Product Characterization and Analysis for Microbials

Brian Belliveau, Ph.D., Health Evaluation Division, PMR

Chris has already elaborated on some of the points that I'm going to discuss now regarding the potency estimation and product guarantee or, what is referred to in the U.S. as, the certification of limits.

The guaranteed amount of the microbial-active ingredient must be expressed in units of potency or another expression of activity, such as colony forming units in the case of bacteria or fungi. Or it could be the counted number of spores per unit weight or volume.

It is very important that the analytical methods used to verify activity of the MPCA be described in sufficient detail, whether they're standardized methods or not, including their limit and level of sensitivity, their reproducibility, and their statistical validity, as well as be supported with representative data.

One or more methods should be used to express the guarantee or verify the certified limits of the microbial agent. If you're planning to express the active ingredient guarantee in colony forming units on your product label, then plate counts would be your obvious choice of method, or units of activity from bioassays if that's more relevant.

One of the things that we like you to do is provide a theoretical discussion on the formation of various other things (unintentional ingredients) that could be associated with your microbial agent such as any allergens which may be specific to your particular active ingredient or have been reported in closely related strains. Any microbial toxins or other toxic metabolites that have been reported in other strains or species closely related to your active ingredient. And this goes back to what Chris discussed about having very good, and usually multiple, methods to distinguish your active ingredient from other microorganisms and not just contaminants, but also to separate your strain from others of the species including mutants.

Microbial contaminants especially the potentially infective or antagonistic forms. Any side products resulting from chemical reactions during the manufacturing process or fermentation residues from the growth of bacteria or fungi should be also discussed.

Any extraneous host residues, for example, from viruses produced in vivo or in vitro cell cultures or any other living forms. And residues of contaminants remaining after purification or extraction as well as any impurities in the chemicals that have been used in the manufacturing process. So if you're not certain of the purity of the ingredients you're using in your formulation, then you should find out.

One of the other things that we require, which has been covered off already in the discussion on manufacturing methods, is details on the analytical methods for the active ingredient, including the methodologies for detection, isolation, and enumeration, as well as quantification of the entire organism or parts thereof or specific chemical components or metabolites.

And apart from the methods used to estimate potency, more than one method may be necessary to distinguish the active ingredient from other closely-related strains or the unmodified forms in the case of genetically engineered microorganisms. As well as to monitor the active or relevant metabolite(s) during production, quantified doses for infectivity and toxicity testing, and enumerate viable forms of the active ingredient in tissues.

So these are methods that should be developed prior to safety testing because they could be relevant to the infectivity component of the tests when you have to monitor such things as clearance of the MPCA from the test animals.

As Chris already stated, the presence of microbials contaminants must be checked during the manufacturing process as well as in the final technical grade of the active ingredient (if there is one) and end-use product.

One of the things that we ask you to check for is the presence of certain indicator organisms which include total heterotrophic aerobes, total coliforms, fecal coliforms such as E. coli, fecal streptococci and enterococci, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella species as well as Shigella and Vibrio, and yeasts and molds.

We also ask that the levels of contaminating microorganisms not exceed those consistent with product safety and performance. It may therefore be necessary for us to set limits for some microbial contaminants. But to emphasize another point that I raised yesterday, is that there must be demonstrated absence of primary human pathogens in your product.

The methods and the criteria for contaminant testing should be consistent with international standards that are employed in the food industry or related industries such as supplements or probiotics. We recommend as a starting point (and we won't necessarily hold you to it) that you use methods that are consistent with the International Commission on Microbiological Specifications for Foods or the AOAC or any other internationally recognized body that has validated methods to screen for and identify contaminant microorganisms in food or other commodities.

The details of the chosen methods, whether it's microspecific or selected-growth media used to assay for these microbial contaminants must be submitted and the validity and specificity, sensitivity and reliability of the detection method must be reported.

As well, any other unintentional ingredients that may be present must be assayed and any toxic or sensitizing substances that may be present at any stage of the manufacturing process must not exist in the final end-use product in quantities that would pose a hazard to either humans at the time of application and post-application or to nontarget organisms.

The precise and detailed methodologies for identification and analysis of these unintentional ingredients must also be provided.

Now, one thing I touched on yesterday in my presentation on submission pitfalls was the requirement for storage stability testing. We require on Canadian labels both a date of manufacture and an expiry statement. And part of the reason for this requirement, again, is to ensure activity of the active ingredient remains at the claimed level (that is, minimum guarantee) to ensure field performance.

As a result, we require that you submit storage stability data to verify any claims of stability, including duration and conditions of storage. In designing a storage test, you must also be able to assess maintenance of the product’s physical properties such as suspendibility, wettability, and viscosity. The primary consideration in the storage stability test, however, is to assess maintenance of the certified limits of activity or potency under the conditions of storage that will be stipulated on the label, including the effects of temperature and light.

And one of the final things we require in the characterization data package is a summary or description of the physical and chemical properties of the end-use product, including its physical state, density or bulk density, specific gravity, viscosity, corrosion character (such as the oxidizing or reducing action), as well as it suspendibility and wettablity or moisture content. Submission of the methods used to measure these properties is also recommended.

And I think that's the last slide.