Micro­bial cont­a­m­i­na­tion moni­toring

Heterotrophic Plate Count (HPC) is the only compen­dial method for micro­bial cont­a­m­i­na­tion moni­toring today. A serious concern for imple­menting a CCS as described in Annex I, is the fact that HPC does not detect all microor­gan­isms in a sample and the time to the result is very long. Only colony forming units (CFU) are detected. Studies have shown that the number of CFU does not reflect the number of microor­gan­isms in a system. To grasp the real micro­bial situ­a­tion in a water purifi­ca­tion system, rapid methods should be consid­ered to gain more control.

Learn about why HPC Analysis results in a perma­nent under­es­ti­ma­tion of the bioburden and how you gain more control over the bioburden mani­fes­ta­tion of your water purifi­ca­tion system by using Rapid Micro­bial Methods (RMM). The EU GMP Annex 1 encour­ages the use of RMM as well.

Annex I mentions in Chapter 2.3 that “a Cont­a­m­ination Control Strategy must be imple­mented across the facility in order to define all Crit­ical Control Points.” This can also be broken down to the water purifi­ca­tion process. Manual sampling and HPC analysis inter­vals are defined throughout the PQ phase. Typical sampling inter­vals are: Daily… Weekly… Monthly. In process control is not regu­lated. But Annex I and other regu­la­tions...

Limi­ta­tions of HPC

Why does hpc not show the real bioburden?

Because only colony forming bacteria can be detected.

The popu­la­tion of microor­gan­isms in a water purifi­ca­tion system is very versa­tile. Microor­gan­isms may exist in different envi­ron­ments and phases of their life cycle.

The popu­la­tion consists of:

  1. Free floating indi­vidual organ­isms
  2. Organ­isms that are attached to parti­cles
  3. Organ­isms that are attached to any surface (Biofilm)
  4. Frag­ments of biofilm freely floating in the water

Understanding conglomerates and VBNC Microbes

Indi­vidual colonies rarely come from indi­vidual cells...
Bacteria in water tend to attach to each other or to surfaces or parti­cles like rouging, rubber, abra­sion from valve membranes e.t.c. They build biofilm or conglom­er­ates. Prelim­i­nary studies (published at Concept Heidel­berg: 2022) have shown that indi­vidual colonies on HPC rarely come from indi­vidual cells but from agglom­er­ates of cells. Knowing this, it’s clear that a colony forming units is not a reli­able measure for the real micro­bial cont­a­m­i­na­tion and thus leads to a perma­nent under­es­ti­ma­tion of the real biolog­ical load of the water.
The majority of the microor­gan­isms are so called VBNC (viable but not cultur­able)
Only colony forming bacteria can be detected. But the growth capa­bil­i­ties of microor­gan­isms depend on various para­me­ters. Viable but non-​culturable (VBNC) is a state of microor­gan­isms which occur espe­cially in phar­ma­ceu­tical water systems.   E.g. trans­fer­ring microor­gan­isms from a low nutri­tion to a high nutri­tion envi­ron­ment is applying high stress. This kind of stress may result in dormancy. Dormancy means that microor­gan­isms that have been exposed to envi­ron­mental stress stop growing and build so called “persister cells” which may be very resis­tant to disin­fec­tion and sani­ti­za­tion. They are not visible within HPC.  
Plate R2A Agar broadway engineering

moni­toring: new methods

Due to the limi­ta­tions of HPC and the increasing demands in micro­bial process control, different tech­nolo­gies for RMM (Rapid Micro­bi­o­log­ical Methods) are under inves­ti­ga­tion. Various suppliers and several phar­ma­ceu­tical compa­nies are looking for solu­tions to receive more infor­ma­tion about the bioburden in phar­ma­ceu­tical water. With the publi­ca­tion of the EU GMP Annex I, it is obvious that also regulatory authorities are interested in new methods that eliminate sampling error, improve process control and produce reliable data in shorter intervals.

As stated in the EU GMP Annex I: "...The adop­tion of suit­able alter­na­tive moni­toring systems such as rapid methods should be consid­ered by manu­fac­turers in order to expe­dite the detec­tion of micro­bi­o­log­ical cont­a­m­i­na­tion issues and to reduce the risk to product. These rapid and auto­mated micro­bial moni­toring methods may be adopted after vali­da­tion has demon­strated their equiv­a­lency or supe­ri­ority to the estab­lished methods..."
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  • Full insight of the current state of bioburden in the pharmaceutical water system
  • Trend monitoring allows for targeted planning of sanitization and maintenance works, due to their necessity (which also protects the heat and chemical sensitive materials)
  • Easy implementation of risk minimization
  • Automated data collection
Flowcytometry  the best RMM for pharmaceutical water systems
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Preview Product Video ASMB

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Learn more about Cont­a­m­i­na­tion Control Strategy and the 4 elements of it...


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