Improving the sterility assurance of the sterility test: How the Steritest® System leads to enhanced controls

Image: Sterility test incubation (by Tim Sandle)

For conducting the pharmacopeia test for sterility the membrane filtration is the method of choice because all of the contents of a small volume product are filtered or at least half the contents of a large volume product are passed through a membrane filter. Therefore, a much larger sample size is evaluated than is so for the direct inoculation method (where the amount of product can vary from 1- 2 ml to half the container contents). 

By Tim Sandle

Furthermore the method is more adept at overcoming interfering factors; for any microorganisms present are far more likely to be separated from potentially inhibitory substances in the product through the act of filtration or, should they remain, they can be eliminated by rinsing the filter.



READ MORE: Raising the bar with sterility test compliance: Merck’s M-Trace® System


However, not all approaches to sterility testing are the same and there are differences in relation to the test method, test environment, test controls and so on. These variances, if not controlled, can adversely impact upon the sterility assurance of the test1. By considering these variances, as discussed in this article, the sterility assurance of the test can be strengthened.

Membrane filtration method

Membrane filtration is the appropriate method for all aqueous, alcoholic, oily and solvent products that can pass through a sterile filter with a porosity of 0.45mm. The standard filter is manufactured from cellulose esters or other similar plastics. The filter acts to separate the product from any microorganisms so that the product passes through the filter and any microorganisms present in the product are trapped within the filter matrix. A rinse solution (such as phosphate buffered saline, saline or Ringer’s solution) is used to remove any product residues (due to the risk of antimicrobial activity posed from the residues)2. This washing process is normally performed three times. The maximum number of rinses permitted in the pharmacopeia is 5 x 100 mL.


To support the membrane filtration method, demonstrably effective culture media must be used. This requires the use of soya-bean casein digest (SCD) and fluid thioglycolate media (FTM) that can support the growth of a range of microorganisms, and which can be introduced into the test environment safely, to avoid cross-contamination and with the containers being sufficiently integral to avoid the seepage of any decontamination agents3.

Achieving improved sterility assurance with the Steritest® System

To avoid cross-contamination from the operator and test environment, the membrane filtration systems needs to be enclosed, such as with the Merck Steritest® System (first introduced in 1974). This system minimises risk of contamination by reducing transfer steps and by minimising the possibility of contamination deposition (Van Doorne et al, 1998). Furthermore, there are no open containers or membrane manipulations, which could increase the risk of contamination, supported by an effective ergonomic design .


While there are different closed systems on the market, not all systems have proven container integrity. The Steritest® System has been developed with robust sealing technology and verified not to allow any ingress from the external environment. It is additionally important that a supplier can provide supporting information for the integrity of the membrane filter. When assessing the filter, the ability of the filter to cope with the expected pressure applied to the system is important (and once established, the selected system should have the ability to carefully control and to program the pump speed). The control of the pump speed not only allows proper product splitting between the 2 pathways, and hence the 2 media for incubation, but it also helps to guard against products from foaming and it reduces the physical stress applied to any microorganisms that might be present to aid their subsequent cultivation.

Strengthening sterility assurance: Test controls

During testing it is prudent to include a negative control. There are different ways to perform a negative control and in a sense there is no right or wrong approach. One common way, with the membrane filtration test, is to filter a bottle of the rinse medium through the same batch of the filtration kit used to conduct the test, and then to add culture media. This provides an indication of the rinse fluid, culture media, test kit and testing environment.


Negative controls are most useful when a sterility test failure occurs. The result of the control may indicate a false positive (in a circumstance where both the sterility test and the negative control fail). Controls should also be trended and out of trend situation should be reacted to even where the sterility test is satisfactory for negative control failures in themselves are indicators of a potential loss of environmental control or poor technique.

Strengthening sterility assurance: Environmental monitoring

During the sterility test, environmental monitoring should be conducted. This is in order to show that the test environment was satisfactory during the time of testing and, should a sterility test fail, to provide a means for assessing whether the contaminant arose from the test environment rather than from the manufacturing process (a complicated process, requiring genotypic microbial identification)4.



The types of environmental monitoring samples will be the same as per the monitoring of pharmaceutical facilities in relation to the viable counting methods:

• Settle plates
• Active (volumetric) air-samplers

Which are taken at some stage during the test session.

• Contact plates. Swabs
• Finger plates of the operator’s gloves (or of the gloveport gauntlet, should an isolator be used)

Which are taken at the end of the test session.

The limit, given the grade of the environment (ISO class 5 or the equivalent PIC/S GMP Grade A) should be ‘no growth’ (effectively 1 CFU - colony forming unit) per sample. The results should be trended. Repeated occurrences of microbial counts from environmental monitoring should trigger an investigation and such results may indicate a loss of environmental control.


Media should be assessed prior to release by conducting growth promotion testing. When testing media, the microbial challenge must be of a low level, at less than 100 colony forming units (CFU). The inoculation challenge must be verified at the time of testing via a plate count. At the same time as conducting the test the use of a previously released batch of the same media as a control provides a useful comparator should anomalous results be seen with the medium under test5.

Particle counting is not normally conducted during the test session, and it not recommended here due to the nature of the activities, which are likely to generate particle counts. It is prudent, however, to periodically ascertain the particle levels within the isolator or mobile unidirectional airflow cabinet within which the sterility test is conducted when such devices are not being used for testing.

Strengthening sterility assurance: Controlled environments

There are two types of environments within which the sterility test can be conducted. These are:


a) A unidirectional airflow (UDAF) cabinet contained within a classified cleanroom;

b) An isolator.


Of the two, the isolator affords better control by virtue of it being a barrier device the analyst is separated from the product under test. However, both types of environments, if they are not properly maintained, or if the methods for transferring materials in and out are not validated, can lead to cross contamination and hence lead to the risk of false positives occurring. Nonetheless, the use of isolators, when used correctly, has reduced incidents of false positive6. Isolators are most commonly sanitised using surface contact disinfectants: hydrogen peroxide vapour (an alternative is ionized hydrogen peroxide) or peracetic acid.

Membrane filtration systems can that be permanently situated inside of the isolator help to minimise the risks of transferring equipment into and out of the controlled environment.

The equipment entering the controlled environment ,including the membrane filtration apparatus and pump, needs to be of an appropriate design (such as the quality of the surface finish) an easily decontaminated using disinfectant agents.

Summary

There are a number of factors that can aid the sterility assurance of the sterility test. The article has looked at the controlled environment, environmental monitoring, negative controls, and the design of membrane filtration equipment and methods. These factors should be considered when building in quality by design into the sterility test process. To support this, there are considerable advantages to be drawn from technology like the Merck Steritest® System.

References

1. Sandle, T. (2013) Sterility Testing of Pharmaceutical Products, PDA / DHI, River Grove, IL, USA

2. Proud, D. W. and Sutton, S.V.W. (1992): Development of a Universal Diluting Fluid for Membrane Filtration Sterility Testing, Applied and Environmental Microbiology, March 1992, Vol. 58, No.3, p1035 – 1038.

3. Ohresser, S., Griveau, S. and Schann, C. (2004): Validation of Microbial Recovery from Hydrogen Peroxide-Sterilised Air, PDA J Pharm Sci Tech. 58 (2): 75-80.

4. Sandle, T. (2012). ‘Environmental Monitoring: a practical approach’ In Moldenhauer, J. Environmental Monitoring: a comprehensive handbook, Volume 6, PDA/DHI: River Grove, USA, pp29-54

5. Sandle, T. (2010): The Media Kitchen: Preparation and Testing of Microbiological Culture Media in Sutton, S. (ed.): Laboratory Design: Establishing the Facility and Management Structure, DHI / Parenteral Drug Association, Bethesda, MD, United States, ISBN 1-933722-46-0, pp269-293.

6. Ackers, J., Agalloco, J. and Kennedy, K. (1995): Experience in the Design and Use of Isolator Systems for Sterility Testing, PDA Journal of Pharmaceutical Science and Technology, 49(3): 140-144


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)