Definitions And Scope

The word bioburden has been ascribed subtly different meanings, not only in scientific texts, but also in official documents. Many authorities use the word simply in a quantitative sense, i.e., in a way suggesting merely a determination of numbers, with little or no specific mention of types of organisms present. ISO 111341, for example, defines bioburden as "Population of viable microorganisms on a raw material, component, a finished product and/or a package." More commonly, however, the word implies both quantitative and qualitative characterization; thus, the glossaries of terms used in FDA2 and the European Community3 guidance on good manufacturing practice (GMP) explain bioburden in an identical manner:

© 2004 by CRC Press LLC 115

"The level and type (e.g., objectionable or not) of microorganisms that can be present in raw materials, Active Pharmaceutical Ingredients (API) starting materials, intermediates or APIs. Bioburden should not be considered contamination unless the levels have been exceeded or defined objectionable organisms have been detected." It is in this latter sense that the word will be used in this chapter.

Characterization of a microbial population could be taken to mean determining the relative numbers of all the different species present, and this implies identification of organisms. Clearly, identification of all organisms comprising the bioburden is not normally practicable, although the regulatory expectation of identification of organisms that regularly appear in successive batches of material, and comprise a major fraction of the bioburden is quite manageable. Unfortunately, details of identification procedures are outside the scope of this chapter, but reviews of the rapid automated methods now widely employed in the industry appear in this publication4 and elsewhere.5

Although bioburden determinations are commonly applied to solid and liquid raw materials, intermediates and finished manufactured medicines, they are also required during manufacture and immediately prior to sterilization of medical devices. Because such devices cannot usually be sampled by the procedures employed for medicines, bioburden determinations require surface-sampling techniques like swabbing and the use of contact (Rodac) plates that are more commonly employed in environmental monitoring.6

2 FACTORS INFLUENCING THE BIOBURDEN AND TESTING REQUIREMENTS

The bioburden of a product will be influenced by a variety of factors including, but not necessarily limited to, the following:

• Microbiological quality of raw materials and components (including containers and packaging)

• The manufacturing environment, i.e., organisms present in the atmosphere, on working surfaces, plant and equipment and on personnel

• The nature of the manufacturing process, which might promote microbial inactivation or, alternatively, support microbial proliferation, depending on exposure of product or components to the various temperatures, pH values, organic solvents, etc. employed

Of these factors, the quality of the raw materials is, for many nonsterile medicines, the one that has the most profound influence on the microbiological quality of the finished product. The high standards required for manufacturing premises, and the application of procedures designed to restrict or eliminate opportunities for microbial proliferation in water-containing materials during manufacture, now largely ensure that little additional contamination is introduced during the manufacturing process itself. While there is a lot of compendial emphasis on bioburden determinations on finished products, investment of effort at the start of the process to fully characterize the raw materials is likely to be very cost-effective for control of final product quality.

While the major compendia describe procedures for both quantitative and qualitative bioburden tests (Table 5.1), it should be emphasized that adoption of these procedures alone, without regard to the impact of the above-mentioned factors that influence the bioburden, may not be sufficient to ensure regulatory approval. The scope of testing and validation required should be determined not merely by the unquestioning application of compendial tests, but by a consideration of the potential impact of all aspects of the manufacturing process, and the intended use of the product on its desired microbiological quality. It is well-established, for example, that "natural" materials of animal, vegetable or mineral origin are likely to possess a higher microbial count than synthetic ones, and in many cases they are more likely to contain potentially pathogenic organisms. On this basis, there would be a regulatory expectation that any excipient of natural origin would be subjected to detection tests for relevant objectionable organisms, regardless of whether the material in question was the subject of a pharmacopoeial monograph.

Potential objectionable organisms in different nonsterile dosage forms have been tabulated in a recent paper in Pharmacopeial Forum1 and are listed in Table 5.2. It should be noted that this table is based directly upon the original paper, and reasons are not clear for the omission of certain potential pathogens from some of the categories, e.g., Pseudomonas aeruginosa is absent in five product categories for which the less hazardous species P. fluorescens is listed.

On this same basis of adapting the testing protocol to the nature and use of the product, it may be necessary, for example, to enumerate the presterilization bioburden of spores prior to a terminal sterilization process and, depending upon the validation scheme for the sterilization process (bioburden or overkill approach), quantify their resistance parameters. Total viable count (TVC) procedures would also need to be modified for a product likely either to contain strict anaerobes, or, by virtue of a low redox potential, support anaerobic growth during use, since standard TVC procedures only enumerate strict aerobes and facultative anaerobes.

Table 5.1 identifies the major compendial, international standard and regulatory documents pertaining to bioburden determinations. The first two of these categories in particular give detailed accounts of the procedures to be adopted for enumeration and detection of specified organisms, and it is not the purpose of this chapter to reproduce this information. Rather, it is intended to identify and explain the issues that impact on the selection of methods, reliability of data and relevant validation.

Table 5.1 Official Methods, Standards and Guidelines on Bioburden Determinations

Compendial Methods

International Standards

Regulatory and Professional Association Documents

EP 2003:

• 2.6.12 Microbiological examination of nonsterile products (total viable aerobic count)

• 2.6.13 Microbiological examination of nonsterile products (test for specified microorganisms)

• 5.1.4 Microbiological quality of pharmaceutical preparations

USP 26:

• <1111) Microbiological attributes of nonsterile pharmacopeial products

• <1227> Validation of microbial recovery from pharmacopeial articles

• <1231> Water for pharmaceutical purposes (microbiological considerations)

• <2021> Microbial limit tests — nutritional supplements

• ISO 8199 (1988) Water quality — General guide to the enumeration of microorganisms by culture

• ISO 6222 (1999) Water quality — Enumeration of culturable microorganisms — Colony count by inoculation in a nutrient agar culture medium

• ISO/TR 13843 (2000) Water quality — Guidance on validation of microbiological methods

• ISO 11737-1 (1995) Sterilization of medical devices — Microbiological methods — Part 1: Estimation of population of microorganisms on products

■ European Commission (2002) The Rules Governing Medicinal Products in the EC Vol 4: Good Manufacturing Practice (reproduced in the Rules and Guidance for Pharmaceutical Manufacturers and Distributors, 2002, U.K. Medicines Control Agency)

■ U.S. FDA Center for Drug Evaluation and Research (2001). Guidance for Industry Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients

■ Parenteral Drug Association (PDA) (1990) Bioburden recovery validation. Technical Report 21

■ ASTM (1991) Standard Practices for evaluating inactivators of antimicrobial agents used in disinfectant, sanitizer, antiseptic or preserved products. Document E—1054—91 U.S. FDA Bacteriological Analytical Manual online (2001) AOAC International

Table 5.2 Potential Objectionable Microorganisms in Nonsterile Dosage Forms*

Organism

Dosage Form

Oral

Oral

Topical

Vaginal

Rectal

Otic

Nasal

Inhalants

Solid

Liquid

E. coli

X

X

Salmonellae

X

X

Aeromonas caviae

X

X

X

Aeromonas hydrophilia

X

X

X

Aeromonas sobria

X

X

X

Plesiomonas shigelloides

X

X

Shigella spp

X

X

Vibrio cholerae

X

X

V. para-haemolyticus

X

X

Yersinia enterocolitica

X

X

Y pseudo-tuberculosis

X

X

Burkholderia cepacia

X

X

X

X

X

X

X

Pseudomonas fluorescens

X

X

X

X

X

X

X

P. aeruginosa

X

X

Serratia marcescens

X

X

X

X

X

X

X

Staphylococcus aureus

X

Staphylococcus

saprophyicus

X

Candida albicans

X

X

X

Klebsiella spp.

X

X

X

Proteus spp.

X

Enterococcus spp.

X

Moraxella catarrhalis

X

X

Aspergillus fumigatus

X

X

A. flavus

X

X

Cryptococcus neoformans

X

X

»Adapted from Cundell (2002)7.

3 SAMPLING

Limited information is provided on sampling by the pharmacopoeial chapters or sections relating specifically to microbiological testing. The USP 26 simply states, in <61> Microbial Limit Tests, "Provide separate 10 ml or 10 g specimens for each of the tests called for in the individual monograph." The EP is rather more helpful; it specifies in Section 2.6.12 the requirement for a sampling plan, mentions some of the factors that might influence plan design, and provides an example of a plan applicable to products in which the bioburden might be not be distributed uniformly. Additionally, the EP indicates the acceptability (or need) to prepare composite samples by mixing the contents of several containers to provide sufficient bulk of material for testing. This practice also facilitates sampling from all parts of the batch. Both pharmacopoeias, however, say little about such aspects as the timing of sample collection, operator training, maximum transportation times, storage temperatures, and validation.

Bioburdens are not static, and there is the potential for microorganisms not only to grow in water-containing materials but also to die in anhydrous materials, due to nutrient deprivation, desiccation, etc. The potential for proliferation is well recognized, and TVCs are usually performed on samples taken at intervals during vulnerable stages of the manufacturing process. The possibility of the count, or a specific fraction of it, declining, is less frequently considered. Gram-negative bacteria in particular are relatively sensitive to desiccation, so they might be recovered from a dry, raw material at a low level after a period of storage yet have been present in higher numbers immediately after receipt. This may have implications for the endotoxin load in parenteral products.

When testing raw materials, bioburden samples should be taken by personnel adequately trained in aseptic techniques in order to avoid contamination of the samples or the bulk material from which the sample is withdrawn. Sterile containers, measuring vessels, etc., and the use of sterile gloves are required along with other precautions including facemasks, hair covering and gowns, depending upon the susceptibility of the material to environmental contamination. To minimize the risk of contamination from airborne organisms, sampling should not take place in areas of high personnel movement or air turbulence. Clearly, the interval between sampling and testing should be minimized, but if transportation time to the laboratory is significant, evidence must be obtained to confirm that the bioburden does not change during that interval. Maximum acceptable transport or storage temperatures and times must be validated. Standard operating procedures (SOPs) should be written by a microbiologist, and, if necessary, they should be prepared in collaboration with laboratory staff familiar with the practical problems that might be posed by sample product. SOPs need to be product-specific. It is easy for a generic SOP describing sampling procedures to fail to address the problems that might arise from the physical nature of the material, or from the container in which it is stored. Aseptic sampling might be problematic if, for example, the product is not a free-flowing solid or liquid, or it is in a container like a sack that is difficult to open without introducing contamination and, possibly, even more difficult to reseal.

Samples should, of course, be representative of the bulk material or the manufactured batch, so it is normal to remove material from different parts of the bulk or from the beginning, middle, and end of the batch. The pharmacopoeias specify the quantity to be taken in the absence of specific indications in individual monographs, but these quantities, typically 10 ml or 10 g, might be reduced if, for example, the batch was a small amount of expensive material. In this case it would be necessary again to demonstrate that the quantity taken was adequately representative of the whole. Samples of manufactured products should be taken after primary packaging to ensure that the bioburden contribution from that source is taken into account.

The subjects of sampling schemes and the statistics that support them can be complex and Kuwahara8 has provided a detailed account written from a microbiological perspective. The EP describes a sampling scheme (2.6.12) whereby five individual samples from a batch are tested independently, and assigned to one of three classes based upon the TVC recorded: acceptable, marginal and defective. The batch passes if none of the five values exceeds the monograph limit by a factor of ten or more (no defectives) and not more than two samples are in the marginal category with a TVC between the prescribed limit and ten times the limit. Despite its inclusion in the pharmacopoeia, it is not commonly used, however, because, for the great majority of raw materials and finished samples, the recorded bioburden is low or absent. This means that there are no samples in the marginal category, and increasing the sample number by a factor of five is an unnecessary waste of time and consumables.

The requirement to avoid extraneous contamination of specimens is just as important during the laboratory investigation of bioburdens as it is during specimen collection, so the facilities required are generally those of a Hazard Category 2 containment laboratory, and the manipulations should be undertaken in a HEPA-filtered, horizontal laminar flow cabinet (or biological safety hood for potentially hazardous specimens). The differences between laminar flow cabinets and biological safety hoods are not readily appreciated by many newly recruited personnel, and these differences, together with a knowledge of when each type of cabinet should be used, are important components of a staff training programme. It is necessary to emphasize:

• That a horizontal laminar flow hood only protects the product from operator-derived contamination and affords no operator protection

• The circumstances when a sample might contain a pathogenic organism. This obviously depends upon a company's product portfolio, but samples associated with vaccine manufacture and mammalian viruses as possible contaminants of certain biotechnology products are two possible generic examples

• The circumstances when a sample might require a biological safety hood for reasons unrelated to microbiology and infection, e.g. when the sample is toxic by inhalation

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