1. Practical Approaches to Depyrogenation Studies
Dr. Tim Sandle
Pharmaceutical Microbiologist

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

3. 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

4. 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.

5. Why depyrogenation is difficult?
Lipopolysaccharide has three distinct chemical regions.
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.

6. 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.

7. 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.

8. 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).

9. 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):

10. 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.

11. 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.

12. 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).

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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).

19. 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?

20. 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.

21. 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.

22. Summary References: 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

23. Dr. Tim Sandle
Thank you