Defining depyrogenation Types of depyrogenation Case study: depyrogenation by dry heat Practical tips Why things go wrong Variations in the approach

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Practical Approaches to Depyrogenation
Studies
Dr. Tim Sandle
Pharmaceutical Microbiologist

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Introduction
• Defining depyrogenation
• Types of depyrogenation
• Case study: depyrogenation by dry heat
– Practical tips
– Why things go wrong
– Variations in the approach

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Defining depyrogenation #1
• Depyrogenation is the elimination of all pyrogenic
substances, including bacterial endotoxin.
– Sterilization refers to the destruction of living cells.
However, the process does not necessarily destroy
microbial by-products and toxins.
• Depyrogenation, like sterilization, is an absolute
term that can only be theoretically
demonstrated.
• Achieved by:
– Removal
– Inactivation

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Defining depyrogenation #2
• Is depyrogentation only:
– Destruction or inactivation of endotoxin?
• Where total destruction or inactivation is theroetically
assumed.
– Or does it include endotoxin removal e.g. rinsing?
• Where the reduction of a significant proportion of the
‘pyroburden’ is assumed.
• Example: rinsing of elastomeric closures.

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Why depyrogenation is difficult?
• LPS can adhere to surface (in nature it
contributes to adhesion of Gram-
negative bacteria to surfaces allowing
them to form as biofilms in aqueous
environments).
• It “attracts” and “entraps” organic
macromolecules from aqueous
environments.
• LPS also increases the negative charge of
the cell membrane and helps stabilize
the overall membrane structure.
• It is strongly resistant to heat and
radiation.
Lipid A, embedded in
the bacterial outer
membrane, is
responsible for
pyrogenicity.
Lipopolysaccharide
has three distinct
chemical regions.

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Examples of depyrogenation
• Ultrafiltration—excluding
endotoxin by molecular weight.
• Reverse osmosis—a size-
excluding filter operating under a
highly pressurized conditions.
• Affinity chromatography (e.g.,
DEAE sepharose or polymyxin-B) -
these binds endotoxin, by using a
positive charge to attract the
negatively charged endotoxin
• Acid or base hydrolysis—this
destroys Lipid-A.
• Dilution or rinsing—endotoxin is
washed away or reduced using
WFI.
• Distillation—turning water from a
liquid to a vapour and then from
vapour back to liquid.
• Adsorption (e.g., activated carbon
beds).
• Hydrophobic attachment—
certain materials.

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Dry heat depyrogenation
• Dry heat (physical destruction):
– Convection (transfer of heat by movement of fluid or air),
– Conduction (transfer of heat from adjacent molecules),
– Irradiation (emission of heat by electromagnetic radiation).
• Depyrogenation of glassware for the production of
parenteral pharmaceuticals (aseptic processing).
– Endotoxin is heat stable and resistant to most conventional
sterilization processes.
– Residual pyrogens could be injected into a patient resulting in an
adverse reaction.

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Dry heat depyrogenation
• Dry heat involves
subjecting the
components to a high
level of heat (e.g.
between 180 and 300∘C)
for a defined time:
– The higher the
temperature, the shorter
the time required).
• Reference cycle is 250oC
for not less than 30
minutes.
Temp. 1-log 2-log 3-log 4-log
T=250oC 5 min. 10 min. 15 min. 20 min.
T=296.4oC 30 sec. 60 sec. 90 sec. 120 sec.
T=342.8oC 3 sec. 6 sec. 9 sec. 12 sec.
At 250°C, the time required to
disintegrate endotoxin 1-log cycle, is 5
minutes (D-value).

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Dry heat depyrogenation
• Depyrogenation starts
by following a pattern
similar to steam
sterilization
• But once destruction
begins it does not
produce a semi-log
reduction and instead it
follows a biphasic
reduction.
Biphasic pattern of depyrogenation kinetics in
wet and dry heat systems. Source: Li et al
(2011 “Kinetics of Hydrothermal Inactivation
of Endotoxins”, Applied and Environmental
Microbiology, 77(8): 2640-2647

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Dry heat depyrogenation devices
• Depyrogenation dry heat
devices include ovens and
tunnel sterilisers.
• A series of parameters
needs to be controlled:
– Unidirectional airflow
controlled by High Efficiency
Particulate Air (HEPA) filters.
• Velocity range
• Grade A particulates.
– Rate of speed (minimum,
maximum and nominal) must
be measured and verified.

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Case study: dry heat depyrogenation #1
• The assessment of
depyrogenation involves:
– Introduction of purified
endotoxin (LPS), of a high
potency,
• Typically a level of >1000
EU/device (former USP
<1211>)
• Sometimes called an
‘Endotoxin Indicator.’
– Post-process testing to
assess if a minimum of a
three-log reduction has
been achieved.

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Case study: dry heat depyrogenation #2
• Depyrogenation devices are biologically challenged using a
known level of a high concentration of Escherichia coli
endotoxin.
– A freeze-dried extract from the Gram-negative bacterial cell
wall: lipopolysaccharide (LPS).
– Similar to Control Standard Endotoxin (CSE) used for routine LAL
testing, but the concentration is far greater.
– A potency determination should be undertaken against
Reference Standard Endotoxin.
• This potency is used to convert the labelled weight of the endotoxin
into Endotoxin Units (expressed as Χ EU/ng).

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Case study: dry heat depyrogenation #3
• Run the depyrogenation tunnel and use
thermocouples to assess for any cold spots.
• Repeat using endotoxin challenges at any
colder areas, alongside thermocouples.
• Two approaches for adding the
endotoxin challenges:
– Use a high potency endotoxin spike
directly onto the surface of the
container to be depyrogenated; allowing
this to dry or to freeze dry and then
placing it into the depyrogenation
device.
– Using vials of high concentration
endotoxin and substituting these for the
containers.

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Case study: dry heat depyrogenation #4
• Endotoxin challenge:
– The endotoxin challenge is typically
1000 Endotoxin Units (EU) or
greater.
– Verified by using control vials that
are not subjected to the
depyrogenation cycle.
• Tested alongside the test vials.
• Endotoxin recovery from glass is
difficult, need to practice to show
reproducibility and typical recovery.
• Care needs to be taken to avoid cross-
contamination.

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Case study: dry heat depyrogenation #5
• Preparation:
– Challenged vials prepared the day before the validation run, in order
to allow for the endotoxin to dry.
– Endotoxin is applied to the base of the vial under a unidirectional
airflow cabinet.
– Positive controls are prepared in the same way.
• Placement:
– Test vials must be identified and placed in known locations in the
depyrogenation tunnel.
• Run:
– On completion of the test run, vials must be collected and transferred
back to the laboratory for LAL testing.

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Case study: dry heat depyrogenation #6
• Testing:
– The endotoxin vials are tested using the LAL assay.
– A known level of pyrogen free water is added to the test vials.
– Volume should be sufficient to cover the base of the vial.
– Different techniques used to recover any endotoxin from the
glass surface:
• Vortex mixing and ultrasonication
• A dispersing agent or buffer in place of pyrogen free water.
– An aliquot is tested against an endotoxin standard series
(consisting of a minimum of three-log concentrations of
endotoxin).
• The standard series should be prepared using the same lot of
endotoxin used to challenge the vials.

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Case study: dry heat depyrogenation #7
• Positive control vials
– Will require dilution prior to testing.
• Negative control vials:
– Two types:
• Uninoculated vials, which are not put into the
depyrogenation device.
– Assess residual endotoxin.
• Vials that have passed through the depyrogenation device.
– Assess device particulates.
– Negative control vials are tested in the same way as
the spiked test vials.

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Case study: dry heat depyrogenation #8
• Acceptable test:
– Positive controls
• Show >1000 EU/vial challenge
– Negative controls
• No detectable endotoxin
– Test vials
• Endotoxin reduction of three-logs (or greater).

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Points for consideration
• Preliminary validation work to determine time between
spiking the vials and placing them into the depyrogenation
device.
– How and at what temperature will vials be stored?
– What is the ‘expiry time’ for vials that have passed through the
depyrogenation device prior to testing?
• How to identify the inoculated vials once they enter the
tunnel?
• The number of challenge vials to use in the study e.g. 10 or
20?

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Why things may go wrong
• Depyrogenation studies do not always
work, because:
– The type of material being
challenged.
• For glass, the type of glass the
challenge vials are made from
(Type I or Type II glass).
– The type of depyrogenation device
and its efficiency.
• Some HEPA filters shed a high
number of particles during
temperature transition.
– Some of these particles
may contain endotoxin or
interfere with the LAL
assay.
• The method used to dry the
endotoxin to the container being
tested.
• The mechanism of the
depyrogenation device.
– e.g. infrared devices are shown
to be are more effective.
• Different manufacturers endotoxin
– Dependent upon how ‘pure’ the
endotoxin is (whether other
cellular components are present)
and whether the endotoxin
containers ‘fillers’, such as
glycol).
• These factors may increase
or decrease the time taken
to achieve heat inactivation.

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Variations to the test
• Number of qualifying runs e.g. three.
• Intervals between qualifications e.g. 6 months? 12 months?
• Only using thermometric data – is a biological challenge
necessary? (see: USP <1228.D>
• Some set the depyrogenation time & temperature
combination beyond a minimum of three-logs to provide an
‘over-kill’.
• Some companies go further and stipulate a minimum of
four-logs, in case of residual endotoxin from
depyrogenation studies.

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Summary
• Defining depyrogenation
• Types of depyrogenation
• Case study: depyrogenation
by dry heat
– Practical tips
– Why things go wrong
– Variations in the
approach
• References:
– Tours, N. and Sandle, T. (2008):
‘Comparison of dry-heat
depyrogenation using three
different types of Gram-negative
bacterial endotoxin’, European
Journal of Parenteral and
Pharmaceutical Sciences, Volume
13, No.1, pp17-20
– Sandle, T. (2011): "A Practical
Approach to Depyrogenation
Studies using Bacterial
Endotoxin", Journal of GXP
Compliance, Autumn 2011

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THANK YOU
Dr. Tim Sandle