It is typical for each site to classify raw materials according to their origin (and likely microbial load or specific species risk) as a means of expressing their risk. Specific organisms can be those identified in the pharmacopeia and by the manufacturer as posing a patient risk for the given product and its route of administration. These organisms will require specialist agars / broths and enrichment test (or the use of a suitable rapid microbiological method).

Within Europe, the specific pharmacopeia chapters of note are:

Ph. Eur. 5.1.4: Microbiological quality of pharmaceutical preparations

Ph. Eur. 2.6.12: Microbiological examination of non-sterile products: total viable aerobic count

Ph. Eur. 2.6.13: Microbiological examination of non-sterile products: tests for specified micro-organisms

All raw materials are tested using two tests for Total Aerobic Microbial Count (TAMC) and Total Yeast and Mould Count (TYMC). When plate counting, it is important to note that the European Pharmacopoeia (Ph. Eur.) requires that all colonies on the Tryptone Soy Agar (TSA) are recorded as the result of the TAMC test and all colonies on the Sabouraud Dextrose Agar (SDA) are recorded as the result of the TYMC, irrespective of whether the colony forming units appear bacteria or fungal.

The test is performed using one of four different methods. The method chosen will depend upon the outcome of method suitability. The choice of method is hierarchical in that method ‘a’ below (membrane filtration) should be the first method attempted and method ‘d’ below (Most Probable Number) the last method attempted. 

a)    Membrane filtration
b)    Pour plate
c)    Spread plate
d)    Most Probable Number (MPN)

MPN is the most limited method because of its accuracy. In addition, the Ph. Eur. states that the MPN result can be used to provide the result of the TAMC test only. This is because the only test medium used is tryptone soya broth. Neutralisation effects can be overcome using buffers and rinses providing they meet the criteria specified in the Ph. Eur.

Other risk factors

As well as the origin of the raw material, there are other risk factors based on the characteristics of the material that can increase or lower the risk:

• Liquid or solid: Liquid samples are often a greater concern than pastes or powders due to water activity levels
• A neutral pH raw material is a greater risk than acidic or basic solution
• Materials with a higher water activity are more likely to support microbial growth

Supplier quality

An additional variable is the suitability of the supplier and whether they claim to be able to provide materials in accordance to a pharmacopeia grade. Raw materials should be purchased from approved suppliers and a microbial risk assessment of each raw material from each supplier should be performed [2].

The microbial limits for incoming raw material should be agreed in advance, with the manufacturer either taking established limits from the relevant pharmacopeia or setting appropriate limits based on pharmacopeia guidance (which in turn will be based on the route of administration for the product that the raw material goes into).

Testing frequency and limits

If microorganism contamination is a concern, the raw material lots should be tested prior to use. Alternatively, some form of skip lot testing can be adopted by risk assessment. Certain types of raw materials may be inherently antimicrobial (such as acids or bases) or show, through historical testing, that microorganisms are not ordinarily present.

Unless there is a reason to question the distribution of the microorganisms contained within individual containers, samples of raw materials may be pooled for testing.

For this, it is common to select the sample(s) at random from the bulk material or from the available containers of the preparation. To obtain the required quantity, mix the contents of a sufficient number of containers to provide the sample of the required weight or volume as per Ph. Eur. 2.612. This is typically 10g or 10 mL of the product to be examined, unless the material is of a very low density.

Unless stated otherwise in a pharmacopeial monograph, or identified by a risk factor, the limit shall be not greater than 103 for TAMC and 102 for TYMC cfu / ml or per g (as per Ph. Eur. 5.1.4.2). Due to the imprecision of the enumeration methods the pharmacopoeia interprets this as:

• 102 : maximum acceptable count = 200
• 103: maximum acceptable count = 2000

Where a different limit has been identified by risk assessment, this is based on:

• Nature of the product, whether the product is growth promoting, growth inhibiting, or antimicrobial
• Theoretical ability of the product to preserve microorganisms
• Material origin and likely presence of starting contamination
• Water activity
• History of the product and the length of the relationship with the supplier (including supplier audit)
• Point in the process where the product is used and any antimicrobial steps

The above may also lead to a consideration of any additional specified microorganisms to test.

The total count methods are to be validated to determine that the raw material - tested in the conditions applied against the method of choice - is not antimicrobial. This involves challenging the raw material with a low-level inoculum (typically less than 100 cfu).

The challenge is of each of the following micro-organisms:

• Staphylococcus aureus subsp. aureus    ATCC 6538
• Bacillus subtilis subsp. spizizenii             ATCC 6633
• Candida albicans                                   ATCC 10231
• Aspergillus brasiliensis                           ATCC 16404
• Pseudomonas aeruginosa                      ATCC 9027

The micro-organisms listed are those recommended in the Ph. Eur. For selective micro-organism testing the following micro-organisms are used for validation:

• Staphylococcus aureus subsp. aureus                               ATCC 6538
• Pseudomonas aeruginosa                                                 ATCC 9027
• Esherichia coli                                                                  ATCC 8739
• Salmonella enterica ssp enterica serotype typhimurium     ATCC14028
• Candida albicans                                                               ATCC 10231
• Clostridium sporogenes                                                     ATCC 19404

Some raw materials are tested for endotoxins using the Bacterial Endotoxin Test. Generally, the raw materials which require endotoxin screening are indicated in a pharmacopoeia monograph.

Inspection and transfer controls

When sampling or inspecting an incoming material, protective measures can be taken. This could include filtering the material prior to its use within the manufacturing area, or other measures such as pasteurisation.

Whether the raw material is filtered or simply inspected and sampled, all handling operations should be performed under clean conditions. If this is within a classified cleanroom, it should be the same grade as the area in manufacturing where the raw material is to be dispensed. In turn, the area where the material is dispensed should be of the highest grade within which the material is to be used. All sampling implements must be of a low bioburden or sterile (where implements are recycled, a suitable cleaning and decontamination process should be in place).

Unless a raw material container is opened for sampling, all materials should be stored in closed containers under controlled conditions. Expiry dates need to be assigned (which could be the manufacturer’s recommended expiry date if a container is unopened). For containers that have been opened, a local expiry date needs to be assigned (this is often more to do with the potential reaction of a chemical within the atmosphere and less for reasons of microbial risk). 

These are necessary steps for achieving contamination control. The personnel performing both sampling and dispensing should be trained appropriately in aseptic techniques. As well as clean air control to protect the material, it may also be necessary to have protective airflows in place to safeguard operators ³

Change control

Changes in raw materials or product formulations should be considered through an appropriate change board. In addition, changes should be reviewed as part of the microbial contamination investigation, and if needed, an updated microbial risk assessment will need to be performed.

Summary

This article has set out some of the risk factors, sampling and test considerations for raw materials. The information contained within the article can be used to assist laboratories in performing risk assessments for incoming raw materials.