VAL-200 Selection and Use of Biological Indicators during Validation Studies

Purpose

The use of Biological Indicators (BI’s) for the validation and in-process monitoring of sterilization cycles is becoming increasingly complex and highly regulated. Therefore, a defined approach to the use and control of biological indicator validation exercises is required. The purpose of this Standard Operating Procedure is to define the selection and usage of Biological Indicator Spore Strips and ampoules when validating thermal processes or equipment.

Scope

The biological indicators defined in this SOP are applicable for the validation and monitoring of sterilization cycles utilized at a GMP site, in the production of biopharmaceutical products. The usage procedures defined in this SOP shall apply to all validation activities carried out at a GMP site using biological indicator spore strips for process and equipment validation or testing work.

Definitions

D value: The time taken, in minutes, to reduce a population by 90% (one Log10). This term is usually followed by a reference to the sterilization modality concerned, in sub-script; thus for steam it is D121, for dry heat D160 and so on.

Z value: The change in temperature required to change the D-value by one Log10. i.e. A Z value of 10°C will change a D value of 5 minutes at 121°C to a D value of 0.5 minutes at 131°C

F₀: The equivalent time, in minutes, at 121°C, calculated from time(s) at different temperatures, assuming a Z value of 10 °C. Used to include heat up and cool down times in cycle lethality calculations.

Time/Temperature Sterilization: A sterilization cycle based on a load being held at a set temperature for a set amount of time. For example, an autoclave cycle of fifteen consecutive minutes at a minimum temperature of 121°C.

Bioburden: The term used to encompass the total biological challenge to a process. Usually, but not exclusively, referring to those organisms recovered from the product matrix prior to sterilization.

Lethality Input (LI): An estimation of the process challenge calculated from the D value and the population of the BI.

Example:

Lethality input = Log10 population of BI x D Value of BI

Example = Log10 1.7 x 106 x 1.95 minutes = 6.23 x 1.95

LI = 12.15

The Lethality Input is to be used to record the challenge to a sterilization process and to ensure that future validation activities use an equivalent challenge to the process.

Lethality Input is also known as F_biological or F_bio.

Sterility Assurance level (SAL): Probability of a single viable micro-organism occurring on an item after sterilization.

Note: The term SAL takes a quantitative value, generally 10^-3 or 10^-6. When applying this quantitative value to assurance of sterility, an SAL of 10^-6 takes a lower value but provides a greater assurance of sterility than an SAL 10^-3.

Probability of a Non-Sterile Unit (PNSU): The probability of a unit (product container) being non-sterile after the application of a lethal agent. The distinction between SAL and PNSU is that non-sterile units may have more than a single surviving micro-organism.

In order to achieve a given PNSU it is necessary to meet several conditions:

– To statistically know the initial bacterial load (bioburdon) of the batch.

– To be certain that the coldest spots inside the units of the batch have received a lethal heat dose sufficient to obtain the required PNSU.

– Capable of performing F_O calculations inside all load locations.

The following equation can be used to calculation the PNSU:

log N_U = -F/D + log N_O

N_U = Probability of a non-sterile unit

F   = F value or lethality of the process

N_O = Bioburdon count per container

D  = Natural resistance of bioburdon

For example, PNSU of 1 in 10⁶ – the probability that a unit is non-sterile is one in one million.

Spore Log Reduction (SLR): The term used to indicate the actual reduction in a spore population. Thus if a population is reduced from 10⁶ to 10³ the spore log reduction is 3.

Responsibilities

– It is the responsibility of Validation personnel to ensure that validation activities are carried out in accordance with the procedures defined in this SOP.

– It is the responsibility of the Validation Manager to ensure that the execution of all thermal validation studies follow the procedures required by this SOP.

Standard

1. General

BI’s are to be used as a part of every sterilizer cycle validation exercise. The intention of any BI validation is to demonstrate a spore log reduction of 5 for glass ampoules and a spore log reduction of 6 for both spore strips and EZ-test ampoules. This is demonstrated by rendering all the BI’s sterile at the end of each cycle.

Spores of Geobacillus Stearothermophilus (for example, ATCC 7953, NCTC 10007, NCIMB 8157 or CIP 52.81) are only to be used. The number of viable spores exceeds 5 x 10⁵ per carrier for glass ampoules and exceeds 1 x 10⁶ per carrier for both spore strips and EZ Test ampoules. The D-value at 121^oC exceeds 1.5 minutes for all three types of ampoules.

Reported D-values for organisms similar to those identified as local environmental isolates are as follows:

Figure 1: Typical Geobacillus Stearothermophilus spore strip

These ampoules contain a known quantity of bacterial spores inoculated onto filter paper and placed inside a plastic culture tube with a crushable glass ampoule containing the culture medium. The appearance of a yellow colour indicates growth while no colour change indicates adequate sterilisation. Typical biological indicator EZ-test ampoules are illustrated in figure 2.

Figure 2: Typical Geobacillus Stearothermophilus EZ-test ampoules

Glass Ampoules are self-contained biological indicators used for monitoring the steam sterilization of liquids. The glass ampoule biological indicator contains Geobacillus stearothermophilus spores and a specially-formulated culture medium that turns a dramatic yellow if spores grow or remain pink/purple if adequate sterilisation occurred.

Figure 3: Ttypical Geobacillus Stearothermophilus Glass ampoules (liquids)

2. Biological Indicators

2.1 Use of Biological Indicators

2.1.1 All BI’s shall be accounted for after execution of the study. Any accountability discrepancies must be treated as potentially serious and investigations must track down lost BI’s to ensure that they have not been left inside manufacturing plant equipment.

2.1.2 The number of BI’s placed in a load depends on many factors. Typically one BI should be placed at the coldest thermocouple/probe positions within the load, usually including one in the drain of the chamber where practicable. BI’s must only be located in close proximity to a probe/thermocouple to allow comparison of F₀ and F Biological results.

2.1.3 Where possible BI’s should be recovered and tested within 4 hours, alternatively BI’s may be refrigerated at 2-8°C within 4 hours of recovery, and then tested within 24 hours of refrigeration, subject to validation of the chosen BI carrier.

2.1.4 Biological Indicator must be placed in the specified locations as defined in the relevant study documentation.

2.1.5 The position of all biological indicators and their associated thermocouple numbers must be recorded.

2.2 Wrapping Procedure of the Biological Spore Strips

2.2.1 The wrapping of biological spore strips is a standardized procedure.

2.2.2 If the BI strips are wrapped around thermocouple wire for the purposes of placement within the load or piping, the BI strips must be wrapped so that the thermocouple wire is parallel to the BI’s longest length. This method is graphically depicted in figure 3. This ensures the greatest surface area of the BI strip is exposed to the sterilisation/sanitisation process. Care must be taken to use as little autoclave tape as possible; the tape must be placed at the highest possible attachment point on the biological indicator to negate a condensate ‘pooling’ effect.

Figure 4: Standard BI wrapping method used during validation studies.

2.2.3 For biological indicators in tubing: Slice into the exact centre of the hose length and insert the thermocouple/biological indicator. The cut must be sealed or you will not be challenging the hose properly. The method of wrapping used will be as described in section 2.2.4.

2.2.4 In the event of the BI location offering restricted space; an alternative wrapping method may be used. This method is used to facilitate locations which will not accommodate the wrapping method described in section 2.2.2. This method is graphically depicted in figure 4. The spore strip is wrapped with the thermocouple wire parallel to the BI’s shortest length. This method presents reduced surface area, however this is deemed a worse case challenge than that described in section 2.2.2. Care must be taken to use as little tape as possible; the tape must be placed at the highest possible attachment point on the biological indicator to negate a condensate ‘pooling’ effect. Use of this wrapping method must be documented in the relevant validation protocol appendix as the use of this method is restricted to reduced space locations.

Figure 5: Reduced space wrapping method used during validation studies.

2.2.5 Use of the Paper Spores Strips during testing in which the BI may be subjected to physical hardship requires a customized approach. The delicate nature of the glassine protective layer must be protected from the extreme conditions. This will be achieved by placing the spore strips in a short piece of silicone tubing. The tubing is closed but not sealed at the ends by tie-wraps. This will protect the spore strips while providing adequate steam penetration to kill the spores. Figure 5 illustrates how this system is put together.

Figure 6: Protected wrapping method used during validation studies.

2.2.6 The BI strips should be examined upon completion of a run and any damage to the envelope or wetness of the strips should be documented the relevant protocol comments section.