
was available in sheets and films and
Figure 2: Steam sterilizable isolator 1946 Figure 3: (1949) Philip C. Trexler (“Trex”),
the search for a suitable germicide (Notre Dame University) on the left with J.A.Reyniers, centre and
led to peracetic acid. Trexler thought (1. Technician, 2. Electrical connection, 3. Air Bob Ervin, Assistant Director of the Lobund
outlet, 4. Mobile truck base, 5. Entrance/exit Institute on the right. Bob is learning on
that it was practicable to be able to autoclave connection) the isolator depicted in Figure 2 above.
make a simple isolator out of PVC and
decontaminate it with a vapour or spray
of peracetic acid (Barrett (12)). The plastic
isolators could be manufactured at one
tenth of the cost of a Reyniers steel unit.
Metal isolators for germ-free animal
use had by that time become very bulky
(see Figure 2) and the opportunity
to have an isolator that was light, easy to
use with multiple gloves and pass through
tanks attached, plus an excellent view to
the interior, was hard to resist. Two such
isolators are shown in Figure 3.
Figure 4: Two typical early Trexler isolators for animal use.
Trexler and Barry (13) in 1958 reported on They are simple units with high visibility.
inexpensive germ-free rearing equipment
and in 1962 Trexler applied for a US patent
– see Figure 6. These were followed by
two more in 1967. All related to medical
uses although some of the concepts
had originated in the design of the
animal isolators.
Reyniers also became interested in
an expansion of isolator technology
outside of the animal world. He believed
the technology could find applications
within industry, hospitals, research
laboratories and various specialised
disciplines. Air hygiene interested
Reyniers who believed that cross-infection

Figure 5: The Reyniers baby cubicle Figure 6: Drawings from one of Trexler’s isolator patents 1962

www.cleanairandcontainment.com Clean Air and Containment Review | Issue 18 | April 2014 15

Main feature

could be considerably reduced using
isolator techniques across a range of
medical sites.
Working with an adoption agency
called The Cradle, with its on-site
nursery in Illinois, Reyniers developed
the Reyniers Baby Cubicle (see Figure
5). This was a two section unit, one side
held the baby, the other an area entered
by a nurse (or visitor) wearing sterile
gowns, mask and gloves. The two sections
were divided by a ‘delivery’ window
through which the nurse’s forearms
could enter the baby section. Temperature
and humidity were controlled and there
was a constant supply of filtered air to
ensure that the airflow was always
outwards so that any infection would
be passed out of the baby chamber.
Trexler and Reyniers, in the late
1950s, had opposing views on the best
way to advance the germ-free animal
technology and the production of
laboratory animals. In 1958 Trexler
started work with S M Levenson at the
Albert Einstein College of Medicine in
New York to explore the use of germ-free
Figure 7: Trexler’s ‘half-suit in a bubble’
surgical isolator
Figure 8: Vickers Medical patient isolator
Figure 9: Barnes Containment Isolator System
isolators in a hospital environment. In
1962 Trexler moved from Notre Dame to
the Albert Einstein College to explore the
transference of his germ-free experience
to the hospital operating room. This led
to the half suit in a bubble concept (see
Figure 7). Used in the Bronx Municipal
Hospital, by 1964 postoperative
infections fell from 14.6 % to 3.8%.
Also in 1962 Trexler was instrumental
in the foundation of the Association for
Applied Gnotobiotics. The Association
instituted standards for the rapidly
expanding technologies of rearing
germ-free animals and a professional
identity for those working with isolators.
By 1964 he was Director of Research
for Charles Rivers Breeding Laboratories
at Boston, Massachusetts. Charles Rivers
used Trexler Isolators extensively and
became a leading global supplier of
laboratory animals.
By the late 1960s germ-free animal
technology had expanded rapidly. At
that stage germ-free animals offered
an opportunity over antibiotic treated
animals for growth and disease control
purposes. The use of antibiotic treated
animals had become controversial. Alan
Betts, a UK veterinarian at the Royal
Veterinary College (RVC), had observed
germ-free pig production in the USA.
This was being carried out by George
Young at the University of Nebraska
using Trexler’s plastic isolators. In
1966 Betts invited Trexler to establish
gnotobiotic facilities at the RVC. The
invitation was accepted by Trexler on
Figure 10: David Vetter, the Boy in the Bubble
Figure 11: David Vetter in a full isolator suit
the promise of new sources of state and
industrial funds that would support
developing his isolators for medical and
veterinary use.
Quite remarkably, within a short
time after arriving in the UK, Trexler
established facilities to produce specific
pathogen free (SPF) piglets. He also
installed enormous isolators at the RVC
for permanent residence of SPF animals
in order to study their prolonged life.
Betts and Trexler worked together on
the gut bacteriology of animals as well
as the relationship between swine and
human respiratory infections. The latter
studies fitted well with Trexler’s desire
to develop isolators for clinical medicine.
Since 1940 isolators had been used
for containment purposes in biological
warfare research. Early in 1970 there
occurred the emergence of new and
apparently highly infectious Marburg,
Lassa and Ebola diseases in Africa. This
coincided with the apprehension of the
increasing resistance to certain antibiotics.
As a result, Trexler was able to obtain
substantial funds for the purpose of
microbial containment in hospitals
through the National Research and
Development Corporation (NRDC) and
the Department of Health (DOH). He
also obtained support from Vickers
Medical Engineering.
Working with Vickers, and based on
his own earlier experiences with surgical
isolators, Trexler developed a containment
isolator suitable for a patient. It was
mobile and could fit into an ambulance.
It filtered both the air entering the
isolator and the air exhausting from it.
A Vickers Medical patient isolator is
shown in Figure 8.
He also designed and developed a
containment isolator. This was originally
intended to protect immunocompromised
patients but it found another use when a
scientist who was accidentally exposed
to Ebola virus was placed in it and treated
successfully. As well as the containment
type of isolator Trexler continued to
work on surgical isolators and reverted
back to access via gloves instead of via a
half suit , making it cheaper to build and
faster for access. (Hutchinson et al (14))
As interest in germ-free environments
and containment increased, a clinical
scientist (R D Barnes) was seeking means
for protecting children with dysfunctional
immune system. With Trexler’s advice,
Barnes and his team had the isolator
system manufactured (see Figure 9).

16 Clean Air and Containment Review | Issue 18 | April 2014 www.cleanairandcontainment.com

 Main feature

In the first trial in 1968 the child placed
into the unit was, after a week found
not to have the immune deficiency but
it was a start of this type of isolator
“It is safe to say that Trexler
was the Godfather of the
isolators that are used today.”
technology. (Barnes ,R.D et al (15) )
without compromising the sterility of
the isolator itself or of the products. He
developed the half-suit concept and also
novel methods for entry and removing
sterile items from the isolator. He also
was one of the initiators in the use of
peracetic acid as a sterilant for isolators.
Trexler was involved in the use of isolation
of immune compromised patients and
later advised NASA on containment
during space exploration. He had many
disappointments, especially with his
patents, as the National Institute of
5. Nuttall,G.H.F., and Thierfelder, H. I
Thierisches Leben ohne Bacterien im
Verdauungskanal I, Ztschr.physiol.Chem.
21:109, 1895
6. Nuttall,G.H.F., and Thierfelder, H. II
Thierisches Leben ohne Bacterien im
Verdauungskanal I, Ztschr.physiol.Chem.
22:62, 1896
7. Nuttall,G.H.F., and Thierfelder, H. III
Thierisches Leben ohne Bacterien im
Verdauungskanal I, Ztschr.physiol.Chem.
23:231, 1897
8. Metchnikoff,E. Sur la flore du corps
humain. Memoirs and Proceedings of the
Manchester Literary and Philosophical Society
45:1 , 1901

In 1971, at the Texas Children’s
Hospital, Houston, USA, a child with
an immune disease was born. This
child, David Vetter, became famous as
the Boy in the Bubble. He grew to the
age 12 years. By that time advances had
been made in the use of unmatched
bone marrow which allowed him to
receive a transplant from his sister.
Unfortunately, undetected, the bone
marrow contained the Epstein-Barr
virus and David Vetter died of Burkitt’s
lymphoma, a few months later, in
February 1984. Figures 10 and 11 show
David Vetter inside his bubble and also
outside it in a full isolator suit.
Parallel with the case in the USA,
a similar case had occurred in France
and the child was raised in an entire
flexible film isolator suite manufactured
by a company called La Calhene. It is
believed that the outcome of this
case was successful and the child was
eventually able to return to the normal
environment. We will start with La
Calhene’s flexible film isolators in the
next paper.
It is safe to say that Trexler was the
Godfather of the isolators that are used
today. From flexible plastic film isolators
have developed the sophisticated stainless
steel isolators with the various methods
for introducing sterile items and product
Health put all of his isolator patents into 9. Metchnikoff,E. Les microbes intestinaux .
Bull.Inst.Pasteur 1:106, 1912
the public domain as they had been
10. Kuster,E. Die gewinung,halting und
obtained with federal funds. aufzucht keimfreier tiere und ihre
He was awarded an honorary doctorate bedeutung fur die erforschung naturlicher
at the University of Notre Dame in 1984. lebensvorgange. Arb.Kaiserl.Geshundheit-
sante 48:1-79, 1914
Born in 1911, he died in 2013 at the age
11. Reyniers,J.A. The use of germ-free guinea
of 102 after a long and remarkable career. pigs in bacteriology. Proc.Ind.Acad.Sci.
42:35-40. 1932
12. Barrett,Jr.,J.P. Sterilizing agents for Lobund
1. Pasteur,L. Observations relatives a la note flexibl;e film apparatus. Proc.Anim.Care
Panel 9:127-133,1959
de M.Duclaux. Compt.rend.Acad.Sc.
100:68,1885 13. Trexler,P.C.& Barry,E.D. Development of
2. Schottelius,M Die Bedeutung der inexpensive germfree rearing equipment.
Darmbakterien fur die Ernaharung II. Arch. Proc.Anim.Care Panel 8:75-77, 1958
Hyg. 42-48. 1902 14. Hutchinson,J.P.G.,Gray,J.,Flewett,T.H.,Emo
3. Schottelius,M. Die Bedeutung der nd,R.,Evans,B.,Trexler,P.C.. The safety of the
Trexler Isolator as judged by some physical
Darmbakterien fur die Ernaharung III.Arch.
Hyg. 67-177. 1908 and biological criteria: a report of
experimental work at two centres. J.Hyg.
4. Schottelius,M. Die Bedeutung der Camb. 81:311, 1978
Darmbakterien fur die Ernaharung IV. Arch.
Hyg. 79-289. 1913 15. Barnes,R.D., Fairweather, D.V.I.,Holliday, J.,
Keane, C., Piesowicz,A., Soothill, J.F., and
Tuffrey, A. A Germfree Infant, Lancet
293(January 25th.): 168-71,1969
Doug Thorogood, Ph.D., studied microbiology and virology
in the UK, Belgium and the USA.
He has many years’ experience in the field of pharmaceutical
and medical research as well as QA/QC Regulatory Affairs
and Production. He started working in the field of containment
in the late 1970s and from that point developed designs, validation
procedures and operational systems for a variety of isolators for
sterility testing and aseptic filling in 19 countries. He is a specialist in the cleaning
and sanitation of enclosures as well as clean rooms and hospital environments.

Save money and cut carbon Gurney-Read

We can help you achieve
savings of typically 20–50%
without compromising
compliance, quality and comfort.
For more information contact:
nigel.lenegan@energyandcarbon.co.uk
Consulting Limited
Containment strategy, audits,
advice, concept design and
awareness training
Email: paul@gurney-read.co.uk
Tel: +44 (0)1428 725715
Web: www.gurney-read.com

www.cleanairandcontainment.com Clean Air and Containment Review | Issue 18 | April 2014 17

Main feature

A history of isolator and containment technology

Part 2: Flexible film isolators
Doug Thorogood

Abstract
This is the second of five papers that will
describe the history and development
of isolator and containment technology
in the fields of research, medicine and
pharmaceutical applications.
The paper commences with the
development of flexible film isolators
based on designs similar to Trexler’s
later models and the expansion of
the use of such isolator designs by
the pharmaceutical industry into
sterility testing, aseptic processing
and, in containment format, for
compounding hazardous products.
Hospital pharmacies also picked up
the technology for dispensing.
The surgeon incised through the
plastic and the skin at the same time.
It is to be noted that the isolator had
been previously decontaminated.
La Calhene’s original work was in
the field of containment for the French
nuclear industry (CEA-French Atomic
Energy Board) commencing in 1960.
They made PVC bags for waste materials,
remote handling systems (mechanical
arms and hands) and a flexible glove
box for hazardous materials. In
collaboration with CEA they also
co-patented the DPTE® (double porte
de transfert étanche) now more commonly
known as an RTP (rapid transfer port).
DPTEs® and RTPs plus other types of
transfer chamber which will be
discussed in a paper later in this series.
La Calhene also built several isolator
systems for immuno-compromised
patients in France similar to the unit

Flexible film isolators

With the introduction of flexible film
isolators by Trexler in the USA, and
the UK interest in such isolators, the
technology developed further. In the
USA a small number of companies
produced similar Trexler isolators for
gnotobiotic animal rearing and research,
but at that time there was a limited
market and the number of manufacturers
dwindled down to two or three. One of
these was Standard Safety Inc.
However there was much interest in
using isolators for developing biological Figure 1: Baby in an early La Calhene isolator (attended by parents)
warfare agents and the business expanded
in that direction in both the USA and
the UK.
La Calhene (now Getinge- La
Calhene) in France also developed
flexible film isolators for germ-free
rodent breeders for animal research.
These were similar in design to the
Standard Safety models from the USA.
Another application for flexible film
isolators at that time was for surgical
procedures such as hip joint replacement.
The unit had several glove ports and an
RTP. The flexible floor of the isolator had
an adhesive outside surface (suitably
protected before use.) The isolator was
placed over the operation area of the
patient, the protective sheet was removed
from the adhesive and the floor of
the isolator was fixed to the skin of the
patient over a fairly large area. Figure 2: A La Calhene sterility test isolator circa 1980

8 Clean Air and Containment Review | Issue 19 | July 2014 www.cleanairandcontainment.com


described in the previous paper of this
series. One example, for a baby, shown
in Figure 1, closely resembles one of
Trexler’s models.
The company continued to expand
their range of isolators for animal
research and hospital use and, at that
time, they were all equipped with canister
type HEPA filters and one blower.
Interest from the pharmaceutical
industry initiated the development of
flexible film sterility test isolators. The
first of these, shown in Figure 2, was
built circa 1980 and may well have
been one of the very first flexible film
isolators used for that purpose. Two later
innovations were the half-suit isolator
and a free-standing automatic peracetic
acid vapour generator.
The early La Calhene isolators stood
on a plastic coated table. The isolator
had a transparent canopy of PVC with
a grey plastic floor. The plastic canopy
was supported on a metal tubular frame.
Above the frame was a plastic covered
board on which were fixed the blower,
the filters and an inclined tube manometer
for measuring pressure inside the isolator.
Pressure was controlled by adjusting the
speed of the blower. A T-junction on the
input air pipe allowed for the connection
of a peracetic acid gassing device.
Large glove-rings were fixed onto
the PVC canopy with metal worm-drive
clamping bands and glove sleeves were
fitted securely to these with rubber
O-rings. Materials entered and exited
the isolator via a DPTE® or via a
circular opening with a cover that
could be removed. Later models
had the floor replaced with a stainless
steel base to which the PVC canopy
was fixed in a leak free manner.
Figure 3: La Calhene half-suit
(demi-scaphandre)
www.cleanairandcontainment.com
The distance that can be comfortably
reached via a standard sleeve and glove
arrangement is about 800 mm, dependant
on the size of the operator, but, as the
size of the isolators increased, as well
as the desire to perform more complex
manipulations, La Calhene offered their
half-suit design as shown in Figure 3.
This was double skinned and sealed
at waist level onto an oval ring set in the
base of the isolator. It was equipped
with a blower and a HEPA filter to provide
respiratory air to the operator’s face and
arms. This air entered via the space in
the double skin, blew into the transparent
head cover and the wrist area and left
via the inside of the suit back to the
room. The half-suit was entered from
beneath the isolator and the floor of the
isolator at that point was usually set at
a slight angle to aid entry.
This in essence is the basic isolator
design that subsequent manufacturers
developed with slight variations
for sterility testing purposes and for
further-germ free animal breeding
and research.
The use of isolators for sterility
testing started to slowly expand in the
late 1980s into the early 1990s, mainly
in Europe, but in 1993/1994 the USP
(United States Pharmacopeia) changed
the criteria for a failed sterility test. The
result was a surge in the use of isolators
for sterility testing in the USA and in
Europe in a variety of systems that
ensured maintenance of sterility.
A few of the larger pharmaceutical
companies used isolators attached to
an autoclave to maintain the external
sterility of the media tubes or bottles.
These were transferred into the sterility
test isolator via a DPTE®. When the tests
were completed, the media tubes were
Figure 4: Typical simple flexible film isolator
– Class Biologically Clean Ltd, USA
Clean Air and Containment Review | Issue 19 | July 2014 9
Main feature
placed into a small transfer isolator,
again by a DPTE® port. The transfer
isolator was then placed into an
incubator room. Thus the media tubes
were never exposed to the external
environment or to the operators
performing the tests. This set-up was
aimed at eliminating the possibility
of false positive growth results.
Other examples of flexible film
isolators are seen in Figures 4 and 5.
At this time Amsco (now Steris
Corporation) had been developing
hydrogen peroxide vapour
decontamination technology and
had produced a hydrogen peroxide gas
generator for use with isolators. They
also started to market an isolator system
called Oasis for sterility testing. These
models were designed and manufactured
by La Calhene and again featured the
DPTE® device.
Parallel with this activity, Millipore,
who had supplied filtration systems for
sterility testing (the open cup method),
developed a closed system with a
piece of equipment that would fit
onto the base of a sterility test isolator
(SteritestTM).
There are now in excess of over 700
sterility test isolators throughout the
world and only a very small number of
sterility test failures have been recorded.
These were found to be due to human
errori. This demonstrates the high degree
of assurance in using isolator technology
plus the security of the DPTE® system.
An example of the latest version from
Getinge- La Calhene (Isoflex) is seen
in Figures 6 and 7.
Figure 5: Typical half-suit flexible film
Isolator – Pharminox Isolation Limited, UK
Main feature
There was also a full suit (scaphandre)
design (see Figures 8, 9 and 10). This
incorporated a special DPTE® for
entering the suit (see Figure 9). Clearly
the diameter of the opening placed a
limit on the size of operator who could
use the suit. The application was in
certain sterile areas such as the loading
and unloading of freeze dryers.
Other types of full suit protection
were developed in the USA during the
period of research into and manufacture
of biological weapons as depicted in
Figure 11. Such types of suit are now in
use during the study and identification
Figure 6: Isoflex flexible film sterility test isolator – Getinge- La Calhene
Figure 7: The Isoflex seen from the inside
10 Clean Air and Containment Review | Issue 19 | July 2014
of highly contagious microorganisms
within a Level 4 biological containment
environment (Figure 12).
Flexible film isolators for germ free
animals and for veterinary use continued
to expand in Europe and also in the USA
through the development of companies
dedicated to breeding such animals for
research purposes and Trexler played a
role in the development of two of these
companies. They continue to provide
gnotobiotic animals today.
Metall + Plastic in Germany produced
flexible film isolators on a grand scale.
One design had a vertical rotating cage
system for germ free animals set inside
a very tall polyurethane plastic canopy,
about 7 meters high. The design used
electric motors and a star-wheel/chain
system to rotate the system in order to
bring one cage at a time down to the
front of the operator. He or she could
then perform cleaning and feeding
operations through a set of gloves
without having to enter the enclosure.
Metall + Plastic also developed doors
with expanding seals for stainless steel
chambers where peracetic acid was
sprayed into the chamber. This technique
was used to decontaminate the exterior
of metal containers holding sterile
antibiotic raw materials. (Peracetic
acid and other chemicals used to
decontaminate isolators will be
discussed in a later paper in this series).
Flexible film isolators were also
adopted in hospitals in certain parts
of Europe, notably in the UK and in
France. One example is shown in
Figure 13. There was a requirement to
compound or mix sterile drugs under
aseptic conditions and this was being
carried out in traditional open fronted
safety cabinets. There were concerns
regarding sterility assurance as well
as operator safety when compounding
certain drugs such as antibiotics and
cytotoxics. One result was a guideline
published in the UK in 1994 with regard
to procedures, quality control and
quality assurance ii. This was completely
rewritten in 2004 as an authoritative
book on the subject iii. Crauste-Manciet iv
gives a good description of the
requirements for high volume preparation
in hospital.
Following on the success of the use of
flexible film isolators there was a rapid
expansion of suppliers of these types of
units. They were (and are) based on the
original Trexler concept but airflow
systems have become more sophisticated
with larger rectangular HEPA filters
replacing the cartridge HEPA filters
where higher air change rates and
unidirectional airflow are required.
Isolator designs also evolved from
Cambridge Isolation Technology in the
UK (now Pharminox Isolation Ltd).
Pharminox continues to make sterility
test isolators but also supplies half-suits,
RTPs, gloves and sleeves for isolators
and other isolator components.
Other manufacturers also followed
including Amercare, Bassaire, Bell,
Bioquell, Contained Air Solutions,
www.cleanairandcontainment.com
 Main feature

Figure 8: Entering an isolator via a Figure 9: Arriving in the isolator in a Figure 10: Inside the isolator in a
Scalhene full suit Scalhene full suit Scalhene full suit

Envair, Extract Technology, Hosokawa

Micron, Howorth Air Technology,
Mach-Aire and Powder Systems in the
UK. Non-UK suppliers include Skan
AG, Telstar, Metall + Plastic, Bosch and
Steriline. This list is far from exhaustive.
The pharmaceutical industry had
seen the various designs and techniques
used for germ free animals, containment
for immune compromised patients,
etc., that demonstrated the possibility
of working in a controlled environment
with a high degree of confidence in
maintaining sterility. The use of positive
pressure was seen as an essential way to
maintain an aseptic or sterile environment
inside the isolator.
It was also found that the methods
and agents used to decontaminate Figure 12: Biological safety suit
animal isolators could be used in the at CDC (Centers for Disease Control
Figure 11: Full suits, USA and Prevention), USA
pharmaceutical industry for sterility
testing on the one hand and for improved
aseptic filling on the other.
In the search to improve testing and
manufacturing environments in the
pharmaceutical industry in the 1980s,
it was found that the methods and
agents used to decontaminate animal
isolators could be used. In addition,
the use of containment techniques
for the containment of highly potent
or hazardous materials that had to
be compounded into a finished
pharmaceutical product was being
explored. The positive-negative debate
is still there in the background but,
to put it into perspective, all isolators,
being total enclosures, provide both an
aseptic environment and containment.
The problem arises if there is any
leakage of contaminated air in or out.
Therefore, where aseptic conditions
are the predominant requirement, then Figure 13: Simple pharmacy isolator – Flexipharm from Extract Technology

www.cleanairandcontainment.com Clean Air and Containment Review | Issue 19 | July 2014 11

Main feature
positive pressure isolators are
favoured and where containment
is the predominant requirement, then
negative pressure isolators are favoured.
It is fair to say that the regulatory
authorities, such as the MHRA, prefer
positive pressure isolators and the safety
authorities, such as the HSE, prefer
negative. However, having said that
there are positive pressure flexible film
isolators in use that handle cytotoxic
drugs and their use will have been
subject to proper risk assessments.
The first pharmaceutical
manufacturing unit in an isolator
system was by Farmitalia in 1978.
The unit was produced by La Calhene.
This was a powder filling operation
and the unit was fitted with quite a
number of gloves. It is believed that
this unit was the first to use a rapid
transfer port (DPTE®) outside of the
nuclear industry.
La Calhene, in the late 1980s also
made an isolator to surround an ampoule
filling line in France. This unit was mainly
flexible film with appropriate metal
areas for exhausting the hot air from
the flame sealing of the ampoules.
A further development from
La Calhene was a Spanish facility using
Who will order the 3000th isolator
produced by Getinge La Calhène?
GETINGE LA CALHENE
1 rue du Comté de Donegal
41100 Vendôme – France
Tél. +33 (0)2 5473 4747
12 Clean Air and Containment Review | Issue 19 | July 2014
a flexible film isolator for filling large freeze dryer was all in one large room v.
volume parenterals. This involved a This type of isolator use has now been
small railway system for moving trolleys expanded by other companies and in
of sterile bottles from an attached the next paper in this series a history
autoclave to the filling position. the use of metal and rigid plastic
The decontamination of the unit was isolators will be presented.
performed from a STAR peracetic acid
unit, also made by La Calhene. Over the
years this unit has been developed i. Personal communication from James Akers
of Akers Kennedy Inc, USA.
further and is now marketed under the
ii. Isolators for Pharmaceutical Applications,
name of ISOVAP. Edited by Gerard Lee and Brian Midcalf,
In the 1990s, a company in south- HMSO, 1994.
west France built a system to compound iii. Pharmaceutical Isolators: A Guide to their
Application, Design and Control. B.
and aseptically fill cytotoxic products.
Midcalf, 2004, Pharmaceutical Press.
The filling line incorporated a dry heat
iv. Isolator design for high volume aseptic
vial depyrogenation oven which was units, S Crauste-Manciet, Hospital
attached to a filler. The line progressed Pharmacy Europe, 2012,Issue 63, July/
August.
to a freeze drier, then onto a capper and
v. New Aseptic Isolator Process for a Cytotoxic
finally to a washer for cleaning the Product, D.Thorogood ,PDA Seminar, Basel,
exterior of the filled and capped vials. Switzerland, 1992.
The entire line was enclosed with an
isolator system made of stainless steel.
A biographical note for Doug
Flexible film isolators were used to transfer
Thorogood is at the end of Part 1
sterilized items from an autoclave
of this series in Issue 18.
to various of the isolator units. The
compounding of the product was
performed in a negative pressure isolator
and the required testing for batch release
was performed in another isolator.
The entire system excepting the
dry heat oven and the body of the
Ask the experts!
www.getinge.com
www.cleanairandcontainment.com
Main feature
A history of isolator and containment technology
Part 3: Non-flexible film isolators including RABS
Doug Thorogood
Abstract
This third paper on the history of isolators
describes the development and use of
non-flexible film isolators including
restricted access barrier systems (RABS).
By non-flexible is meant that type of
unit that has a metal or a rigid plastic
structure. These are referred to as
conventional isolators.
Many of these models have been
designed to enclose various types of
filling machines for processing vials,
ampoules, cartridges or syringes and
are the descendants of glove boxes or
safety cabinets for aseptic processing.
RABS appear to have been largely
devoted to aseptic filling of various forms
of vial and syringe or filling products for
terminal sterilisation and are dependent
on unidirectional airflow as in
conventional clean rooms.
Other such isolator models have been
used for handling, processing and
compounding pharmaceutically active
Figure 1: Aseptic filling process circa 1930
(courtesy J Agalloco and Associates)
Figure 2: Aseptic process in a glove box type of
enclosure (courtesy J Agalloco and Associates)
14 Clean Air and Containment Review | Issue 20 | October 2014
materials and for enclosing small tanks,
washing utilities, analytical instruments,
etc. Some of the flexible film units such
as sterility testing or for hospital pharmacy
use have also been replaced by rigid
versions. One addition to mention is
the preparation and compounding of
radio-active products.
Aseptic filling
As can be seen in Figure 1, early aseptic
filling was primitive by today’s standards.
It was in the 1930s and afterwards that
glove boxes came into use for similar
purposes as shown in Figure 2.
Glove boxes continued to be used
with modifications that eventually
included some form of air filtration.
It was not until after World War II that
HEPA filters became available and the
traditional cleanroom concept emerged.
The cleanrooms were based upon
unidirectional airflow over the filling
area and have remained so ever since.
Improvements to avoid the effect of air
displacement by operator movements
led to screening with sheets of hanging
flexible plastic, see Figure 3.
Figure 3: Early aseptic filling in a clean room
(courtesy J Agalloco and Associates)
Figure 4: Flexible film aseptic process
isolator (courtesy Nova Laboratories)
“The cleanrooms were based
upon unidirectional airflow
over the filling area and have
remained so ever since.”
Early flexible film process isolators
Flexible film isolators continued to be
used for raising germ-free animals and,
as described in the previous paper,
the pharmaceutical industry used the
technology for sterility testing.
Attempts to use flexible isolators for
aseptic filling were successful but there
were problems related to robustness and
cleaning before and after a filling process.
This also applied to units used to prepare
the product prior to filling, especially
highly active or hazardous drugs such
as hormones or cytotoxics.
An example of such a flexible film
process isolator is shown in Figure 4.
This is a large eight-glove unit (8 gloves)
with a plastic film envelope and a stainless
steel base. The plastic envelope was fixed
to the metal base with a continuous
spring-pressure seal. Filling or processing
equipment was located inside the
enclosure and a single batch was
processed, adding suitably wrapped
sterile containers, equipment and test
items prior to a decontamination
process with peracetic acid/hydrogen
peroxide vapour.
Conventional isolators
versus RABS
There was much hesitation in the industry
to move to isolators fixed onto a filling
machine. The move was largely driven
by the need to aseptically fill products
that were heat labile such as vaccines
and blood products. Another consideration
was the preparation of certain products
that were hazardous to operators such
as hormones or cytotoxic preparations.
The dilemma was that nobody wanted to
be first but nobody wanted to be third!
Parallel with these moves, filling
instrument manufacturers also viewed
the use of isolators as part of the
www.cleanairandcontainment.com

development of their equipment and
gradually filling lines became more
‘isolator friendly’. Air leakage was
viewed as a problem when using positive
pressures due to the design of early fillers
and also the escape of decontaminating
agent into the surrounding environment,
but gradually these problems were
addressed by the equipment manufacturers
and most of the modern isolator/filler
combinations showed very little leakage of
air or decontaminating agent when sealed.
Isolator manufacturers also looked at
the requirements of placing an isolator
onto a filling machine and developed
the typical (as seen today) stainless steel
shell with doors. Windows in the doors
and in other panels surrounding the filling
machine were equipped with sleeves
and gloves for handling purposes. These
were suitably placed for appropriate
manipulations within the enclosure.
Other rigid models appeared using
plastic materials moulded and shaped,
with typical window and glove fixtures.
Many had attached pass-throughs that
could be ‘sterilised’, either together with
the isolator or separately. The use of
the term ‘sterilised’ is for simplicity.
In practice a sporicidally active chemical
decontaminant is normally used. The
types of chemicals and the apparatus
used to ‘sterilise’ isolators will be
discussed in a later paper in this series.
As aseptic filling processes had,
by tradition, been carried out under
Figure 5: Variety of RABS (courtesy of Robert Bosch GmbH)
Figure 6: Air return ducts from inside
the isolator to the plenum
www.cleanairandcontainment.com
unidirectional (laminar) airflow which
was the only real protective mode for
such a process (Grade A conditions),
it followed that for particle control and
for microbiological purposes the rigid
isolators had to be built to use the same
principle. This type of design became
the conventional isolator for aseptic
filling and processing the active principles
into finished product.
In parallel with these developments,
due to the cost of some of the isolator
systems and the size and complexity
of filling machines at that time, a new
concept of a barrier system evolved.
Rigid sheets of plastic fitted with gloves
were suspended around the critical
filling area as it was the operating
personnel who posed the greatest risk
of generating airborne contamination
in the critical filling area.
Further development led to a variety
of RABS used for aseptic filling which
closely resembled conventional isolators.
It is important to note that the main
protective mode of the RABS is still
the use of unidirectional airflow with
a barrier to prevent intrusion by the
process operators during the filling
process. As can be seen in Figure 5,
a variety of different modes of RABS
evolved, with differences in the air
handling systems inside and outside
the RABS.
Further developments were made so
as to be able to biologically decontaminate
Figure 7: Isolator syringe filler combination
with in-line plunger inserter, inspection and
labelling line
Clean Air and Containment Review | Issue 20 | October 2014
Main feature
a RABS in a similar way to a
conventional isolator.
In nearly all cases, with the design
of a conventional isolator or a RABS,
the HEPA filters, in essence, became the
‘ceiling’ of the isolator. In the early days
this type of design using unidirectional
airflow became the norm for filler/isolator
combinations and has continued but
there are now debates about the use of
turbulent airflow as another method.
In the case of isolators used to transfer
sterile product or equipment into the
filler/isolator, then in many cases turbulent
airflow was and is used, either with
cartridge or with box type filters. The
debate has largely centred on the
microbiological aspects of conventional
isolators as it has been confirmed that
the status of the isolator, after a suitable
validated biological decontamination
process, showed no microorganisms
present on all of the surfaces exposed to
the chemical agent used. This led PIC/S
inside a recommendation(1) to state the
isolation of one colony forming unit inside
a ‘sterilised’ isolator indicated the failure
of the system and a thorough investigation
should take place. PIC/S also made
recommendations regarding assaying
the microbial status of the ‘sterilised’
isolator without placing any bacteriological
test equipment inside the unit.
With the use of unidirectional
airflow within a ‘sealed box’ it was
necessary to have the larger part of the
airflow return back to above the HEPA
filters with a little make up air from the
surrounding environment. The return
systems varied in shape, size and
complexity and were, in some cases,
demountable for ease of cleaning. Air
return ducts with slotted apertures on the
edge of the floor of the filling machine
were common with associated pipe work
outside the enclosure returning air to the
plenum above the HEPA filters.
Later developments for air return
included the sophisticated design of a
double window where the small space
between the windows in the isolator or on
the isolator doors allowed for the return
air to the plenum. This technique largely
removed the need for external air return
systems and was much more elegant.
Cleaning was obviously essential
especially with the use of decontaminating
or sanitising agents such as peracetic
acid/hydrogen peroxide mixtures and
later hydrogen peroxide either as a vapour,
aerosol or ultra-sonic mist. Figure 7 shows
a typical isolator syringe filler line.
15
Main feature
As with the early flexible film
isolators other process equipment was
added onto the rigid type isolators to
enable a continuous flow of product in a
sterile environment. Examples include:
• Dry heat tunnels to deliver sterile
endotoxin free containers direct onto
the filling line. These could be bottles,
vials and syringe barrels.
• E-beam tunnels to deliver tubs of
sterile syringes directly into the
filling zone. The e-beam system is
used to decontaminate the external
surfaces of the syringe tub.
• Autoclaves for delivering sterile
media into sterility test isolators,
product contact components of a
filling line into the isolator or sterile
containers for filling.
• Freeze driers, attached to the filling
machine isolator for the aseptic
transfer of partially stoppered vials
into the freeze drier and also
recovering the stoppered vials back
out of the freeze drier for capping to
seal etc. This form of the technology
involved resolving issues of reach for
the operators. Some systems were
made to work automatically (loading
Figure 8: Freeze drier attached to a
process isolator using a half-suit for
loading and unloading
Figure 9: Freeze drier attached to an isolator
– side view of Figure 8
16 Clean Air and Containment Review | Issue 20 | October 2014
and unloading) but for manual work released product was aseptically filtered
it was usual to employ a half suit. into sterile mobile tanks that were in
Figures 8 and 9 show a freeze drier turn aseptically connected to the filling
interfaced with an isolator equipped isolator. See Figure 13.
with a half suit.
Radio-active compounding
Freeze drier interfaces with filling
and isolators
isolators have become very sophisticated
Since the advent of the nuclear industry
and in some cases fully automatic by way
some form of isolator has been used
of loading and unloading.
to protect operators from the intense
Downstream from the filling isolator,
radiation. These were referred to as
depending on the product, other units
“hot cells” and were used to manipulate
could be and were added including particle
various radioactive materials. See Figure 14.
inspection, labelling and packing into
As mentioned in a previous issue
primary cartons. In the case of syringes
of this series, La Calhene figured
also a unit for plunger insertion, see
largely in the French nuclear industry
Figure 7. This shows the filling isolator
for manufacturing remote handling
followed by a series of isolators for
equipment and also transfer systems.
inspection, plunger insertion, blister
The development of isotopes that
pack and final pack.
were used for diagnostic and treatment
Some isolator systems became
purposes in human medicine led to
extremely large, see Figures 10 and 11.
the use of aseptic filling isolators for
Many isolator/filler combinations
such isotopes as iodine131 which has
were used for the aseptic filing of vials.
a half-life of 8 days. Such a unit was
Figure 12 depicts a typical modern
built for Amersham and was a fully
system made by Metall + Plastic
automated filling system.
(Optima) Germany.
For later developments such as in
A complete set of isolators used for
positron emission tomography (PET) mini
aseptically filling cytotoxic compounds
isolators were fabricated that met the GMP
with the compounding and analysing
requirement for aseptic manufacture but
for release to fill in the same area. The
worked at negative pressures.
Figure 12: Optima (Metall+ Plastic) vial
Figure 10: (courtesy of J Agalloco filling isolator/filler system
and Associates)
Figure 13: Entire suite of isolators for
various purposes. (Pierre Fabre, France)
Figure 11: Part of a 46 m long filler/freeze
drier complex for vial filling, with over
60 glove ports (courtesy MSD, Riom, France / Figure 14: ‘Hot Cell’ type isolator
Getinge La Calhène) (Isotope Technologies Dresden)
www.cleanairandcontainment.com
 Main feature

One example is the production of
Technetium99m. This isotope is one the
most widely used in diagnostic medicine
and has a half-life of 6 hours. A typical
negative pressure isolator for the aseptic
production of this product is depicted in
Figure 15. Radiation for Technetium99m
is such that normal glass and plastic
materials are safe barriers to its radiation.
In the maintenance of sterility when
using isolator technology, the most
important factors are the aseptic transfer
into the ‘sterile’ environment of the
aseptic process isolator and the removal
of sterile items without breaking sterility.
With positive pressure regimes, the
exit of the isolator can be an aperture that
is only slightly larger than the size of the
container being filled. This is sometimes
called the mouse-hole and there is usually
sufficient outwards airflow through it to
prevent incoming air entering. However
some manufacturers provide a small
unidirectional airflow source over the hole
as additional protection.
Throughout the genesis of
containment and isolators the use of
gloves appeared to be mandatory.
Gloves of various materials were used
and are now mainly materials such as
Hypalon or Neoprene. These are
resistant to the various ‘sterilising’
agents used.
However gloves presented a problem
by way of breaching the sterility of the
isolator through pin-holes and minute
tears. Various types of glove testing
devices are available for use prior
to processing to check the integrity of
the gloves.
In one report it was stated that
well-trained operators were better
in detecting flaws than the testing
equipment and this highlights the area
of training operators thoroughly when
using isolator technology.
As gloves may be a potential hazard
as far as sterility assurance is concerned
and to avoid their use various robotic
devices have been developed both in
the USA and Japan that allow
manipulations within an isolator. This
technology, shown in Figures 16 and 17,
may well be the next stage in the
development of glove free isolators.
In the next of this series the use of
various techniques and apparatus to
make a sterile transfer into an aseptic
process isolator will be discussed.

References:
1. Isolators used for Aseptic Processing and
Sterility testing, Pharmaceutical Inspection
Convention & Pharmaceutical Inspection
Co-operation Scheme: Recommendation .
Pl.014-3,25th September 2007

Figure 15: Technetium99m preparation Figure 16: Double arm robot in an isolator
isolator (Envair) (courtesy of J Agalloco and Associates)

Doug Thorogood, Ph.D., studied microbiology and virology

Figure 17: Operator using the double arm
robot (courtesy of J Agalloco and Associates)
in the UK, Belgium and the USA.
He has many years’ experience in the field of pharmaceutical
and medical research as well as QA/QC Regulatory Affairs
and Production. He started working in the field of containment
in the late 1970s and from that point developed designs, validation
procedures and operational systems for a variety of isolators for
sterility testing and aseptic filling in 19 countries. He is a specialist in the cleaning
and sanitation of enclosures as well as cleanrooms and hospital environments.

Clean Air Test & Certification

cGMP Compliance & Validation Support
GAMP Compliant Continuous Monitoring Systems
Cleanroom Design, Construction & Project Management
www.validair.com www.diamondscientific.co.uk www.fmonsys.com

www.cleanairandcontainment.com Clean Air and Containment Review | Issue 20 | October 2014 17

Main feature
A history of isolator and containment technology
Part 4: Transfer devices
Doug Throrogood
Abstract
The discussion in this part of the history
of isolator and containment technology
reviews the development and use of
various devices that permit the aseptic
transfer of components such as sterile
vials, syringes, bottles and other types
of container as well as the actual product
itself into and out of the aseptic filling
area of an isolator or RABS (restricted
access barrier system).
Introduction
Some of the systems that permit aseptic
transfers, such as pass-through hatches
or devices 1, autoclaves and dry heat
tunnels, used in current cleanroom
technology, have been adapted by isolator
and RABS manufacturers and users.
With the advent of isolators and RABS,
other devices have been developed
as well as new techniques for aseptic
transfers. Some of the devices are
shown in Figure 1.
Note: RTP is rapid transfer port.
The discussion of the various devices
will be divided into three sections:
1. The use of systems during the
performance of sterility testing.
Figure 1: Diagrammatic representation of devices that may be used for the aseptic
transfer of materials into and out of a ‘protected area contained environment’
1. Editor’s note: Throughout this paper, the author has used the term ‘pass-through’.
When discussing isolators, alternative terms are ‘transfer chamber’ or ‘transfer device’.
14 Clean Air and Containment Review | Issue 21 | January 2015
2. The use of systems for the
compounding and transfer of
a pharmaceutical product into and
out the aseptic area of an isolator
or a RABS. Non-sterile compounding
of hazardous product is also included
as an illustration of the containment
aspect of the technology.
3. The use of systems for the entry and
exit of sterile containers, components
and testing equipment into and out of
the aseptic area of an isolator or RABS.
Early pass-through technology
In aseptic manufacture using the classic
cleanroom approach the problem of
getting sterile materials and components
into the cleanroom was to use a double-
door autoclave or a dry heat sterilising
tunnel. This enabled the transfer of
items of filling equipment (autoclave)
and sterile containers (dry heat tunnel).
For other items, usually to replenish
stocks of sterile gloves, garments,
etc., as well as items forgotten in the
preparation for the filling process,
simple double door pass-throughs
were used, see Figure 2. Some of
these were equipped with HEPA filters
and had an air over-pressure profile.
Wrapped sterile items were placed in
the pass-through and then sprayed with
70% filtered alcohol or another approved
disinfectant and allowed to dry. The
inner door to cleanroom was then opened
and the items were removed. Reliance
for asepsis depended upon correctly
observed procedures and the effectiveness
of the alcohol spray/disinfectant.
Later versions included a diluted
peracetic acid spray system to treat the
surfaces of stainless steel containers
etc. placed in the pass-through. Such a
system was developed by Metall + Plastic
in Germany, using their expanding seal
technology for the doors, an automated
peracetic acid spraying system and an
appropriate aeration system to remove
the vapours after the exposure period.
This method was used to decontaminate
the external surfaces of sterile stainless
steel containers that had to be passed
into the sterile filling area, in this
particular case for the bulk packing
of antibiotic products.
Fedegari in Italy also developed
a low temperature decontamination
system based on the same concept,
using hydrogen peroxide as the
decontaminating agent. Furthermore
Figure 2: Simple two-door pass-through
www.cleanairandcontainment.com

Fedegari has also introduced a new
generation of H2O2 vaporizers controlling
its concentration within the chamber with
a feedback control loop, see Figure 3.
One could also argue that the
changing facilities for the operators
who were to work in the aseptic filling
area could also be considered as pass-
throughs. Reliance for maintenance of
asepsis was placed on compliance with
correct dressing procedures with sterile
garments and accoutrements and
with the use of sanitising agents prior
to entering the aseptic area. An
air-pressure ‘cascade’ ensured that air
flowed from the cleanroom into the
changing room and thence to the area
outside the entry to the changing room.
Figure 3: Fedegari low temperature
decontamination chamber
Figure 4: DPTE® mode of connection
www.cleanairandcontainment.com
Transfer systems used in
sterility testing isolators
As mentioned in an earlier part of this
five-part history, sterility testing using
isolators became popular and this saw
the introduction by La Calhene (now
Getinge La Calhene) of an RTP branded
DPTE® (double porte à transfert étanche:
double door for leak-tight transfer). This
device had been developed initially for
use in the French nuclear industry for
the safe transfer of radio-active materials.
La Calhene saw that the same device
could be adapted for use with flexible
film isolators as a novel way to maintain
the integrity of the sterile isolator
while making transfers into and out
of the isolator.
The DPTE® allowed the connection
of a sterile container or bag or even
another isolator to the test isolator for
the transfer or exit of materials without
loss of ‘sterility’ in the test isolator or
the connected items.
The basis of the DPTE® action
is simple. There are two parts to the
system: an alpha port section and a beta
port section. The alpha port is usually
installed in a surface such as a wall or
the floor of an isolator. It comprises a
flange, a seal and a door. The beta port
also has a flange, a seal and a door and
is connected to a container, another
isolator or a suitable device for transfers,
ŽŶƚĂŝŶĞƌ
ĂƉƉƌŽĂĐŚ
>ŽĐŬďLJ
ZŽƚĂƚŝŽŶ
;ϲϬΣͿ
KƉĞŶƚŚĞ
ŽƵďůĞĚŽŽƌ
Clean Air and Containment Review | Issue 21 | January 2015
Main feature
e.g. a bag. The seal of the DPTE® is
usually referred to as a lip-seal and
this is an important component of the
entire assembled system.
The alpha and beta sections are
connected and, by rotating the beta
section approximately 60 degrees,
the doors are locked together as one.
The alpha side of the unit is then opened
with access into the isolator. The external
surfaces of both the alpha and the beta
section doors remain firmly locked
together until the alpha door is closed
and a reverse rotation of the beta unit
takes place, separating the two doors.
These actions are shown in Figure 4.
A Getinge La Calhene alpha port is
shown in Figure 5 and different sizes
of container with beta ports from the
same manufacturer in Figure 6.
Obviously the beta container or
attached device has to be internally
sterile like the isolator. To effect
sterilisation of the alpha door a simple
beta port with a plastic cap is docked
onto the alpha door which is then opened
and exposed to the decontaminating
agent during the ‘sterilisation’ of the
isolator. At the end of the process the
alpha door is closed and the beta cap
removed.
Early in the use of the DPTE® on
sterility test isolators, a group in the
USA coined the phrase ‘ring of death’
Figure 5: Getinge La Calhene alpha port, inside
view (the beta port docks onto the outside)
Figure 6: Getinge La Calhene beta ports
with plastic containers attached
15
Main feature
as they found that a very small (0.1 to
0.5 mm) peripheral band on the lip-seal
exposed to the environment outside
the isolator was also exposed inside the
isolator. This observation raised some
concern in the industry.
The author of this current article has,
over the years, run many tests to show
if any contamination could be transferred,
even using deliberately contaminated
seals, but under normal GMP conditions
it was found not to be a problem.
Additional security could be provided
by wiping the seals with 70% alcohol
or another approved disinfectant.
Lubrication was needed occasionally
and this was provided by using
sterilised silicone oil.
However there remained much
concern about the ‘ring of death’ in the
USA and Central Research Laboratories,
Chicago produced an RTP where the
seal could be heated to above 100 °C
in order to ‘sterilise’ it. They subsequently
reverted to a normal design of RTP which
is marketed by DE-STA-CO. In common
with other RTP manufacturers they
offer alpha door diameters of 105,190,
270 and 350 mm.
As mentioned in previous parts of
this history, there are over 700 sterility
test isolators in use throughout the World
fitted with the DPTE® units and there
have been no reports of any sterility
failure due to the transfer door.
While the DPTE® beta section was
usually fitted with a stainless steel or
Figure 7: Optima Alpha port, Cape Europe Ltd.
Figure 8: External view of the SART system,
Sartorius GMBH
16 Clean Air and Containment Review | Issue 21 | January 2015
plastic container and the entire unit Transfer of product into and
sterilised internally, other uses included out of process isolators
connecting two isolators together and The containment achieved by using an
also fitting sterile waste bags to the isolator in the compounding of active
isolator to hold any materials after pharmaceutical ingredients worked in
the sterility tests had been completed. two ways:
In many cases, the rotation of the 1. Aseptic processing in EU Grade A
beta section when connecting to another conditions by filling previously
isolator was overcome by the use of a sterilised product into sterile
flexible sleeve that could accommodate containers.
the rotation. Early DPTE® or RTP units
2. Non-aseptic processing under
were not fitted with a locking device
negative pressure and, usually,
and it was possible to remove the beta
EU Grade B or Grade C conditions,
while the alpha port was still open, thus
in the compounding of hazardous
losing containment and ‘sterility’. Later
active pharmaceutical ingredients.
models were fitted with a locking device
In this case the finished product was
so that unless a lever was moved inside
filtered through 0.22 µm filters into
the isolator the beta section could not be
sterile vessels for subsequent transfer
removed. Following the expiry of the
to an aseptic filling isolator.
patent on the La Calhene DPTE®, other
similar devices followed.
There were three ways to achieve these:
Cape Europe offer Optima alpha and
1. Directly piped into or out of the
beta RTP units and they claim that their
isolator using fixed piping in place,
RTPs are compatible with the DPTE®
subsequently cleaned and sterilised
of La Calhene. See Figure 7.
– CIP (clean in place) and SIP (sterilise
M + W Group, Germany, also offer
in place). This type of processing was
similar RTP designs based on the alpha
for large volumes of product prepared
and beta unit approach. Dynamic
on a regular basis.
Design Pharma, USA, developed a beta
port that was reported to be compatible 2. Very small batches of product actually
with the La Calhene DPTE® alpha port compounded and filled in adjoining
and the novelty of this design was that isolators, and transferred in small
the beta port rotated but not the attached vessels inside the isolators.
container. However not all available
3. More commonly by the use of RTP
RTPs are compatible with La Calhene
technology where the alpha port
models and this was reported by
was installed in the wall or floor
La Calhene in a recent report.i
of the isolator and the beta port
The use of sterility testing isolators
with an appropriate container was
and the associated transfer units
equipped with filters and tubing.
demonstrated the efficacy and the safety
This unit would be sterilised in an
of the DPTE®/RTP and the industry
autoclave. The beta port would be
adopted these devices for the safe aseptic
attached to the alpha port, the door
transfer of product into process isolators.
opened and the enclosed sterile
Figure 9: Sterile disposable filling system, Bosch /Sartorius, Germany
www.cleanairandcontainment.com

tubing fixed to the filling head.
The product would be sent from
an external vessel (sometimes a
mobile tank) via a sterilising filter
attached to the beta port container.
This method allowed for the
compounding and filling of various
types of product including vaccines,
hormonal and cytotoxic drugs.
As with all systems the components
that came into contact with the sterile
product to be filled had to be sterilised
by a recognised sterilisation process.
This meant that filler components had
to be sterilised outside of the isolator
and introduced via the DPTE® method,
or wrapped, placed in the isolator and
exposed to the ‘sterilising’ agent during
the decontamination of the isolator.
Recently a new form of aseptic
transfer has been developed by Sartorius
known as the SART system (Sartorius
Aseptic Rapid Transfer), see Figure 8.
This basically was an alpha/beta
type port where a sterile line capped
at the end by a special closure was
inserted and the special end closure
removed inside the isolator. Tubing from
the filling machine was attached to the
exposed entry tube after the removal of
the special closure. The asepsis of the
system when inserted into the port was
provided by knife edge seals similar to
the DPTE® seal system.
This type of system has now been
superseded by a Sartorius/Bosch
disposable filling line where the system
is pre-sterilised with filters in place and
is complete with balancing sections and
filling needles. It requires a special
peristaltic pump section, one pump
for each filling needle. Again an alpha/
beta port type of connection is used,
see Figure 8.
As mentioned previously containment
is required when compounding hazardous
products into tablet or injectable
form. Powder handling under such
Figure 10: Double valve system,
ChargePoint Technology, UK
www.cleanairandcontainment.com
circumstances requires special pass-
through systems and a common feature
is the use of sterile product in bulk,
held in a large vessel, connected to an
isolator for transfer through to a powder
filling system. In the early days, this
was a 25 kg sterile small container
(usually an antibiotic product) which
was connected manually via a simple
aseptic connection consisting of a large
diameter flexible tube. Later saw the
development of larger powder holding
vessels and special docking valve systems.
One example is the Charge Point
Pharmasafe® double valve, see Figure
10. It is in a sense the same concept as
an alpha/beta port but the components
are two parts of a single valve that,
when connected, form the whole valve.
This is then opened and product allowed
to flow through. Powder Systems Limited
also offers a similar design. The system
offers a very secure and safe way to
transfer hazardous powder product
with containment claims of < 0.1 µg/m3.
The transfer of product after being
aseptically filled in an isolator is usually
carried out by two methods:
1. Small batch sizes can be filled and
held in the isolator or an adjoining
isolator.
2. For larger batches the isolator
filling line is in a sense continuous
throughout the batch size.
In the second method, sterile
containers, usually from a dry heat tunnel
attached to the filling isolator are fed
onto the filling line. The product is filled
Figure 11: Two syringe box transfer isolators, Baxter Healthcare, USA
Clean Air and Containment Review | Issue 21 | January 2015
Main feature
and the container capped. The capped
container then exits through a small
aperture, known as the ‘mouse hole’ (see
previous articles in this series). Asepsis
at the ‘mouse hole’ was maintained by
the over pressure within the isolator
causing the air to exit at speeds of up to
2-3 meters per second. Some producers
also placed a small unidirectional air flow
unit over the exit of the ‘mouse hole’
This type of filling was adapted for
a wide range of sizes of bottles and vials
for various heat labile products and also
syringes filled with vaccines.
Syringes could be supplied through a
dry heat oven or via boxes of pre-sterilised
syringes. With the latter it was important
that the surfaces of the boxes were sterile
before entering the isolator and also
when the syringe ‘nest’ was placed back
into the box after the filling and stoppering
process. The exit was a modified ‘mouse
hole’.
Decontaminating the outer surface
of the syringe boxes was originally
accomplished by the use of large transfer
isolators where up to 100 boxes were
Figure 12: E-beam system for attaching
to an isolator, Getinge La Calhene
17
Main feature
Figure 13: UV system for stopper transfer
(outside of isolator), Millipore, USA
Figure 14: UV system for stopper transfer
(inside isolator), Millipore, USA
decontaminated with hydrogen peroxide
vapour, see Figure 11. The transfer
isolators were then connected to the
main filler isolator via a DPTE®.
A later development was the use of
e-beam technology to decontaminate
the exterior of the syringe boxes. This
method has the advantage of speed and
also simplicity. La Calhene successfully
developed a unit compatible with isolator
use, where three small e-beam units
were arranged around the conveyor
system so that all the external surfaces
of the syringe boxes were exposed to
a sterilising dose of electron radiation.
The syringe boxes were then moved
directly into the filling isolator. This
type of system, shown in Figure 12,
has also now been adopted by other
isolator manufacturers.
Entry and exit of containers,
components and equipment
Finally there is the introduction of
items into the aseptic filling isolator.
These mainly consist of filler containers,
components and also testing equipment.
The transfer of filler containers has been
described earlier but testing equipment
is usually wrapped and placed in the
isolator prior to a ‘sterilising’ process.
As particle counting is normally dealt
with by having in-built detection and
measuring systems, the main equipment
introduced is for microbiological testing.
18 Clean Air and Containment Review | Issue 21 | January 2015
The door with UV tubes was closed, the
cap of the bag introduced through the
circular opening and fixed in place. The
UV source was activated for 3 minutes
during which the surface of the cap was
bathed in UV radiation at about 1 to 2
mm. distance. The door was then
opened and the cap seal removed
allowing the contents of the bag to be
emptied into the feed hopper. The
system is shown in Figures 13 and 14..
Other methods of stopper transfer
evolved around a single large vessel filled
with stoppers and attached to a system
by which the stoppers were sterilised,
treated with silicone and dried. The
vessel was detached and moved to the
isolator where with a lifting device it was
up-ended and attached to the isolator,
the exposed section of the connection
was decontaminated during the cycle
Figure 15: Bioquell Port for rapid bio-
used for the isolator. Companies such as
decontamination transfers, Bioquell, UK ChargePoint Technology offer this type
of equipment.
The closure of aseptically filled vials Finally going back to the original
and bottles needs components such decontamination pass-through at the
as stoppers and caps plus plunger start of this paper and with the advent
plugs for syringes and, sometimes, of rapid sterilisation methods, various
the separate needles. small-pass through devices have been
The main method of transfer of developed where, using hydrogen
components is to use the RTP system peroxide vapour technology, items
and dedicated disposable plastic bags placed in the pass-through can be
for the pre-sterilised stoppers, caps decontaminated very rapidly, in as little
plugs and needles. The plastic beta ports as 20 minutes, depending on load. One
that are integral with these bags are also such device is shown in Figure 15.
disposable. Different types of chute Such ‘sterilisable’ pass-throughs are
devices inside the isolator allow direct now placed between two sterility testing
transfer into the feed hopper bowls. isolators and are used to introduce and
One unique transfer system, remove sterile items as and when required.
developed by Millipore, utilised intense Bioquell also offers a full size transfer
UV radiation technology. It required a isolator based on the same principle as
dedicated disposable bag of stoppers, described at the start of this paper.
etc. (pre-sterilised) fitted with a short
cylindrical sealed cap. On the isolator
was fitted a stretcher on which the bag i. Rapid Transfer Port Systems- A comparative
study by Getinge La Calhene: C.Mounier &
could rest opposite a small circular C.Guimet, Clean Air and Containment
opening. Inside the isolator was a small Review, Issue 20, October 2014, p.26-29
door fitted with 6 or 8 small UV tubes.
Doug Thorogood, Ph.D., studied microbiology and virology
in the UK, Belgium and the USA. He has many years’
experience in the field of pharmaceutical and medical research
as well as QA/QC Regulatory Affairs and Production. He
started working in the field of containment in the late 1970s
and from that point developed designs, validation procedures
and operational systems for a variety of isolators for sterility
testing and aseptic filling in 19 countries. He is a specialist in the cleaning and
sanitation of enclosures as well as clean rooms and hospital environments.
www.cleanairandcontainment.com
2021/3/4 A History of Isolator and Containment Technology, Part 5: Development and use of sterilising agents with associated devices - Sterilize

Mail: info@sterilize.it  – Linkedin: sterilize.it Member Area Registration

A History of Isolator and Containment Technology, Part 5: Search

Development and use of sterilising agents with associated
Enter your keyword
devices
Search …
Abstract
In this penultimate section the development of the ‘sterilisation’ of isolators Processes

used for sterility testing and aseptic processing will be reviewed. All Processes
This will include the chemical agents used, the equipment to deliver and
Applications
remove that agent (in a gaseous form), as well as agents used for  manual wet
processes and also fogging techniques with associated equipment. All Applications

  Author
Sterilising All
As mentioned in previous sections of this history the term ‘sterilising’ has
been used when related to the ‘sterility’ of isolators1 . A better term perhaps Search
would be ‘biological decontamination’ but the end result of any such
decontamination must be to achieve surfaces and other areas such as lters,
apertures and other items placed in the treated environment that should be
essentially free of microbial life (i.e. sterile).
Recent articles
While some of the agents that will be described have been reported as broad
A History of Isolator and
spectrum biocides, their action was initially tested and reported in the liquid
Containment

https://www.sterilize.it/applications/a-history-of-isolator-and-containment-technology-part-5-development-and-use-of-sterilising-agents-with-associated-devices/ 1/5
2021/3/4 A History of Isolator and Containment Technology, Part 5: Development and use of sterilising agents with associated devices - Sterilize

phase not as a gas or vapour. The sole exception is formaldehyde which has a Technology, Part 5:
long history as a vapour ‘sterilising’ agent for clean rooms, safety cabinets Development and use
and animal housing. Sterilisation methods in the realm of medical or of sterilising agents
pharmaceutical uses and which have regulatory approval are all described in with associated
some detail in the various pharmacopoeias. There are four main methods: devices
• Moist steam
A history of isolator and
• Dry heat
containment
• Irradiation (gamma & e-beam) technology Part 4:
• Ethylene oxide gas Transfer devices
As none of these have practical use for isolator sterilisation, other agents
were sought and these are listed below in an approximate order of their use. A history of isolator and

It would be appropriate to mention two other sterilising methods, as agents containment

used for instrument sterilisation have been used in isolators: technology Part 3: Non-
exible lm isolators
1. Low temperature with formaldehyde was used to rapidly sterilise certain
including RABS
instruments, at temperatures ranging from 50°C to 80°C utilising steam.
Getinge o ered a chamber device based on this type of treatment.
A history of isolator and
2. Gas plasma sterilisation where the chamber is lled with peracetic acid or containment
hydrogen peroxide vapour under vacuum. After a required exposure time technology Part 2:
radio frequency energy is applied to the chamber to induce the plasma state Flexible lm isolators
in which the active agent is broken down quickly into innocuous parts. This
technique is used mainly for instruments and one advantage is that the Importance of risk
instrument packs are dry at the end of the cycle. Such a steriliser is the assessment for aseptic
Sterrad® system. […] transfer in
A courtesy of ‘Clean Air & Containment Review’ – pharmaceutical
 www.cleanairandcontainment.com compounding

 author: Thorogood Doug

https://www.sterilize.it/applications/a-history-of-isolator-and-containment-technology-part-5-development-and-use-of-sterilising-agents-with-associated-devices/ 2/5
Main feature
A history of isolator and containment technology,
Part 6: Development of the validation of isolators
Doug Thorogood
Abstract
This final section is devoted to the
development of various validation
methods conforming to GMP when
applied to isolators and also a review
of the methods in relation to validating
the processes used in preparing for
aseptic processing and sterility testing.
There are brief comments on what the
regulators expect.
Early aspects
Validation, as far as the pharmaceutical
industry is concerned, started in the
1970s, initially in the field of large volume
parenterals. Up until that date no
organised regulatory approved validation
processes had been developed.
If we look at the isolator usage up to
that point, it focused on the rearing of
germ free animals, protection of patients
with compromised immunological
systems and also protection of staff in
treating patients with highly contagious
diseases.
Germ free animal technology
depended on HEPA filtered air and air
exchange rates with disinfection, either
manually or, later, by the use of gaseous
hƐĞƌƌĞƋƵŝƌĞŵĞŶƚƐ
;hZ^Ϳ
hƐƵĂůůLJƉƌĞƉĂƌĞĚďLJĐůŝĞŶƚ
&ƵŶĐƚŝŽŶĂůƐƉĞĐŝĨŝĐĂƚŝŽŶ
hƐƵĂůůLJƐƵƉƉůŝĞĚďLJ
ǀĞŶĚŽƌ
ĞƐŝŐŶĂŶĚďƵŝůĚ
ƐƉĞĐŝĨŝĐĂƚŝŽŶ
^ƵƉƉůŝĞĚďLJǀĞŶĚŽƌ
ƵŝůĚƉƌŽĚƵĐƚ
&ĂĐƚŽƌLJĂĐĐĞƉƚĂŶĐĞƚĞƐƚƐ
;&dͿ
&ŝŶĂůƐLJƐƚĞŵďƵŝůƚĂŶĚƚĞƐƚĞĚ
;ƚĞƐƚŝŶŐǀŝĞǁĞĚďLJĐůŝĞŶƚŽƌ
ƌĞƉƌĞƐĞŶƚĂƚŝǀĞŽĨĐůŝĞŶƚͿ
^ŝŐŶĞĚŽĨĨŽŶĂĐĐĞƉƚĂŶĐĞ
Figure 1: Typical pattern of an isolator validation pathway.
14 Clean Air and Containment Review | Issue 23 | July 2015
formalin or peracetic acid. These were
validated, in a sense, by the success or
failure of raising and holding germ free
animals in a germ free environment.
For protection of compromised patients
again reliance was placed on air filtration
and exchange rates plus appropriate
disinfection. Here some environmental
microbiological testing was undertaken,
essentially to demonstrate freedom of
bacteria and fungi.
For protection of operator, when
dealing with seriously infected patients,
there were barrier nursing techniques
and also the use of validated disinfectants
for decontaminating any items removed
from the environment of the isolator
and for the washing of the nursing staff
as a routine.
Initial validation attempts
With the introduction of isolators into
the pharmaceutical industry (circa
late 1970) for sterility testing and then
aseptic processing, the focus of
the validation centred on tests
such as freedom of leaks (positive
pressure profile), efficiency of the
biodecontamination (sterilisation)
WĞƌĨŽƌŵĂŶĐĞƋƵĂůŝĨŝĐĂƚŝŽŶ
;WYͿ
KƉĞƌĂƚŝŽŶĂůƋƵĂůŝĨŝĐĂƚŝŽŶ
;KYͿ
/ŶƐƚĂůůĂƚŝŽŶƋƵĂůŝĨŝĐĂƚŝŽŶ
;/YͿ
WƌŽĚƵĐƚƐŚŝƉƉĞĚƚŽĐůŝĞŶƚƐŝƚĞ
^ŝƚĞĂĐĐĞƉƚĂŶĐĞƚĞƐƚ
;^dͿ
process and the beginning of the
now established installation and
operational qualification process.
When the validation of isolators was
first contemplated, previous applications
for aseptic processing and sterility testing
were examined:
1. Those based on the use and
function of safety cabinets
2. Those based on the use and function
of ethylene oxide sterilisation in
a dedicated chamber.
3. Those based on conventional
clean room aseptic processing.
It was thought possible to take some
of the validation test procedures used by
some or all of the three applications
above and blend into a validation package
for sterility testing and for aseptic
processing.
Where there was a need for
containment but not sterility the focus
was upon the ability to validate that no
dangerous active chemical agent could
escape from the isolator (usually by
using negative pressure profiles) and to
ensure that the exposure to personnel
operating the process was within the
safe prescribed exposure limits.
What eventually evolved was the
classic V-shaped approach for the
validation process for isolators (see
Figure 1) that had been developed after
1970 for pharmaceutical manufacturing
and testing equipment.
Quality risk management process
Another aspect that was added was the
development of risk analysis in order to
identify any of the problems that may
be encountered during the qualification
processes and to identify the risks
requiring validation (see Figure 2).
This was a very useful tool to bring
together representatives of Production,
QC, QA and Engineering to debate and
agree the problems that may be found
during the testing programmes and to
decide the actions required for
validation with the acceptance criteria.
www.cleanairandcontainment.com

Previous manufacturing
and testing processes
Sterility testing, in general, had been
performed in conventional safety cabinets
using a unidirectional air flow for asepsis
during the testing and a robust training
programme to validate the operators.
Physical validation included airflow rate
and direction and also the cleaning and
disinfection of the environment within the
cabinet prior to gassing with formaldehyde
vapour. The latter required the use
of a biological indicator to determine
the effect of the formaldehyde cycle.
The issue of using biological indicators
during the validation of isolators also
arose at that time (mid-1970s) from the
established validation techniques used
in ethylene oxide sterilisation processes.
Here the biological indicator was used
to test the combined effect of temperature,
humidity, vacuum, time of exposure and
gas concentration that are the essential
aspects for an effective and reproducible
ethylene oxide sterilisation cycle. It
was reasoned that the same biological
indicator approach (and model) could
be used for the sterilisation of isolators.
As the sterilisation cycle could be observed
(most isolators were transparent to some
degree) it was thought that some form
/ŶŝƚŝĂƚĞ
YƵĂůŝƚLJZŝƐŬDĂŶĂŐĞŵĞŶƚWƌŽĐĞƐƐ
ZŝƐŬƐƐĞƐƐŵĞŶƚ
ZŝƐŬ/ĚĞŶƚŝĨŝĐĂƚŝŽŶ
ZŝƐŬŶĂůLJƐŝƐ
ZŝƐŬǀĂůƵĂƚŝŽŶ
ZŝƐŬŽŵŵƵŶŝĐĂƚŝŽŶ
ZŝƐŬŽŶƚƌŽů
ZŝƐŬZĞĚƵĐƚŝŽŶ
ZŝƐŬĐĐĞƉƚĂŶĐĞ
KƵƚƉƵƚͬZĞƐƵůƚŽĨƚŚĞ
YƵĂůŝƚLJZŝƐŬDĂŶĂŐĞŵĞŶƚWƌŽĐĞƐƐ
ZŝƐŬZĞǀŝĞǁ
ZĞǀŝĞǁǀĞŶƚƐ
Figure 2: Quality Risk Management Process
www.cleanairandcontainment.com
of chemical indicator would be useful in
observing the presence of the sterilising
agent as well as its distribution.
By the mid-1980s several isolators
had been built (mainly by La Calhene).
The Rapid Transfer Port (RTP) was
introduced in these and also early versions
of gassing devices using mainly peracetic
acid. Much debate evolved around
the validation of the RTP and also the
integrity of the gloves used on the isolator.
In the late 1980s Amsco (now Steris),
actively marketing La Calhene isolators
in the USA, introduced a hydrogen
peroxide gas generator. This technology
led to the use of chemical indicators,
other types of biological indicators and
eventually methods to measure peroxide
concentrations inside an isolator.
Later the use of chlorine dioxide was
also offered by Johnson & Johnson
as an alternative to hydrogen peroxide.
Validation from the 1980s
It is now perhaps appropriate to examine
the history of the various validation tools
used and the results obtained, especially
for the validation of sterility test isolators
and various types of aseptic processing
isolators. It is assumed that the correct
installation qualification studies had
ZŝƐŬDĂŶĂŐĞŵĞŶƚdŽŽůƐ
ƵŶĂĐĐĞƉƚĂďůĞ
Clean Air and Containment Review | Issue 23 | July 2015 15
Main feature
been completed satisfactorily. Many
of the tests described below were used
both in the operational and the final
process qualification process.
1. Pressure hold tests
As almost all of the isolators used for
sterility testing and aseptic processing
were used in a positive pressure profile,
usually 25 to 50 Pa above ambient
pressure, some form of pressure holding
or leak test was utilised. In most cases,
for the test, the unit was over-pressured
to around 250-300 Pa and the decay
rate was measured over time. This was
a problem for flexible film isolators as
changes in the ambient pressure and
temperature affected the decay rate.
However a major leak could be detected
by the rapid loss of pressure.
Later models of aseptic processing
isolators had a pressure hold test built
into the computer control system so that
a sterilisation cycle could not start if the
unit failed the leak test.
To detect a leak initially, ammonia
fumes were used, either from a cylinder
of ammonia vapour or from a dish of
0.880 ammonia placed inside the isolator
filling the volume with ammonia vapour.
Over-pressure was applied and a pH
sensitive cloth (yellow in colour) was
passed over the surface of the isolator,
especially around the RTP and the
various seals on the unit. A leak of
ammonia was detected by the cloth
changing colour to blue.
A more searching test was to inject
helium into the isolator and scan with
a helium detector that gave an audible
signal if helium was found to be leaking
out. It was found with both test methods
that leaks in gloves could also be detected.
Helium was eventually used mainly for
detecting leaks from RTPs and around
the seals of penetrations for equipment
in the isolator.
Gloves and gauntlets present certain
challenges with regard to leaks and leak
testing. The earlier types of gloves used
were based on latex composition and
worked well with formaldehyde but due
to the presence of peroxide in peracetic
acid it was found that the latex became
sticky and also degraded causing leaks.
Various other compositions appeared
which included vinyl and Hypalon.
The latter composition worked well with
peroxide vapour and is now the main
form of glove and gauntlet composition
offered and used. Sleeves were made of
Main feature
PVC and also polyurethane with a cuff
system where the glove could be attached
and if required changed safely without
compromising the sterility of the isolator.
Glove leak testing covers a wide
range of methods but the most common
test today is an automatic pressure hold
test either on a single glove in situ or a
test system that can encompass several
glove fittings at the same time. Edwards i
gives a useful review of test methods
and observed that trained operators
were better at detecting leaks visually
than the testing methods described.
2. Air exchange rates and airflow rates
The conventional clean room specification
of a minimum of 20 air changes per hour
was targeted as a goal in early flexible
film isolators fitted with an inlet and
exhaust filter in the ceiling of the unit.
The air pattern inside was seen to be
mainly turbulent but with some direct
streaming from the inlet to the exhaust
filter. This led to lengthy sterilisation
cycles, as the vapour only circulated
slowly around the inside of the isolator,
and was resolved by the introduction of
internal fans for aiding the distribution
(see later). Also an inlet manifold was
used to bring the entry of the vapour to
be base of the isolator to encourage a
better gas distribution. Many sterility
test isolators used one of these methods
of gas distribution i.e. internal fans or a
different entry mode for the gas.
With the introduction of the rigid
type of isolator for aseptic processing,
the use of unidirectional air flow was
considered and adopted because that
is how it was for clean rooms. The target
for airflow velocities was usually the
norm of 0.45 m/second ±10% with air
exchange rates of not less than 20 per
hour. Thus many isolators of this type
had a ceiling of HEPA filters to achieve
the desired effect through the entire
volume of the isolator. The air return
systems were varied in design but it was
found that very high rates of air
exchange were achieved , especially in
the small sterilisable transfer chambers,
used to introduce items into the main
isolator, where such air exchange rates
exceeded over 500 to 800 per hour
With the introduction of Restricted
Access Barrier Systems (RABS), in their
earlier forms, reliance was still placed
on unidirectional air flow but the physical
barrier between the aseptic process
and the operators gave an additional
16 Clean Air and Containment Review | Issue 23 | July 2015
advantage. Gloves fitted to glove ports
were utilised for any manipulations
required, although these are rated as
‘intrusions’ in the scoring system for
safe aseptic filling. Later forms of RABS
were totally enclosed and could be
biologically decontaminated.
Current thinking is directed back
to turbulent airflow over aseptic filling
areas within a process isolator on the
premise of low particle generation, high
air exchange rates and easier and cheaper
design for the isolator, plus the additional
advantage of distribution of the sterilising
vapour. These considerations would lead
to smaller more compact isolators that
are easier to clean and disinfect prior
to sterilising.
3. Pressure and temperature
For aseptic processing and sterility
testing the pressure in the isolator has
always been positive and greater than
the environment surrounding the unit.
Generally the trend was to follow the
classic pressure cascade by being higher
in the aseptic area (Grade A) than in the
surrounding environment, either Grade
B, C or D, and the environmental pressure
higher than in the areas external to areas
surrounding the isolator. Generally the
area surrounding the isolator for sterility
testing was Grade C as it was for some
aseptic processing units, but many
isolators were, and still are, sited in
Grade D areas. Usually the positive
pressure in the isolator was between
25 and 50 Pa above the ambient
pressure. Any value higher than 50 Pa
tended to make hand and arm insertion
into the glove and sleeve difficult.
Temperature was also considered
and, depending on the process was
usually set at 18 to 20°C, although
in later aseptic processing models
the temperature was raised as part
of the sterilising cycle. Where a low
temperature was necessary, e.g. for
aseptic compounding of cytotoxic or
antibiotic drugs, a refrigerator was built
into the isolator for storage of the drugs
and the compounded final product.
Temperature studies were also
required when contemplating sterilisation
with hydrogen peroxide in order to locate
potential cold spots and to validate a
reasonable degree of temperature stability
and value, usually within 2 to 5°C for
each thermocouple.
4. Chemical indicators
With the introduction of peracetic acid
as a sterilant, the indication of the acid
vapour being present was based on
litmus paper, the blue paper changing
to red. Later a peracetic acid dip stick
(Merck) was used but these were only
bleached to a beige colour and in both
cases the test was qualitative. The dip
stick method required moisture and the
early method of evaporation of peracetic
acid (40-50°C.) precluded the presence
of water. Later spraying or aerosol
techniques with peracetic acid showed
the dip stick changing to a deep blue
colour from white very quickly.
Amsco (Steris) introduced a chemical
indicator (VHP® Indicator) for hydrogen
peroxide vapour which worked well but
over time faded so it was not possible
to retain them in batch records. A later
version (Steraffirm®) overcame this
problem and also included a reference
colour section on each strip. The test
was based on a beige colour turning to
blue when exposed to peroxide vapour.
Other test strips for peroxide were
developed e.g. Bioquell HPV-CI®.
The strips are not quantitative but
are used to show the presence of the
vapour. They were also used for peracetic
acid ii. The test strips were very useful
to indicate not only the presence of the
sterilising agent but also to roughly
determine the distribution of the vapour
in the isolator. As isolators became
larger in design, with additional
equipment placed inside such as filling
machines, gas distribution became an
important facet of the validation process.
Initially the gas entry was usually from
one point above an inlet filter but later
developments found the gas being
introduced from a point under the inlet
filters. In both cases reliance was placed
on the air flow through the HEPA filters
and the rate of flow had to be reduced
to around 0.1 m/s to avoid gas streaming
and also to encourage turbulence.
Other devices to aid distribution
were a rotating nozzle inside the isolator
(Bioquell) and multipoint entry across
the length and breadth of the isolator.
However, even in sophisticated isolators
with excellent unidirectional air flow,
the use of small fans to distribute the
gassing agent was sometimes required,
particularly when the isolators were
attached to product incoming conveyors
and exit sections with automatic opening
and closing doors.
www.cleanairandcontainment.com

5. Biological indicators (BIs)
For early validation studies to determine
the reproducibility of a sterilisation cycle
a typical biological indicator for ethylene
oxide was chosen. These were paper
strips inoculated with spores of Bacillus
atrophaeus, formerly Bacillus subtilis var.
niger (ATCC 9372, NCTC 10073 and
NCIMB 8058). These BIs proved to be
successful when using peracetic acid but
required some humidity to be present.
Also as the active agent adsorbed into
the paper strip a suitable inactivator was
required to be added to the recovery media.
Amsco (Steris), as manufacturers of
BIs, tested a wide range of them against
hydrogen peroxide and suggested that
among the most resistant a suitable
validation model would be Geobacillus
stearothermophilus (ATCC 7953 or ATCC
12980, NCTC 10007). This type of BI
was made by inoculating a suspension of
spores onto a small saucer shaped
stainless steel disc that was then enclosed
in a Tyvek envelope iii. Initially Amsco
prepared a 105 population of spores on
the carrier and stated that the Tyvek
barrier represented a 101 resistance
Image 1: G. stearothermophilus spores
encased in thick media components on
a commercially available BI
Image 3: Areas of dense microbial load
on a commercially available BI
Figure 3: Images of various defective BIs, courtesy of Bioquell (these images are not of Bioquell products)
www.cleanairandcontainment.com
equivalence thus totally the carrier
represented a 106 challenge. It is believed
that the 106 challenge was adopted as
this was used for conventional steam
sterilisation cycles.
Subsequent studies showed that this
approach was erroneous and that the
Tyvek did not represent any challenge
at all and thus it became the norm to
use 106 spore carriers in Tyvek.
Other carriers for the spores were
examined and reported on iv. These
included glass, plastic materials and
materials used for the manufacture of
the gloves. Variable results were cited
using hydrogen peroxide vapour as the
sterilising agent as it was thought that
these carrier materials may influence
the response to the sterilising effect.
The variation may have been due to
the preparation of the carrier material
and also the density and purity of the
spore suspension used. See comments
on rogue BIs later.
Over the years D-values for hydrogen
peroxide vapour concentrations have
been cited but, as discussed by Agalloco
and Akers v, there is no method available
Image 2: Uneven surface finish of carrier
disc causing formation of spore aggregates
on a commercially available BI
Image 4: Areas of dense and uneven
microbial load (several layers thick)
on a commercially available BI
Clean Air and Containment Review | Issue 23 | July 2015 17
Main feature
to determine the D-value under a state
of fixed parameters comparable with
a Bier vessel for steam sterilisation
D-values. In vaporising hydrogen peroxide
there develops a melange of air, water
vapour and hydrogen peroxide vapour
that is usually changing over time.
The changes are under the influence
of temperature, humidity and the
concentration of hydrogen peroxide
so that it is difficult to fix an exact value
for these various parameters.
Many tests have been made by
exposing multiple BIs inside the isolator
and removing them at timed intervals
in order to determine a D-value. This
is known as the Limited Spearman
Karber Method.
In many cases the measured gas
concentration of peroxide is in excess
of 1 mg/Litre (720 parts per million)
and is usually regarded as an overkill
concentration. That value only exists for
that particular time and environmental
conditions and there is no guarantee that
the next day or week the circumstances
will be exactly the same. Agalloco et al
also mention the desirability of attaining
a state inside the isolator where
micro-condensation can take place,
as it is suggested that there would then
be a very high concentration of peroxide
developing on surfaces (around 70%)
which results in rapid and effective
sterilisation. They also suggest using
BIs with a lower population count of
circa 104 as an example. The reason for
this comment is based on two factors:
1. In a cleaned isolator the challenge
to the sterilisation cycle would be
extremely low. The author of this
paper found, over the years, that
in a properly cleaned isolator, the
Image 5: Even distribution of clean
G. stearothermophilus spores across
a stainless steel carrier disc
Main feature

environmental air and surface

counts were always <10 cfu/ft3 or
<10 cfu/25cm2 respectively and the
organisms identified were very
susceptible to peracetic acid, hydrogen
peroxide and chlorine dioxide. In
many cases the actual count was
extremely low. Any spore bearing
isolates, and these were few, were
fungal in origin and again very
susceptible to the sterilising agents.

2. In the late 1980s and early 1990s

there appeared a number of BIs on
the market dedicated to use with
hydrogen peroxide. Many users
found inexplicable results with many
positive growth events occurring
when none was expected. A group
in the UK in the 1990s started to
investigate the problem and found,
using scanning electron microscopy,
Figure 4: Guided Wave’s bench top Model 412 Fiber Optic Spectrometer that many of the BIs offered were
substandard by way of the presence
of gross organic matter (media),
actual vegetative examples of the
spore source used and the piling up
of spores in clumps or layers that
defied penetration of the sterilant
vapour. The stainless steel carriers
were also found in some cases to be
scratched and allowed spores to
build up in the scratches. The images
in Figure 3 illustrate these problems.

These findings led to a plea to the

manufacturers to improve their
quality and from this there emerged BI
manufacturers with a good reputation
for reproducibility and quality such as
Bioquell with their HPV-BI. A number
of these manufacturers have now been
brought into Mesa Labs, Inc. in the USA.
Nevertheless a lot of effort was made
to account for the rogue BIs that may be

Figure 6: Dräger Sensor ® H 2O2LC for

Dräger Polytron 7000 gas detector,
Figure 5: Dräger Polytron 7000 gas detector, copyright Dräger 2015 copyright Dräger 2015

18 Clean Air and Containment Review | Issue 23 | July 2015 www.cleanairandcontainment.com


present in a batch. Much elaborate testing
was done to determine if a batch or lot
would be acceptable and a theory evolved
to allow for a certain number of positive
growth BIs in a sterilisation cycle vi.
Furthermore, based upon this theory,
it has been suggested that cycle
development should be carried to using
three BIs per site in the isolator.
Prior to the problems encountered
with BIs used for hydrogen peroxide
decontamination the acceptance criteria
was that all BIs placed in the isolator
would be rendered sterile and this was
the norm for regulators. Today it appears
that under the new regime of using
multiple BIs at each test point a number
of positive growth samples would
be permitted.
Now there are also theories with
regard to allowing a cycle to reach as
close to condensation as possible so that
a very low level of hydrogen peroxide
concentration (circa 250 to 350 ppm)
would be very effective, especially if
using a low challenge of a BI population
such as 104 which may remove many
of the rogue BI issues. This may be
an aspect worth pursuing as this
phenomenon was found by the author
in the late 1980s with surprising but
gratifying results.
Figure 7: Dräger hand operated vacuum device for gas sampling tubes, copyright Dräger 2015
www.cleanairandcontainment.com
6. Instruments for measuring
gassing agents
In the early period of peracetic acid
sterilisation the only method available
to measure gas concentration was to
withdraw a large volume of the air
in the isolator (100 to 200 ml volume)
and inject it into water and perform a
titration. This method is obviously full
of potential errors but it gave a rough
indication of the concentration of
peracetic acid in the isolator.
With the introduction of hydrogen
peroxide a number of instruments
were tested and the most accurate
device was found to be an infra-red
spectrophotometer. This device
had the additional advantage of
also measuring the concentration
of water vapour present.
The unit consisted of a
spectrophotometer with fibre optic
cables for positioning a light path unit
inside the isolator. Whilst the device
was accurate to 0.1 mg/l peroxide, the
light path unit could not be moved
during the cycle so that measurements
from various sites within the isolator
meant repeat cycles. The fibre optic
cables were also subject to inadvertent
damage. An important aspect of this
device is that it can be calibrated at the
start of each cycle.
Clean Air and Containment Review | Issue 23 | July 2015 19
Main feature
Other options focused on electro-
chemical method devices marketed by
Dräger, Bioquell and ATI. The early
models were calibrated against sulphur
dioxide but recent models are now
calibrated against hydrogen peroxide.
Dräger offers an electrochemical
sensing device with direct readout (see
Figure 5) and peroxide sensor heads
with ranges of 0-1ppm up to 300 ppm
(LC) and 0-1,000ppm up to 7,000 ppm
(HC). The LC sensor head (see Figure 6)
has a default of 5 ppm and the HC
sensor head 4,000 ppm. Many isolator
systems now have this type of unit built
in and by using a small vacuum pump
vapour from inside the isolator can be
drawn over the sensor head. Readings
can be sent direct to the control panel
of the isolator and also saved for
record purposes.
ATI offer a similar unit and, when
fitted with the low level range of peroxide
(0-20 ppm), either the Dräger unit or
the ATI unit can be mounted in the
room surrounding the isolator as a guard
against peroxide leakage into the area.
The calibration of these devices is
usually by replacing the sensor head
with a calibrated one and sending
the old head back to the supplier for
re-calibration or re-calibrating in-situ.
The re-calibration interval depends
largely on the frequency of use and also
the concentration of peroxide measured.
The Guided Wave unit is expensive
and is usually used to establish a desired
concentration of peroxide vapour during
cycle development.
The cost of the electro-chemical type
of measuring unit is reasonable and, with
the sensor mounted on a long cable to the
recording unit, it also has the advantage
that it can be moved around the inside
of the isolator. The main advantage of
the Dräger and ATI systems is that the
cycle can be recorded each time and
the values compared with the original
validated and approved cycle so that
a degree of reproducibility is possible.
7. Aeration
Having gassed the isolator, enclosure or
room the next stage was to remove the
sterilising agent.
When formaldehyde was the
sterilising agent, ammonia vapour
was used to neutralise it and the
air-handling system was used to
purge the neutralised gas. This method
Main feature

can create a difficult to remove white
deposit of paraformaldehyde.
With the advent of peracetic acid the
conundrum was the residual smell of
acetic acid and many users used a low
level of acetic acid as a marker for
indicating that the enclosure had been
sufficiently aerated.
There was much investigation
into the potential effects of residual
acetic acid in sterility testing and
the manufacture of aseptically filled
product but it was found that the
effect was negligible.
In using hydrogen peroxide, the
hydrogen peroxide acceptance level for
aeration was 1 ppm. Again there were
instances of much testing of product
and of sterility tests when reaching a
The problem of using electro-chemical
sensor devices for this purpose was
that the high sensitivity unit would
be overloaded if exposed to the high
concentrations of a normal peroxide
gassing cycle and give erroneous
readings.
Some safety concerns have been
expressed for workers in isolator rooms
being exposed to peroxide vapour and
a limit of 0.5 to 1ppm per time weighted
average has been adopted and the
devices described can be used for this
purpose too.
Due to the sensitivity of certain
products, aeration acceptance criteria
could be as low as 0.1 ppm peroxide
vapour and in this case other forms
of instrumentation are required. The
microbiology laboratories. There is
a reference to barrier technology in
their quality assurance manual xi, xii.
References
i. Edwards L. Glove Leak Testing in Isolators
and Rabs. ISPE Isolator & Barrier Conference.
USA, 25th February 2014
ii. Depaquy C. et al. CHI Poissy, Saint
Germaine-en-Laye, France. Hydrogen
peroxide indicators for validation of isolator
sterilization with peracetic acid
iii. Rickloff J.R. Key Aspects of Validating
Hydrogen Peroxide Gas Cycles in Isolator
Systems. J of Validation Technology,
5:61-71,1998
iv. Sigwarth et al. Effect of carrier materials on
the resistance of Bacillus stearothermophilus
to gaseous hydrogen peroxide. PDA Journal of
Pharmaceutical Science and Technology. July/
August 2007, 61 ($), 255-275

level of 10 ppm as it appeared to take
a very long time to reach 1 ppm or
even lower without the use of catalytic
converters in the air handling system.
Some mobile isolator units not
attached to an exhaust system were
equipped with catalysts mounted after
the exhaust filters and these proved to
be extremely effective for allowing the
aeration phase to be performed in the
room where the isolators were situated.
No peroxide was detected in the room
and the aeration phase was of a reasonable
time (45 minutes) for processing purposes.
Dräger’s gas sampling tubes for
hydrogen peroxide are often used to
spot test atmospheres for the presence
of hydrogen peroxide. The tubes are
used in conjunction with Dräger’s hand
operated vacuum device (see Figure 7).
To use the Dräger sampling system,
the ends of the tube are removed and
the non-calibrated end is inserted into
the hand operated vacuum pump device.
The open end of the tube is inserted into
the isolator (via a convenient dedicated
inlet tube). The hand device is pumped
20 times (equal to 1 litre volume). The
peroxide is drawn over the chemicals
in the tube (it is based on an iodine/
iodide reaction) and the brown colour
that develops is read against the
graduations that are marked 0 to 3 ppm.
The air drawn into the tube has to have
a moderate degree of relative humidity
for the reaction to take place. Dry air
leads to a yellow colour. Dräger can also
supply an electrically operated pump,
the Dräger X-act® 5000, which is an
automatic version of the handheld pump.
Picarro hydrogen peroxide sensor has v. Agalloco P. & Akers J.E. Overcoming
Limitations of Vaporized Hydrogen Peroxide.
been found to be useful for developing Pharmaceutical Technology, September 2, 2013
aeration times requiring an acceptance vi. Templeton P. et al. A Strategy for Cycle
criteria of <0.1 ppm. Development, Validation and Re-validation.
Proceedings of the 14th Annual Barrier
Isolation Forum. Washington, USA, June 2005
8. GMP issues and the regulators
There are various Regulatory Authorities vii. EudraLex. The Rules Governing Medicinal
Products in the European Community: Annex
and their comments with regard to GMP 1. European Commission, 2009
that affect the design and use of isolators.
viii. PIC/S. Recommendation: Isolators used for
1. In the EU GMP Guide isolators are Aseptic processing and Sterility Testing: PI
014-3. Pharmaceutical Inspection
referenced in Annex 1 vii.
Convention/Pharmaceutical Inspection
2. PIC/S has references to isolators Co-operation Scheme. Geneva,
Switzerland,2007
in their inspectorate guidelines.
ix. US Department of Health and Human
Services, Food and Drug Administration.
3. FDA Aseptic Filling guidance also
Guidance for Industry: Sterile drug products
includes sections on isolators ix. produced by aseptic processing – current
Good Manufacturing Practice. 2004, Code of
4. There is no specific reference Federal Regulations. Sterility Testing. 610.12
to isolators in the European Federal Register/Vol 76, No.119/June 21, 2011
Pharmacopeia although a reference x. US Pharmacopeial Convention. General
Chapter <1208> Sterility Testing – Validation
is made in the biological indicator of Isolator Systems, USP 30-NF 25
section in Chapter 5 x. xi. WHO. Good Practices for Pharmaceutical
Microbiology Laboratories. WHO Technical
5. World Health Organisation (WHO) Report Series No.961. World Health
has no GMP documentation for Organisation, Geneva, Switzerland, 2011
isolators but there is reference to xii. WHO. Quality Assurance of Pharmaceuticals.
A compendium of guidelines and related
using them for sterility testing in
materials, Volume 2 Good Manufacturing
their guide for pharmaceutical Practices and Inspection. World Health
Organisation, Geneva, Switzerland, 2007
Doug Thorogood, Ph.D., studied microbiology and virology
in the UK, Belgium and the USA. He has many years’
experience in the field of pharmaceutical and medical research
as well as QA/QC Regulatory Affairs and Production. He
started working in the field of containment in the late 1970s
and from that point developed designs, validation procedures
and operational systems for a variety of isolators for sterility
testing and aseptic filling in 19 countries. He is a specialist in the cleaning and
sanitation of enclosures as well as clean rooms and hospital environments.

20 Clean Air and Containment Review | Issue 23 | July 2015 www.cleanairandcontainment.com