Acceptance Criteria In the Validation of VPHP Decontamination

Dr. James E. Akers

A3P 22nd Congress, October 2010.

Isolator’s have been subjected to antimicrobial treatments to destroy

environmental contamination since their introduction to the

pharmaceutical industry in the 1980’s.

Initially, Peracetic acid/H2O2 was the sporicidal chemical used, but it was

replaced by Vapor Phase Hydrogen Peroxide (VPHP) in the early 1990’s.

Currently, almost all isolators for product testing or production are

“decontaminated” with VPHP.

The majority of firms define success in decontamination as the complete

kill of BI’s with a population of 106 G. stearothermophilus spores.

Occasionally, an unexpected BI positive occurs which is deemed a failure

and this has been deemed the result of “Rogue” BI’s.

In the early years after isolator introduction the sporicidal

treatment of isolators was called “sterilization”.

Some regulators argued that sterilization of isolators was not

a reasonable objective.

Many surfaces inside an isolator are not easily treated with

VPHP.

Experience has shown that penetration of VPHP is limited.

Although named decontamination, the process validation

looks very much like sterilization.

It has become a standard expectation that biological indicators must always

have a population of 106.

This is most likely because inspectors or auditors perceived some kind of

linkage between the SAL of 10-6 and a BI population of 106.

Unfortunately, this idea is scientifically incorrect!

It is not necessary to use a BI with a population of 10-6 to prove a SAL of 10-

6.

In fact neither 10-6 nor 106 have anything to do with the attribute of sterility.

106 spore population has even less significance in the decontamination of

isolators.

Why 10-6 as a target

SAL?• It is important to consider that SAL and sterility are two

distinctly different things.

• Also, a “6 log spore reduction” and complete kill of a BI with a

population of 106 are two very different concepts.

• There is NO probability associated with the attribute of sterlity-

a material is either sterile or it isn’t.

• A SAL (or more accurately probability of non-sterility) of one in

a million is a measure of acceptable RISK.

• Because sterility is not the target in isolator decontamination

the risk based concept of SAL does not (or should not) matter.

To summarize this section 10-6 SAL is a measure

of acceptable risk. 106 as a spore population on

BI’s has nothing to do with a 10-6 SAL . It has

never been suggested that isolators SHOULD or

MUST have a 10-6 SAL, which would be impossible

to prove anyway!! Decontamination is not

sterilization!!

So, with these facts in mind why do we stipulate

that BI’s used in isolator decontamination must

have a population of 106?

Does the requirement for BI’s with a population of

106 play a role in the so-called “Rogue” BI?

BIs are meant to challenge sterilization processes or to evaluate the

efficacy of a sporicide.

BIs were never intended to provide the central design criterion for

sterilization processes.

In sterilization process design the user should define how much

lethality is necessary for their process

BIs are biological evaluation that such lethality has been delivered.

Unfortunately- in the case of VPHP there is no standard definition of

lethality.

In steam sterilization we have defined BIER conditions and a

standard measure of lethality the Fo.

BIER=Biological Indicator Evaluation Resistometer.

The lethality value of Fo= 1 minute of heating at 121oC (which is

the metric conversion of 250oF, the temp. of steam at 15 PSIG.)

The BIER standards allow us to define process D and Z values.

The D value is the time required to achieve a one spore log

reduction at defined processing conditions.

Z value is a measure of rate of change of D value as kill

conditions change (in moist heat this means temperature).

110 103 154.5

115 32.6 49

118 16.3 24.5

121 8.2 12.3

124 4.1 6.2

D is defined relative to process temperature i.e. D121.

Process challenge is a product of D value x time.

A process with a lethality of 12 minutes could be evaluated with: a BI with a

population of 104 and a D121 of 3 minutes or a BI with a population of 106 and a

D121 of 2 minutes.

It both examples you would expect statistically 63% of the Bis to be positive.

The challenge afforded by both of these BIs is exactly the same! Thus, both

SHOULD be equally suitable for validation purposes! (reference USP <1211>)

It is only regulatory and compliance convention that limits BI selection to those

with a population of 106 scientifically 103 or 104 could for most processes work just

as well.

VPHP and D Value and

Determination• There are no standard conditions for the evaluation of VPHP

BI’s- BIER conditions have not been defined.

• The multifactoral nature of VPHP processing means that

BIER conditions are difficult to define.

• Therefore, none of the BI manufacturer supplied D values are

tested against a recognized standardized set of conditions.

• In VPHP “D-values” are most properly considered measures

of relative resistance.

• D values are only roughly comparable only to other lots tested

by the same manufacturer under the same conditions.

D values and BI Spore

Population

• There should be no observable change in D value with respect to increasing or

decreasing spore population on a BI.

• This is true of BI’s for dry heat and moist heat and is common to chemical and radiation

sterilization as well.

• The survivor curve should be log linear to r> 0.95.

• BI kill in VPHP is single hit or first order kinetics.

• Many VPHP BI survivor curves I have seen or personally analyzed do not follow first order

kinetics and are not log linear

• Non-linear survivor curves are indicative of multiple rates of reaction- this most probably

correlates to issues relating to the BI and spores being “accessible” to VPHP.

• Studies have shown that D value in VPHP processes can vary with respect to spore

population on BI’s.

Unusual BI performance

• Is is common to use 30-100 BI’s in VPHP decontamination

trails.

• It is not uncommon to hear of reports of three such tests being

done with only one or perhaps two positives.

• Repeat tests find that kill is observed reproducibly at the site

of a positive BI.

• In many tests several BI’s have been placed at a positive

location and all of these test BI’s are killed.

• These observations led to the term “rogue” BI!

Bacillus spores 106

SEM 4000x

Solid plastic coupon

G. stearothermophilus

105 population

Stainless Coupon

SEM 8000X

Typical Clump

Likely factors in extreme

BI resistance• BI agglomeration or clumping- this may protect spores in the

center of a clump resulting in slow or no kill.

• In BI’s deposited on solid surfaces hundreds of clumps both large

and small may form.

• We have also seen in our studies that spore preparations that

have high levels of residual media or salts can be abnormally

resistant.

• Reports indicate that in some cases resistance can be 100 fold

higher than the mean value for a population.

• We have found that high levels of biomass can also result in the

appearance of extreme resistance of some mold to VPHP

Are super resistant BI’s

indicative of failure?• No! They are more likely the result of a misplaced focus on

106 BI populations.

• This is probably a result of an over emphasis placed upon the

creation of “worst case” conditions.

• Also, it relates to a misunderstanding regarding the

“relationship” between 106 and 10-6 “SAL”- actually there is

NO relationship.

• It is appropriate to carefully consider what we are trying to

prove in the validation of VPHP decontamination.

The purpose of VPHP treatment is to eliminate bioburden

In 20 years of isolator testing experience has shown that

bioburden is typically <20cfu/25cm2 on isolator surfaces.

The typical bioburden is also much less resistant than G.

stearothermophilus spores.

It is necessary to kill each organism only once!

Extreme worst case conditions do not reduce risk and do not

really accomplish enhanced product safety.

Therefore it is not necessary to overreact to rare BI positives.

Avoiding the problem

• It should be possible to use 103 or 104 BI’s, even where parts

hoppers are treated during decontamination.

• Most guidelines on isolator decontamination require a 4 to 6

“spore log reduction”

• This is a very different thing than complete kill of a BI with a

population of 106 spores.

Summary

• Super resistant BI’s are not “Rogues”, the occur naturally for predictable reasons.

• These super resistant BI’s are not the result of a genetic phenomenon and have

nothing to do with worst case.

• Super resistant BI’s do not indicate high process or product risk.

• They are instead the result of over-aggressive and unnatural test conditions.

• There is nothing magical, scientifically special, or statistically meaningful about a

106 spore population.

• It is possible to use lower concentration BI’s to reduce the likelihood of clumping.

• Decontamination is to eliminate bioburden which in cleaned isolators is very low.

• It is necessary to kill each bioburden organism only one time!