In addition to the operating parameters specified in the cycle development study, the required Fh value

and the bioburden/ pyroburden levels of the components being treated must be determined. Any over-
sterilization concerns (for heat-labile products) should be also addressed.

Proper cycle development according to the USP can be achieved by verifying a microbial survival
probability of 10-12 for sterilization. If the cycle is required torender the container free of pyrogens as
well as viable microbes, the cycle must demonstrate a 3 log cycle reduction of bacterial endotoxin
(1/1000 of the original amount is inactivated). The pyrogen challenge can consist of inoculating an
article with a minimum of 1x103 USP EU of bacterial endotoxin. A number of guidelines addressing the
cycle development and validation of sterilization cycles are referenced in the USP from the PDA, PMA,
HIMA and the AAMI

If overkill cycles are utilized (based on bio-chal-lenges), it is generally not necessary to evaluate the
bioburden of pyroburden present on the incoming glass containers or equipment. The usual assumption
considers that the relatively low numbers of organisms, or minute concentration of contaminants
present on the surface of glassware for parental use, are well within the challenge destruction of 106
spores of B. subtilis or a 3-log reduction in endotoxin. The enumeration and identification of bioburden
and pyroburden are considered of little value.

For sterilization processes only, the presence of high numbers of gram negative organisms on a
component before sterilization can raise concerns about the presence of endotoxin on the components
prior to processing, since the overkill sterilization cycle will only inactivating the pyrogenic activity of the
endotoxin. As glass containers are molded at high temperatures (1500oC) and shrink wrapped by the
supplier for shipping, they are unlikely to have a problem with gram-negative organisms. Extensive
studies performed on the glass containes as received in the shrink wrap and before washing, were
tested as free of endotoxin or having very low levels per container volume (< 0.003 EU/mL). washing of
the articles will decrease the endotoxin levels, if any are present.

The biological indicators of choice for validating and monitoring dry heat sterilization are commercial
spore strips of B. subtilis spores. When the strips are utilized, the manufacturers D value can be used.
Spore strips can be purchased prepared with 104 to 109 spores. There are certain grades of strips which
can be utilized for temperature between 250 C and 450 C. any browning of the paper without crumbling
does not interfere with usage or testing.

Alternately, a spore suspension can be inoculated on a part of the load (on a container or piece of
equipment) to closely represent the surface bio-challeenge. If the spores are dried on the surface of the
article to be sterilized, the user must establish the D value for each type of component to be tested.
Heat-resitant organisms have D values of only a few seconds at temperatures generally used for
endotoxin inactivation and for this reason a spore challenge is not required in any process where
depyrogenation is demonstrated.

D and Z values
The microorganism used as a biological indicator must have resistance characteristics (D and Z values)
that are documented and appropriate for the sterilization or depirogenization cycle. The D value is
defined as the time required to reduce the microbial population by 90% (one logarithm). The relationhip
of lethality to temperature is expressed in the Z value. The Z value studies will define the number of
degrees that are required for a change in the D value by a factor of 10. Laboratory studies are used to
define the D value of the typical bioburden and/or pyroburden. The microorganism used as a
bioindicator must have resistant characteristics (D value) that are documented and appropriate for the
cycle. The bioburden data, D and Z values, are used to calculate the minimum Fh value required.

Sterilization-Bioburden Calculations Fh

The Fh value for sterilization is the integration of lethality at a reference temperature of 170 C. the Fh
value for depyrogenation is the integration of lethality at a reference temperature of 250 C. A
conservative approach to determining a minimum sterilization Fh would utilize the heat resistance
spores of B. subtilis (globigii) and assume a D170 value of three minutes (at a reference temperature of
170 C) and a Z value of 20 C. the lethal rete determines the increment of lethal heat effect obtained over
various temperatures using the Z value (as compared with a reference temperature). The Fh value is
derived by integration of the lethal rate with respect to time. The Fh value (equivalent time at the
reference temperature) accumulates the total tethality. When sterilization temperatures other than 170
C are used, the Fh value is reported as process equivalent time at the reference temperature of 170 C.

The wquations used for equivalent time are as follows:

Fh = D170(Log a Log b)

Where a is the bioburden per item, b the probability of survival, D the time at 170 C to reduce the
population of the most microorganisms in the product by 90%, and Fh the equivalent time in minutes at
170 C and a Z value of 20 C and

Ft = F170/L

Where Ft is the equivalent time at temperature t delivered to a container for the purpose of sterilization
with a specific Z value, F 170 is the equivalent time at 170 C delivered to a container for the purpose of
sterilization with a specific Z value (when Z= 20 C, then F170 = Fh) and L is the lethal rate.

L=log-1 (to-tb)/Z

Or L = 10 (To-Tb)/Z

Where T0 is the temperature within the container or item and Tb is the base temperature of 170 C.

As a supplement to temperature data, spores of B. subtilis are used to monitor the lethality of dry heat
sterilization during the validation runs. Heat-labile products rwquire strictly controlled sterilization
cycles, since under-processing will result in a non-sterile product while over-processing may cause
degradation of the product. The cycle development will determine the minimum amount of dry heat
required to ensure that the probability of survival of the bioburden is less than 10-6. The equivalent
sterilization time and temperature can be described by the F value with a reference temperature of 170
C and assuming a Z value of 20 C.