BY DR TIM SANDLE | 8 September 2023
Introduction
Cleanrooms and controlled environments are essential for all forms of pharmaceutical processing. To maintain a clean environment, contamination must be minimised and controlled. To do this requires an understanding of the different sources of contamination and the types of contamination associated with them.
There are five common sources of contamination within cleanrooms:
Figure 1: Major contamination sources in pharmaceutical environment
Often these contamination sources occur in combinations. For example, in a cleanroom contamination could potentially arise from air, personnel and equipment at different proportions during the same event.
In this RSSL series, we’ll look at each of these sources in turn, commencing with personnel.
Sources of contamination: Personnel
Microorganisms may be transferred to pharmaceutical preparations from the process operator. This is undesirable for sterile products, as it could result in patient harm. In the case of tablets and powders, such contamination could result in the spoilage of solutions or suspensions. A level of protection is afforded by personnel wearing masks, gloves and cleanroom suits, as well as through protective airflow and physical barriers from unidirectional airflow devices.
Our understanding of the microorganisms found on and in the human body has been advanced through the work of the Human Microbiome Project (HMP). The HMP has revealed a rich diversity of microorganisms carried on the human body. Prior to the adoption of genotypic methods, identification depended upon microbiological culture techniques, which presented a problem because many varieties of bacteria did not grow with culture methods. This, as the HMP research has now revealed, led to a significant underestimation of the different species and types carried on the skin.
The outcomes of the HMP research have shown that there is a high population on, and a considerable diversity of microbial species across, the outer layer of the skin. With bacteria there are around 1000 species upon human skin from 19 phyla. Of these, most bacteria can be categorised into just four phyla:
- Actinobacteria (51.8%) - a group of gram-positive bacteria with high guanine and cytosine content, such as Micrococcus, Corynebacteria and Propionibacteria
- Firmicutes (24.4%) – including the genera Clostridia and Bacillus
- Proteobacteria (16.5%) - a major phylum of bacteria, which includes a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera
- Bacteroidetes (6.3%) - composed of three large classes of gram-negative, non-spore-forming, anaerobic and rod-shaped bacteria
Surface materials and types
The type of surface and its relative smoothness is also a factor in bacterial adhesion. Surfaces differ in terms of surface morphology, such as fractal dimensions, Z ranges, and roughness (5). Roughness is irregularities in the material’s surface topography, and it is typically measured by Ra values, with values of <0.8 µm being considered optimal. Of the different materials available, electropolished stainless steel creates a surface of low-level of roughness, allowing fewer bacterial cells to attach (without electropolishing, when metal surfaces are machined, ground or lapped, an amorphous layer forms and this sustains the trapping of bacteria). The quality of the fabrication is also important - corners and sharp angled equipment fabricated of stainless steel often contains welds. Welds need to be smooth, otherwise they can influence bacterial accumulation and colonisation (6).
The grade of stainless steel is an important consideration. 316 Stainless steel is smoother than the type 304 material, and 316 grade has fewer microscopic surface scratches, grooves and associated deformations, making it superior for critical surfaces (7). However, even 316 can become damaged or corroded.
With the four phyla, the microbial diversity across the skin is not evenly distributed as the density and composition of the normal flora of the skin varies with anatomical local. The distribution of microorganisms varies by topography and there are three main ecological areas of the skin: sebaceous, moist and dry. Examples of microbial divergence include:
- Propionibacteria and Staphylococci species dominating the sebaceous areas (with a high oil content)
- Gram-positive cocci, primarily the Micrococcaceae, are found on the arms and legs (dry, calloused areas)
- Gram-positive rods are found in high numbers on the torso
- Staphylococci and Coryenbacteria are found together with some Gram-negative bacteria in moist areas
Table 1: Regions of the skin and the types of bacteria recovered
Surface damage and increased increased risk
From the above table, the common skin flora are usually non-pathogenic and either commensals (which are not harmful to their host) or mutualistic (offer a benefit). However, in relation to pharmaceutical manufacturing, the presence of such organisms remains problematic. The potentially pathogenic Staphylococcus aureus is found on the face and hands in individuals who are nasal carriers. Such individuals may auto-inoculate themselves with the pathogen or spread it to other individuals or food.
Another observation is that the ratio of the microorganisms recovered from the skin is relatively evenly divided between the aerobic and the anaerobic. The aerobic microorganisms tend to live on the outermost layers of the skin and the anaerobic microorganisms live in the deeper layers of the skin and hair follicles. The high numbers of anaerobes (notably Cutibacterium acnes) raises a potential implication for environmental monitoring, which tends towards the screening for mesophilic aerobic bacteria.
A further observation of interest is temporal, where longer-term data indicates that the microbiome alters over time. Furthermore, the species associated with the skin fungi change more often than bacteria. Moreover, variation occurs not only across different body locations, but between individuals, with men carrying more microorganisms than women. A further complication to understanding the skin microbiota is that the human skin microbiome may not be stable and may change over time as a person ages.
Notably the high moisture content of the axilla, groin and folds between the toes supports the activity and growth of relatively high densities of bacterial cells. High levels are also found on regions of the skin close to mucosal openings (like the mouth and eyes). Here there are some areas of the skin with higher moisture content, especially occluded regions where sweat does not easily evaporate (such as toe webs). Furthermore, with these occluded areas there are larger densities of microorganisms when compared with dry areas.
The density of bacterial populations at most sites are relatively low, generally in 100s or 1000s per square centimetre. The highest numbers of microorganisms are isolated from the sweat gland. With the numbers of microorganisms located on a given area of the skin, the numbers present at any one time are limited.
Control measures
An element of personnel control is achieved by protection of critical activities through unidirectional airflow and isolators and by using the correct cleanroom grade clothing and discipline. The following instructions can control contamination through personnel:
- All workers who are directly associated with product manufacturing and handling must be thoroughly trained on hygiene standards
- Mandatory use of Personal Protective Equipment (PPE), including hand gloves, face masks, overshoes and hair caps inside the manufacturing unit must be standardised
- Ensure workers maintain high levels of personal hygiene
- Practice proper and regulatory-compliant workwear cleaning and disinfection measures
- Avoid direct contact between the worker’s hand and any part of the product
- Regulate access to production areas to ensure that only the correct and trained personnel can enter the manufacturing unit
While we cannot detect every type of microorganism found on the human body, we can ensure that our personnel related controls remain suitable for the cleanroom environment and understand the risks associated with operator gowning and behaviours.