An article that appeared in October edition of Cleanroom Technology Magazine 

The trouble with biological indicators. Understanding the challenge. The solution to continuous monitoring of “fragile” hydrogen peroxide decontamination processes.

The trouble with biological indicators.

For nearly forty years the pharmaceutical and healthcare sectors have used cold gaseous processes to deliver a decontamination of environments – be it cleanrooms, isolated processes or any of the many other environments and facilities around the globe, multi-millions have been spent on developing these decontamination technologies in order to reliably deliver cold decontaminations outside of an autoclave. Most notably and perhaps palatable for pharma and healthcare would be the hydrogen peroxide decontamination cycle, when delivered correctly hydrogen peroxide offers excellent biological inactivation and is by far the most environmentally friendly of all the gaseous decontamination process. Hydrogen peroxide can routinely deliver a very robust decontamination, more than the industry norm of an inactivation of a million geobacillus stearothermophilus bacteria.

This article isn’t about the technology delivering the decontamination cycle. This article is going to focus on the trouble with the validation of the decontamination cycle.

For the last forty years decontamination cycle performance validation has been based on a 19th century principle. The principle is to place typically stainless-steel carriers or tokens containing that million bacteria into the decontamination process, to run the decon cycle, to recover those bacteria carriers, to place those tokens into a suitable growth media and to then incubate the bacteria for up to seven days and see if you can make any of them grow! Now it sounds pretty robust in terms of a process, but it isn’t. In fact, this validation process is fraught with dangers, peril and difficulties. Let me explain;

First off, creating and delivering a million bacterial on to a carrier. Its technically possible but not practically. The fermentation process that creates the bacterial in the first place – growing bacteria, its biology not chemistry, every batch behaves differently, similar maybe but identical no. Then deposition onto the carrier, each deposit in every batch has a different amount of bacteria, that lands on the carrier in a unique way. So, every BI, yes every single BI is at best slightly different to the next and at worst totally different. Batches of biological indicators are so different, so routinely different that manufacturers indicate the population of bacteria typically found within that batch and the associated D value on each batch.

D value? What exactly is a D value? Well that would be the time it takes the million bacteria to reduce by one zero when exposed to a controlled dose of hydrogen peroxide in a repeatedly stable environmental condition (1,000,000 to 100,000 CFU’s of bacteria all the way down to zero CFU’s). This qualification process being set by the BI manufacturer typically, oh and the dose and environmental conditions are a secret that isn’t delivered with the BI and can’t be replicated outside of the manufacturers facilities! So, we really can only say the D value is a measure of resistance from one manufacturer’s BI batch to another. i.e. This one is harder to kill than the other.

So, what does that look like simply?

In very basic terms, if the environmental conditions and dose is the same then then the time to kill all the bacteria for a low resistance (low D value) BI could be just less than 6 minutes through to a highly resistance BI that is something over 20 minutes.

Ladies and Gentlemen – This is supposed to be the same test! Yet as you can clearly see it isn’t.

In addition to a validation tool that differs sometimes dramatically from one batch to another we have to contend with a host of manufacturing, storage and even use and incubation hurdles that can all create difficulties in the challenge.

Rogue BI syndrome. What is it? Basically, when dealing with viable bacteria it is impossible to predict how they will behave, sometimes they clump when being deposited. Sometimes they layer. Sometimes some small contamination entrains into the manufacturing process and the contamination effects how the bacteria behaves and causes unexpected issues. Storage of BI’s is critically important, as is how you present the BI for exposure to the oxidisation process as well as recovery, the storage and use of growth media and even the incubator. The picture here is that using biological indicators is hard work at best and many factors can create a rogue result, one that instantly creates hours, days, weeks or even months and years of work.

Understanding the challenge.

Let’s put all of that to one side and assume everything is good, lets go beyond good and say if the BI performs perfectly – what is the result? BI’s take a long time to work with (up to seven days incubation), the result is binary and very expensive in terms of operational impact (many companies use three BI’s in each position and on some isolated production lines hundreds and even thousands of positions are studied!).

Some production facilities I know have an annual usage of over one hundred thousand biological indicators and teams of people and facilities to administer the qualification process. It costs millions even when it goes perfectly and routinely fails.

But today after forty years of going in one direction people are conditioned and have belief that it is the only direction available. The guidance and publications on the subject are not specific and don’t really give any prescribed and clear markers. Log 6 Bacillus BI’s… It would be very helpful for all if some of these questions where answered clearly. What exactly is the expectation?

Thus, the most critical part of the model is to really understand the challenge. What resistance BI will you use? How will you design your cycle? Success is pinned on defining the challenge;

The acceptable resistance range of the biological indicator. Its critical to specify to the supplier the acceptable range of D value – if you don’t you run the chance of receiving either low or high resistance BI’s and that makes the challenge on the outlying area of the scale and one way or another you are headed into potential difficulties.

One way of limiting risk is to design a decontamination cycle that is very robust – many do. The cycle is designed to inactivate a high resistance BI and this is safe, however the drawback, it’s also a very long cycle, lengthy injection phase, lengthy dwell phase and massive aeration cycles to remove the gas or vapour. Safe but long cycles reduce risk but also impact productivity – especially if the product you intend to work with is sensitive to the decontamination gas and you need to reduce the cycle to levels into the parts per billion range!

The manufacturer of the BI is also something that needs to be defined and not changed. BI manufacturers are not equal. They are totally different challenges. The BI from supplier A is usually totally different to supplier B! Select a BI supplier and stick to it. Move at your peril.

What type of BI do you use? In Tyvek? Naked Ribbons? Wrapping your bacteria in a blanket of Tyvek creates a different challenge, sometimes more robust, but not always. Dependent upon the type of decontamination technology used. Some people specify and use naked BI’s others Tyvek. Some manufacturers use a different grade of Tyvek to others, the Tyvek itself is impregnated with a backing material that also differs from one supplier to another. All of these are the “SAME” Log 6 BI! But totally different in terms of the challenge.

By defining the challenge very accurately we can minimise the impact the validation process has on delivering an effective cycle.

Just to talk about that decontamination cycle for a second. Many factors impact the delivery of efficacy; Temperature, Humidity, Dewpoint and Saturation, Concentration, Delivery mechanism – Vaporised (evaporated), Nebulised (spray or ultrasonic), Ionised (with a plasma arc) to name just a few and combinations of all the above and more, Hot spots, Cold spots – the list goes on.  Some say it’s a black art. Well for many years that has been the perception.

The solution to continuous monitoring

Welcome to the 21st Century and a digital validation tool called an Enzymatic Biological Indicator. The EI is everything we need to deliver successful decontamination validation quickly, simply and in a way that is palatable.

The EI is a biological challenge. Its an enzyme from a very hardy thermophile. Many times, more resistant to inactivation that the current Geobacillus norm. The enzyme inactivates biologically. Just like a BI the EI delivers a measure of decontamination efficacy rather than an indication of “chemical in the vicinity of the test” such as a chemical indicator. Even the very best chemical indicators only deliver a measure of chemical in the area rather than a measure of biological inactivation such as a BI or EI can.

The enzyme on the EI is used to catalyse a bioluminescent assay. Put simply the enzyme before inactivation can deliver a known and measurable volume of luminescence. Exposure the oxidisation processes such as hydrogen peroxide and ethylene oxide degrade and inactivates the enzyme in a time and dose responsive way and as such less luminescence is produced.

The EI efficacy measurement process consists of four parts;

The EI test strip, the Luminescent Assay, A reader called a Luminometer and a digitally delivered automatic reading process and results generation from a software platform called Athena.

The EI test process has been engineered to deliver a robust test. It is not binary, rather the EI test is quantitive. Each EI delivers a measure of efficacy that correlates simply to Log reduction that ranges from <2.5Log to >9 Log from a simple, singular test. The EI does not need to be wrapped in a protective Tyvek blanket to protect it. So positioning of the EI is easy, the test is more usable.

The reader fully automates the reading process. If you can press one button you can be a master of the EI process in less than an hour’s training and the best part. The test takes only sixty seconds. Yes, in just 60 seconds EI’s deliver accurate process efficacy results.

The time that EI’s save is incredible. The value EI’s deliver is clear and the technology is rapidly becoming the validation tool of choice for pharmaceutical manufacturers around the globe.

EI technology has been developed by the UK’s Public Health England over the last fifteen years and Protak Scientific is the globally exclusive licensee of the technology. Protak have engineered the technology specifically for use in cold oxidisation process decontamination applications, with imminent certification to ISO 13485 and pending FDA registration the technology can be relied upon to deliver a qualification tool that meets all expectations of todays forward thinking industry.

It really is very simple, EI’s measure decontamination efficacy simply and easily. EI’s take away all the hard work, all the guesswork and deliver total cycle mapping and understanding on the very first cycle. EI’s deliver qualification in a way that totally radicalises the industry and does so with a high degree of reliability. EI’s are not inoculated like BI’s they are manufactured with a very high degree of accuracy and batch variance is less than 5% VS the 350% for BI’s.

Now is the time to say Bye bye to the BI and hello to the EI. Now is the time to move from a 19th century process to a 21st century technology that has been engineered for the process and task at hand.

Protak Scientific is working with the UK’s MHRA to establish an industry focus group to meet with the MHRA at its new offices in Canary Wharf to discuss with the regulator directly how the new EI technology can be introduced and implemented within industry. If you would like to know more about EI technology or how you could go about joining the industry group, please contact Protak directly.

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