Bioburden Testing

Viroxy helps manufacturers enumerate the population of viable microorganisms in medical devices for effective validation and control of sterilisation process.

For a sterilisation process to be effective on a newly manufactured pharmaceutical product or medical device, manufacturers must be aware of the number, properties and characteristics of microorganisms that occur in the products naturally or while going through the production process. This is a crucial step prior to setting sterilisation parameters such as dosing, temperature etc. to ensure that the process is effective and the product safe when used. The purpose of bioburden testing is to enumerate the quantity of viable microorganisms on a pharmaceutical product, medical device, component, raw material, or package before sterilisation. Bioburden testing is also an important indicator of problems in the production process which could lead to product recalls later.

In theory, the bioburden testing process is quite simple and can be illustrated as follows:

Step 1 Prepare Sample

A sample of the test product is prepared depending on the physical characteristics of the product:

Water soluble products, non-fatty products that are not water soluble, fatty products, fluids or solids in aerosol form and transdermal patches each require different type of solvents.

Step 2 Isolate Microorganisms

Samples are typically tested through membrane filtration method or plate-count method. Other methods include most probable number (MPN).

At Viroxy, we rarely conduct tests using MPN method as it is least accurate.

Step 3 Culture Microorganisms

The incubation period, temperature and culture media can each affect the growth rate of microorganisms such as aerobic and anaerobic bacteria, yeast and mold. This in turn can affect the test result.

Step 4 Enumerate Microorganisms

Viroxy’s enumeration methods include:

1. Total Aerobic Microbial Count (TAMC)

2. Total Yeasts and Molds Count (TYMC).

Step 5 Prepare Report

The findings are recorded and a report is prepared, detailing the outcome of the test.

In practice however, bioburden testing is a tedious process that must be undertaken by qualified microbiologists using verified methods. At Viroxy, bioburden testing is mostly conducted using membrane filtration method or plate-count method outlined in USP 61 and ISO 11737-1:2006.

Membrane Filtration Method

In membrane filtration method, the sample is filtered separately through 2 membrane filters with pore size no greater than 0.45µm. One of the membranes is the transferred to the surface of Soybean-Casein Digest Agar and incubated to determine the TAMC. The remaining membrane is transferred to the surface of Sabouraud Dextrose Agar and incubated to determine the TYMC. Soybean-Casein Digest Agar and Sabouraud Dextrose Agar are culture mediums prepared specifically to promote the growth of aerobic microorganisms and yeasts respectively.

Plate-Count Method

Plate-count method can be further divided into pour-plate method and spread-plate method. Both methods are the same in principle but contain a minor variation in technique. In the pour-plate method, the sample is mixed with Soybean-Casein Digest Agar or Sabouraud Dextrose Agar before incubation. In the spread-plate method, the sample is spread on the solidified surface of Soybean-Casein Digest Agar and Sabouraud Dextrose Agar before incubation.

The microbial content of a product or device is expressed in colony forming unit (CFU) and this number is used to determine the bioburden level. Pharmaceutical products typically have an acceptable range and type within which the bioburden content should fall.

Bioburden testing is useful to:

Validate and control sterilisation process
Monitor the quality of raw materials, components and packaging
Highlight manufacturing process weaknesses
Evaluate the effectiveness of cleaning measures