Lecture 11 validation of

sterilisation cycles_part 2_process

indicators
Deirdre D’Arcy References: BP/PhEur, USP, Codex, Quality in the manufacture of medicines and
other healthcare products (Sharp). Biopharmaceutics 2nd ed., (Gary Walsh).

http://ec.europa.eu/health/files/eudralex/vol-4/pdfs-en/v4an15_en.pdf
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInf
ormation/Guidances/UCM070342.pdf
(General info! FDA guidance: Process Validation: General Principles and Practices)
 Recall Chapters on sterilisation

➢ Recall that the chapters you read in Unit B

block 1, concerning thermal, gaseous and
filtration sterilisation, had information
relevant to sterilisation cycle validation also.
 Physical qualification
• Sterilisation conditions must be achieved in every
part of product/packaging
• Measurements required depend on the process.
• Need separate sensors: control of equipment and
recording equipment. Recording equipment
calibrated.
• For both physical and biological methods, need to
ensure that most difficult to sterilise part of
load/container is identified for validation and
monitoring
 Physical qualification
(Codex)
Some Physical methods
• Measurement of heat distribution: thermocouples
(Copper/Constan (Type T)).
• Pressure: Pressure transducers or bourdon type
pressure gauges
• RH: Difficult to measure physically – dew point
hygrometers can’t stand the temperatures and
pressures of a sterilisation cycle.
 Physical qualification (codex)
Some Chemical Methods
• In general chemical indicators undergo melting and/or colour
change
• Process indicators: eg. Autoclave tape*
• Single variable indicators: designed to react to attainment of
one critical variable eg a specific melting point with a specific
colour change on melting
• Multi-variable indicators: Respond to attainment of two or
more critical conditions eg Browne’s tubes*: require exposure
to a specific time at a specific temperature to produce a
colour change.
 Indicators
*Used in online autoclave video (practical 5), along with TST
spots (Time, temp and steam exposure).
 Physical qualification
Some Chemical Methods
• Integrating Indicators: Respond to defined
combination of critical variables: quantitative
indicators. Eg. Liquid (acidified solutions of ferric
ammonium sulphate) and solid (strips of
polymethacrylate (perspex)) dosimeters for
radiation sterilization: respond to radiation by dose
related changes in UV spectra or Perspex changes
from colourless to red.
• Specific Indicator: eg Bowie Dick test. Standard test
for rapid and even steam penetration. Uniform
colour change at end of cycle if it is satisfactory.
 Biological methods – biological indicators
BP
• Biological indicators are test systems containing
viable micro-organisms (usually spores of bacteria)
that provide a defined challenge to verify the
required effectiveness of a specified sterilisation
process.
• E.g. – inocculated carriers, self contained indicators
• Physical validation –physical conditions delivered to
all parts of the load. Biological validation: correlates
physical conditions and biological effect
 Biological methods – biological indicators
• Biological indicators have very specific information:
e.g. name, strain number, number of viable spores, D-
value, recovery medium...(see section 2.2 appendix
XVIII BP)
• Placed in locations known to be difficult to sterilise
e.g. cold spots, vial stopper interface, areas difficult to
penetrate
• After sterilisation the indicator carrier must be
aseptically transferred to culture media. Can use self-
contained indicator where no aseptic manipulation is
necessary
 Sterilisation protocols:
Winfield and Richards and Codex

Reference organism selection – Biological indicator (BI):

• Consider ease of cultivation, identification and
reproduciblity.
• Standardised: viability/resistance, cultivation and
storage conditions…
• Bacterial spores usually used. BI resistance usually
great compared to resistance of micro-organisms
contaminating the product
• Should be able to determine meaningful D and IF
values from survivor curves; strict quality data
relating to the BI used are required (BP).
 Biological methods – biological indicators

Consider:
-process conditions (e.g. temp/time/gas
concentration/radiation dose)
-characteristics of the pharmaceutical product
(e.g. a tube, final container…)
-type of indicator required (inoculated
carrier/self contained/custom-made…)
 Biological methods – Recommended (BP)
biological indicators
• Steam sterilisation:
Spores of Geobacillus stearothermophilus.

• Dry heat sterilisation:

Spores of Bacillus atrophaeus.

• Ionising radation sterilisation:

Spores of Bacillus pumilus. NOTE: Don’t usually use Biological indicators in
radiation sterilisation

• Gas sterilisation:
Use of B.I.s necessary for all procedures: cycle validation and routine
operation.
Spores of Bacillus atrophaeus recommended for ethylene oxide.

• Filtration: microbial challenge test –

Brevundimonas diminuta
 Sterilisation process validation-
measurements required:
• Steam and dry heat sterilisation: All items being
sterilised must reach the required temperature for
the required time to result in sterility. Moist heat
also chamber pressure monitoring. Physical and
biological indicators required.
• Radiation Sterilisation: physical tests with
dosimeters to ensure that required dose is delivered
with a particular load configuration. Biological
indicators not always necessary –depending on dose
validation methods.
 Sterilisation process validation-
measurements required (Sharp):
• EtO: As success of sterilisation depends on many
variables i.e. Humidity, temperature, time,
pressure/vacuum, and concentration of EtO, many
measurements are required. These include heat
sensing thermocouples, humidity sensors, pressure
gauges and separate recorders for each measuring
device. Also timer controls and eg. Chromatography
to determine EtO concentration. Biological
indicators also required by pharmacopoeias in both
validation and routine use for gas sterilisation.
➢ Recall that for all sterilisation methods sterilisation is
validated for a specific loading pattern