Biological Process Validation of Dry-Heat Sterilization Cycles

If a dry-heat process is claimed to produce sterile commodities, micro-organisms
known to be most resistant to dry heat must be used to prove the ability
of the dry-heat cycle to destroy them at the coolest location in the load. If the
dry-heat process is claimed to produce both sterile and pyrogen-free commodities,
validation studies must be done using both micro-organisms and microbial
endotoxins. It is the strong opinion of many, including the authors, that biological
validation of dry-heat cycles should be based on the destruction of endotoxin
rather than on the destruction of microorganisms because of the enormous dryheat
resistance of endotoxin compared to micro-organisms [23]. To satisfy the
FDA, however, microbial challenges continue to be done.
With both micro-organism and endotoxin challenges, the cool spot identified
in the heat-distribution and heat-penetration studies will be the logical location
to run the microbial challenge tests. Containers inoculated with microbial
cells or endotoxin will be situated adjacent to identical containers into which
thermocouples are secured to monitor temperature. Temperature profiles must
not deviate from temperature data obtained in earlier studies.
The goal of the biological validation procedure depends on the nature of
the process. If the process is intended to sterilize only, the probability of survival
approach is used. In this case, validation studies must determine a dry-heat cycle
that will assure that the probability of survival of the microbial indicator is not
greater than 10−6. If the process is intended to sterilize and depyrogenate, which
occurs when the materials can withstand excessive heat, the overkill approach
is used. The goal here is to validate a heating cycle that can produce a 12-log
reduction in the biological indicator population.
Equations that apply for determining log reductions or survival probabilities
are Eq. (11) and Eq. (12), respectively. Information that must be known
prior to initiating biological validations include the D value of the biological
indicator to be used, the change in its heat resistance as temperature is changed
(Z value), and the presterilization microbial load on the commodity being steri-lized. Methods for obtaining these values have been adequately described with
ample references in the Parenteral Drug Association technical report on dry-heat
validation [13].
The most widely used biological indicators for dry heat have been spores
of B. subtilis; however, spores of other bacterial species may be used if they are
shown to have greater resistance to dry heat. At 170°C, even the most resistant
microbial spore form will have a D value of 6 to 10 min. At temperatures required
to depyrogenate, microbial spores will have D values of only a few seconds.
The acceptable Z value for microbial dry-heat resistance is 20°C [13].
This value is used primarily in programming computerized temperature-detection
devices, which take temperature data from thermocouple monitors and compute
F values as seen with Eq. (6). A suggested Z value to be used for endotoxin
dry-heat resistance is 54°C [24]. The greater Z value for endotoxin demonstrates
the greater resistance of endotoxin to dry heat.
A suggested step-by-step sequence in the microbial validation of a dryheat
process for sterilizing and depyrogenating large-volume glass containers by
a convection batch oven is presented. Procedures for the validation of a tunnel
sterilization process have been reported by Wegel [25] and Akers et al. [26].
1. The overkill approach is selected for the validation study. This eliminates
the need for bioburden and resistance studies. The objective is
to ensure that the coolest area in the loading pattern, as determined in
earlier heat-penetration and heat-distribution studies, receives sufficient
heat to cause a 12-log reduction in the biological indicator
chosen.
2. Select the type of biological indicator to be used in monitoring process
lethality. Calibrate the biological indicator in its carrier medium
(strip or suspension).
3. Place spore carrier in approximately 12 glass bottles located at the
previously determined coolest area of the oven. Bottles adjacent to
the inoculated bottles should contain thermocouples for monitoring
purposes.
4. Run a complete cycle using the desired loading pattern for future dryheat
overkill cycles.
5. After the cycle, aseptically transfer the spore strip to vessels of culture
media. If spore suspensions were used, aseptically transfer the inoculated
bottles to a laminar airflow workstation and add culture media
to the bottles. Use appropriate positive and negative controls.
6. Determine the number of survivors by plate-counting or fraction negative
methods [13].