FOCUS ON...
DISPOSABLES
Reducing Microbial Contamination
Risk in Biotherapeutic Manufacturing
Validation of Sterile Connections
Christopher J. Mach and Donna Riedman
T
he risk of contamination
(especially microbiological) is
always an area for special
attention in biopharmaceutical
processes. No matter the process stage,
whether upstream of a bioreactor or in
the final filling of a sterile product,
effective contamination control
continues to be a critical requirement,
so any opportunities for improvement
may justify further investigation. Even
with established validated processes,
demands for higher purity and
increased sterility assurance may
require manufacturers to reassess their
procedures and technologies. New
processes present an even greater
opportunity to introduce innovative,
enhanced technologies that can
improve contamination control to
satisfy regulatory requirements not only
today but in the foreseeable future.
Introducing a new technology into
a process may, however, bring concern
over possibly time-consuming,
demanding process qualification and
validation studies and ultimate
regulatory approval. We address these
issues by using as an example new
technologies that can reduce
contamination risk when making
aseptic connections. Here, validation
data on a single-use aseptic connector
is presented based on an extreme
interpretation of worst-case
20 BioProcess International September 2008
conditions, to confirm the effective
control of sterility during connection.
Core validation data of this type can
help make the introduction of
enhanced technologies easier, faster,
and safer.
Disposable Connectors
and Fittings
Almost every biopharmaceutical
www.pall.com
process involves making sterile
connections between fluid pathways.
Such connections may be part of
relatively simple systems, such as a
sampling point for quality control
assays or a link from a sterile fluid
container to a filling lines.
Traditionally, reusable fittings have
been used, particularly those made of
stainless steel, but the recent trend has www.pall.com
been toward single-use disposable
connectors. In some cases, connectors assurance and reduced cross-
may be integral to more complex, contamination (1). Before embarking
single-use systems, often involving on any qualification and validation
flexible bags, pre- and sterilizing or program for disposable connection
virus-removal capsule filters, devices, it is important to recognize
chromatography purification capsules, the major features of such devices and
tangential-flow filtration modules, how they may or may not be suitable
bioreactors, and so on. for a specific process.
According to the Bioplan Major Features of Disposable
Associates’ 2006 survey of Connectors: It is not our purpose here
biopharmaceutical manufacturing, to review in detail the range of single-
two primary reasons stated by use connection products currently
biopharmaceutical manufacturers for available. It is, however, important to
using disposable systems were sterility recognize their features that can
provide benefits in contamination
control and would influence the
content of a validation program.
These connectors typically incorporate
the practical benefits of quick-connect
fittings, such as speed and ease of use.
However, a unique feature (and one of
the most important) to some recent
developments is elimination of the
need for connection under Grade A
(Class 100, ISO 5) laminar-flow
conditions. The validation approach
described here is specific to these
types of aseptic or sterile connectors
and focuses exclusively on controlling
sterility of the fluid pathway.
Aseptic/Sterile Connectors
and Connections
Much discussion has occurred about
the semantic distinction between the
terms aseptic and sterile in regards to
connectors and connections. Aseptic
is defined as “(a), free of pathogenic
microorganisms, as in aseptic
surgical instruments,” and “(b) using
methods to protect against infection
by pathogenic microorganisms: as in
aseptic surgical techniques.” Sterile
is defined as “free from live bacteria
or other microorganisms, as in a
sterile operating area; sterile
instruments” (2).
Although the dictionary definition
of aseptic leaves open the possible
presence of nonpathogenic
microorganisms (hence nonsterility),
in practice the term is often applied
interchangeably with sterile to describe
the process of connecting two sterile
components in a controlled (not
necessarily sterile) environment, such
that sterility of their interconnected
pathways is maintained. In this sense,
the two terms are used synonymously.
As a means of distinguishing them,
we can think of aseptic as describing
the process of making a connection
that can be validated as being sterile.
So aseptic refers to the connection
process, whereas sterile refers to the
validated connection result.
The ability of disposable
connection devices to make sterile
connections in less-controlled
environments places a strong
dependence on the sterile barrier
within the connector and on the
22 BioProcess International September 2008
Table 1:  Summary of qualification test methods for single-use connectors per BPSA component
quality test guide
Test Type General Description Reference Source
Bacterial challenge and Identifies bacteria’s ability to breach a seal Connector
soiling test manufacturer
Biological reactivity in vitro Evaluates response of mammalian cell ISO, USP, or EN
cultures to extracts of polymeric materials
Biological reactivity in vivo Evaluates biological reactivity of animals USP or ISO
to polymeric material or interaction of
medical devices with blood
Particulate matter Evaluates presence of particulates in or on USP or ANSI/AAMI
a sample
Physicochemical tests Measures the physical and chemical USP
properties of plastics and their extracts
Water flow rate and Measures flow through connector at ISO
pressure drop tests differential pressure
Sterilization process Confirmation of connector manufacturer’s Connector
compatibility specified performance claims after manufacturer
sterilization process
Figure 1:  Simplified schematic for bacterial soiling test on disposable connectors
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availability of validation data to validation program can now draw
demonstrate the security of this upon several sources.
alternative technology. Taking these Bio-Process Systems Alliance
concepts and features into account, we (BPSA) Guides: Established in 2006 by
must consider how this new the Society of the Plastics Industry to
technology can be validated to meet the challenges of emerging
demonstrate that it will consistently single-use bioprocesses, the BPSA has
maintain a sterile fluid pathway. issued a guide on qualification test
methods for connectors (3). This
Validation Approach guide provides a good starting point
At first sight, the validation approach for a validation program, because it
may seem daunting to potential users describes not only the types of tests
of disposable connectors, especially in that may be appropriate but also the
companies currently using traditional, sources of reference tests (e.g, ISO,
manual aseptic connection methods ASTM, USP). Table 1 summarizes
for validated, sterile processes — as the qualification tests for connectors
well as people who are unfamiliar referenced in the BPSA guide.
with new technologies. The reality is Core Validation Data from
that biopharmaceutical companies, Connector Manufacturers: A
suppliers, and organizations have substantial amount of data is now
addressed this task in recent years available from connector
with considerable vigor and speed, manufacturers to demonstrate the
resulting in publication of industry efficacy and safety of their products.
guides, recommended test methods, Summarized in supplier validation
and generically applicable validation guides, these methods and results can
data. The basic framework of a form a significant part of the
Table 2:  Serratia marcescens aerosol challenge test on Kleenpak connectors
Total Average in
Aerosolized Settle Dishes
Assay (cfu/ft3) (CFU/mL/dish)
1 3.9 × 104 3.4 × 102
2 9.8 × 104 7.8 × 102
3 7.2 × 104 7.5 × 102
4 5.3 × 104 5.3 × 102
validation support users submit to
regulatory authorities.
Process-Specific Validation Data:
To help confirm the suitability of a
connector in a given process, vendor-
supplied data may be supplemented by
additional validation tests, often
performed in conjunction with the
connector manufacturer. In many
processes, however, only a limited
number of tests may be required: e.g.,
when simple aqueous fluids are
involved or connectors form part of a
presterilized disposable system with its
own validation support. In such cases,
a supplier’s process qualification data
can form the major part of the
validation package.
A speedy and successful validation
is achievable by
• following recommendations issued
by connector manufacturers and
organizations such as the BPSA
• using appropriate reference tests
• working closely with connector
manufacturers and their core
validation data.
Typical Validation Data
The following examples provide
insight into two of the most
demanding tests of sterility assurance
for a connector. The product tested
was a Pall Kleenpak connector, a two-
component connector that incorporates
protective barrier peel-away strips. It is

EJTIFT 
sterilizable by gamma irradiation or
autoclave. After sterilization, the
barrier strips protect the sterile fluid
pathway before and during connection.
And an irreversible locking mechanism
prevents reopening.
Two functional bacterial challenge
tests were performed on these
connectors: a liquid challenge soiling
test using Geobacillus stearothermophilus
and an aerosol challenge test using
Serratia marcescens. These tests were
24 BioProcess International September 2008
guide (4). Figure 1 shows simplified
S. marcescens schematics of the test systems.
Recovered from S. marcescens in TSB After the connection was complete,
Binding Control Transferred through
(CFU/set) Connector (CFU) sterility of the fluid pathway was then
2.0 × 102 0 assessed by flowing sterile bacterial
2.1 × 102 0 culture broth (e.g., trypticase soy
1.1 × 102 0 broth, TSB) through the connector,
1.1 × 102 0 incubating that broth for seven days
and checking for turbidity, then
filtering it through a sterile 0.2-µm
based on qualification tests described
analytical membrane filter disc. The
in the BPSA component quality test
disc was plated onto agar, incubated
guide, as shown in Table 1 (3).
for an additional seven days, and
Functional Liquid Bacterial
finally examined for sterility or
Challenge Soiling Test: Representing
recovered bacterial colonies.
perhaps the most extreme
A total of 29 tests were performed,
interpretation of worst-case conditions,
and in all cases sterility of the fluid
this test involves immersing the
pathway was confirmed. Negative
presterilized components of a
controls with noninoculated,
connector into a liquid bacterial spore
presterilized connectors also gave
suspension to coat all externally
sterile results in all tests. Positive
exposed surfaces of each connector
controls (in which the protective
with >106 G. stearothermophilus spores.
barrier strips were removed before
Because aseptic/sterile type connectors
inoculation and connection) showed
are designed to operate under dry
broth turbidity and confluent bacterial
conditions, these severely contaminated
colonies on the analysis membrane
connectors are then allowed to dry
filter discs in all tests and confirmed
before subsequent connection and
validity of the bacterial challenge and
sterility testing of the connected fluid
recovery procedure.
pathway. Use of desiccation-resistant
Functional Bacterial Aerosol
bacterial spores ensures high bioburden
Challenge Test: A second test
viability during connections made after
complemented the liquid bacterial
drying. Full details of the procedure
challenge soiling test in providing an
can be found in a published validation
aerosol challenge. We chose Serratia
Figure 2:  Simplified schematic for bacterial aerosol challenge test on disposable connectors
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The significance of
these data can be
demonstrated by
comparing the
results with
environmental
microbial limits in
GMP guidelines.
marcescens for this test (rather than the
smaller Brevundimonas diminuta
commonly used in filter challenges) to
maximize sedimentation onto the
connectors during use and provide a
worst-case bacterial challenge
environment.
This study simulated a sterile media
fill similar to those performed in
process-specific validation studies
conducted by users. In this core
validation study, however, worst-case
environmental conditions for microbial
levels were introduced to assess the
safety margin and sterility assurance
attainable when the product is used in
a highly contaminated uncontrolled
environment: outside a Grade A/Class
100/IS0 5 environment or even a
Grade C/Class 10,000/IS0 7 or Grade
D/Class 100,000/ISO 8 environment.
Figure 2 provides simplified
schematics of the test systems.
After exposure of unassembled
sterilized connectors (binding
controls) to aerosolized bacteria, the
connectors were joined, and sterile
TSB was passed through them into a
collection vessel. The liquid was then
incubated and assayed for bacterial
content, as described above. Suitable
positive controls were included, with
their peel-away strips removed before
the aerosol challenge, as well as
negative controls with no aerosol
challenge. Table 2 summarizes the
results obtained.
Aerosolized bacteria conditions
represent a severely uncontrolled
environment exceeding Grade D/
Class 100,000/ISO 8 particulate
26 BioProcess International September 2008
levels by >100× and microbial aerosol
levels by >10,000×. In all tests,
sterility of TSB transferred through
the completed connections was
maintained. The negative controls
(sterile media transferred and filled
with no aerosol bacterial challenge)
also gave sterile results in all tests.
And as expected, the positive controls
(with protective barrier peel-away
strips removed before bacterial aerosol
challenge and connection) gave
bacterial counts (nonsterility) in the
transferred TSB.
The practical significance of these
aerosol challenge data for
pharmaceutical manufacturing
environments can be demonstrated by
comparing these results with
environmental microbial limits in
GMP guidelines. The levels presented
in Table 2 are more than 106× higher
than the maximum permitted microbial
limits for Grade A/Class 100/ISO 5
environments, according to EU Annexe
1 and US aseptic processing guidelines
(5, 6). As noted above, the levels are also
more than 104× higher than Grade
D/Class 100,000/ISO 8, the
environment in which these connectors
are designed to function. So we
concluded from the bacterial aerosol
challenge functional test that sterile
fluid pathways can be maintained
during connections, even under worst-
case environmental conditions.
Easy, Fast, and Safe
Reducing the risk of bacterial
contamination and achieving high
sterility assurance levels are major
driving forces behind the application
of disposable connectors. In 2002,
Richard Friedman of the US FDA’s
Center for Drug Evaluation and
Research (CDER) presented data
showing a substantial increase in
product recalls associated with lack of
sterility assurance over a three-year
period (7, 8). At the same time, the
FDA also recognized that “enhanced
technologies” such as isolators and
form–fill–seal machines could
contribute significantly toward
improving sterility assurance. Indeed,
revisions or appendices were
specifically added to subsequent FDA
and EU GMP guidelines in
recognition of the special nature of
these technologies and to encourage
their use wherever applicable.
Manufacturers of enhanced aseptic
processing technologies, which include
single-use aseptic/sterile connectors,
can assist by providing comprehensive
validation data on the functionality
and safety of their products. The data
presented here constitute an example of
core validation studies which, together
with other published data, can help to
make the introduction of such new
technologies into processes easier,
faster, and safer.
References
1 Langer, E. Fifth Annual Report and
Survey on Biopharmaceutical Manufacturing.
Bioplan Associates Inc.: Rockville, MD, 2006.
2 The American Heritage Dictionary of the
English Language, Fourth Edition. Houghton
Mifflin Company: Boston, MA, 2006.
3 BPSA Guidelines and Standards
Committee. Part One: Bio-Process Systems
Alliance Component Quality Test Matrices.
BioProcess Int. 5(4) 2007: 52–67;
www.bpsalliance.org/bpsamatrices.pdf.
4 Publication USTR 2232a: Validation
Guide for Pall Kleenpak Connector. Pall Life
Sciences: East Hills, NY, 2005.
5 EU Guide to Good Manufacturing
Practice, Annex: Manufacture of Sterile Medicinal
Products. European Commission: Brussels,
Belgium, 14 February 2008; http://ec.europa.
eu/enterprise/pharmaceuticals/eudralex/vol-4/
pdfs-en/2008_02_12_gmp_annex1.pdf.
6 CDER/CBER/ORA. Guidance for
Industry: Sterile Drug Products Produced By
Aseptic Processing — Current Good Manufacturing
Practice. US Food and Drug Administration:
Rockville, MD, September 2004; www.fda.
gov/cder/Guidance/5882fnl.htm
7 R. Friedman. Presentation on Sterility
Assurance Issues. PDA Spring Conference,
March 2002, Orlando, FL.
8 Stoedter W. Aseptic Processing: How
Good Science and Good Manufacturing
Practices Can Prevent Contamination. PDA
Digest25.htm. c
Letter 38(8) 2002: www.teknopakonline.com/
Corresponding author Christopher J.
Mach is global product manager, and
Donna Riedman is a project leader in the
scientific laboratory services department of
Pall Life Sciences, 2200 Northern Boulevard,
East Hills, NY 11548; 1-516-801-9546, fax 1-
516-484-5228; chris_mach@pall.com.