The Importance of Not Being Too Attached: Pharmaceutical Equipment Characteristics and Microbial Attachment

Wednesday, October 20, 2021

Tim Sandle Head of Microbiology and Sterility Assurance Bio Products Laboratory Limited, Hertfordshire, UK

Introduction

An important factor for consideration when cleanroom equipment is selected is the surface finish and surface roughness, particularly in relation to stainless steel given the commonality of this material, along with anodized aluminum, to construct pharmaceutical processing items. Stainless steels such as types 304 and 316 have no antibacterial properties; therefore, specifying appropriate finishes and roughness should form part of facility specifications and be part of Quality by Design. Durability and resistance to corrosion represent some important reasons as to why material grade, finish, and roughness matter. In addition, these specifications are also probable determinants of the likelihood of microbial attachment and hence material specifications need to be considered within a facility contamination control strategy. Focusing on microbial attachment, this review article assesses available research into surface finish and roughness and uses the outcome to set out the important considerations when developing a facility material surface specification.

Pharmaceutical Equipment and Surface Specifications

Different types of austenitic stainless steel are used around the world. The differences relate to composition, strength, resistance to corrosion, versatility, and price. Generally, in pharmaceuticals, stainless steel composed of chromium-nickel alloys is used; commonly these are 304 and 316 grades, as per the AISI/SAE standard. The smoothness of the finish is generally achieved by electropolishing with the aim of ensuring that friction is reduced (this produces stainless steel with a finish that appears highly reflective and smooth). While both grades are of equivalent strength, when corrosion is a direct concern (as with a pharmaceutical water system or where specific disinfectants are used) the 316 grade is the grade of choice as this type is more resistant to corrosion than 304 grade). Rouging is the most common form of corrosion, as small spots on the surface begin to rust as water molecules oxidize some of the iron. There is a not inconsiderable cost difference between these grades and for both grades compared with other stainless-steel grades since it is difficult and expensive to control surface roughness during manufacturing. Electropolishing and grit 4000-polished steel also add a level of corrosion resistance, when compared to grit 80- and 120- polished surfaces (higher grit scores correlate with lower surface roughness).1  Electropolishing is achieved using an electrolyte bath in which the metal acts as an anode and the process removes a layer of metal and smooths the surface. Microorganisms themselves can cause corrosion especially when a biofilm develops, with corrosion caused by microbial metabolic activities or secreted metabolites. Stainless steel is not immune from such corrosion, especially in the form of ‘pitting’ (indentations occurring in the surface), although this is to a lesser degree compared with other forms of steel, such as carbon steel. Microbial-induced corrosion fosters conditions for future bacterial attachment unless the surface is treated and re-passivated using dilute nitric acid.2

Finish and grade are not, however, commensurate with surface roughness.3 There are many different roughness parameters in use, but the Ra measure is by far the most common.4  Hence, finish and roughness are necessary factors to specify when outlining hygienic design requirements. Despite the importance of these measures, it remains that too few facilities specify for both surface finish and surface roughness.

Surface roughness is associated with microbial attachment and adhesion, as well as entrapment. Attachment occurs when cell appendages stick to a surface; adhesion is a non-specific mechanism that occurs when adhesive molecules expressed on the bacterial surface bind to the material surface (the association adherence is related to a specific bacterial ligand-receptor interaction).5 It follows that increased surface roughness is associated with increased bacterial attachment.6,7 Further supporting experimental data, stainless steel specifically has demonstrated that the possibility of bacterial contamination and the potential for early biofilm formation on stainless steel surfaces can be reduced provided that a combination of mechanical and electrochemical treatments are performed, and that the resultant surface morphology and chemical reactions are optimal.8 With stainless steel achieving this is rarely ubiquitous as slight variations occur due to the random nature of the surface structure.9

Limitations of Ra

The most commonly reported surface roughness parameters are average and root mean square (RMS) roughness (Ra and Rq respectively), which are both measures of the typical height variation of the surface. Of these, Ra is the most common measure. Ra is the average of a set of individual measurements of the peaks and valleys of a surface (‘roughness’). There are different methods for assessing the Ra value, such as using the stylus method. Here, as a stylus travels across the surface, the movement of the stylus is amplified and the signal recorded. The resulting Ra value (or “microns Ra”) is the arithmetic average value of the deviation of the trace above and below the centerline.10 Criticism of measures like Ra include the measure offering no insights into the spatial distribution or precise shape of the surface features like peaks or valleys, as well as pointing out that different measures of roughness can produce very different results when the same material is examined.11 Nevertheless, Ra remains the  accepted industry marker for surface roughness and is favored by engineers as it informs about wear and tear (the higher the Ra value the greater the friction force and the faster a material will wear).12 Ra is also important for those concerned about contamination control.

Characteristics for Surface Attachment

Most microorganisms prefer being attached to surfaces (the sessile state), one reason is that this provides more opportunities to obtain nutrients. Microorganisms will remain present on surfaces by mechanisms such as attachment, adhesion, adherence, or by entrapment. Adhesion and adherence in particular are the product of material type and prevailing conditions, although roughness of the surfaces is also a contributing factor. Entrapment is perhaps the variable of greatest concern when considering surface roughness. In relation to Ra values, studies have demonstrated a stronger possibility of microbial entrapment at 0.9 μm compared with 0.8 μm.13,14 Therefore, the point at which surface roughness is deemed significant appears to be around Ra = 0.82 μm. This is why an Ra of ≤0.8 μm has become the common standard for hygienic design considerations.15 There are variations in terms of microbial type. For example, Staphylococcus aureus (cells 0.5–1 μm diameter) are retained in higher numbers on surfaces with microtopography pits of Ra ≥0.8 μm, along with Streptococcus oralis (0·5–1·0 × 1·0–2·0 μm),16 compared with Pseudomonas aeruginosa (cells 1 μm × 3 μm), which are less often retained.17 This is not only a product of cell size, but it also relates to different morphological cell shapes, which possess different surface energies, as is the case with rod and coccoidal shaped bacteria. Even within the same microbial species, there are variations of which the most important appears to be bacterial cell surface hydrophobicity (repulsion). This determinant can be shown using a hydrocarbons test and contact angle measurement test and it appears to be of particular importance during the first 30 minutes of microbial cell contact and the commencement of adhesion.18 The formation of a biofilm community leads to the microbial population becoming very difficult to remove. A biofilm is a community of microorganisms embedded in extracellular polymeric substances that function to protect sessile cells from the outside environments.

It also stands that the risk becomes lower the smoother the surface is at below 0.8 μm along a sliding scale (at descending 0.1 μm increments) and this continues to a point where anything smoother ceases to make a difference.19 Research places this marginal point as 0.16 μm.20 Moving in the other direction, roughened stainless steel of an Ra of >1.6 Ra μm has a higher association with bacterial surface reliance; and an Ra of 5.38 μm and higher has been shown to be completely unacceptable for any form of effective cleaning with the hope of removing microorganisms or appreciable levels of soil.21 With the Ra being an average value there may be variations with the substratum surface roughness in different locations across the surface.22 Nevertheless, the margin between 0.8 μm and higher values conforms to the international standard for surface roughness: ISO 4287.23 Under the standard, an N6 notation corresponds to an Ra of 0.8 μm; whereas the next lowest grade, N7, corresponds to an Ra of 1.6 μm.

Surface finish

Surface finishes can range from smooth and shiny to rough and non-reflective. Generally, surface finish is a factor that increases or decrease the likelihood of corrosion. The method of finishing is also of importance, with a correctly polished stainless-steel surface showing a better resistance to corrosion than a surface that is roughly or poorly polished. The type of surface finish can contribute to reducing the likelihood of microbial attachment together with the grade of the stainless steel. Chemical analysis has found that the difference in the compositions of the passive surface layers on grades 304 and 316L stainless steel is the presence of molybdenum for the type 316 steel, with 316L recording lower levels of microbial attachment when both grades of steel are subject to equivalent microbial challenges under the same conditions.24 While finish is a factor, the majority of studies indicate that surface finish is not the primary factor in relation to microbial attachment, unlike the factors of roughness or damage.25,26 This is provided that the surface finish is electropolished and uniform. For example, one study looked at the difference between surface finish and surface roughness by testing 304 grade stainless steel composed of nine different surface finishes, in relation to ‘cleanability’. The finishes included: 1D hot rolled and pickled (a relatively rough surface), 2B cold rolled and mechanical polished, and electropolished. Cleanability was assessed by using both Geobacillus stearothermophilus (spores) and Pseudomonas sp. biofilm. A milk powder was used to represent a soiling substance. The sanitization agent was 100 ppm hypochlorite.

The results analysis indicated that the number and size of surface defects was the optimal determinant for bacteria remaining on the surface rather than finish type.

Surface roughness

Surface roughness is the measure of the finely spaced micro- irregularities on the surface texture which is composed of three  components, namely roughness, waviness, and form. The assessment of roughness of a surface relates to the design of the process used to manufacture the material, such as certain milling processes being designed to produce smoother services than others.27 As well as being more important than surface finish, the influence of surface roughness appears to be more influential than other physicochemical properties, such as the surface free energy (which is the excess energy that the surface has compared to the bulk of the material).28

The reason why surface roughness is important is because an increased degree of roughness generally leads to an increase in the contact area. This is because the larger the contact area is, the larger the attachment force will be.29 This is a generalization because microbial cells exhibit flexibility in terms of shape and variations in physiological shape in relation to attachment.

Within a controlled environment, roughness is not a static concept for surfaces will be subject to periodic cleaning and disinfection and ineffective cleaning will be influential for ensuring microorganisms remain, especially when soil remains.30 This is because microscopic observations demonstrate how the influence of the shape and size of surface irregularities affects the level of residual soil after cleaning,31 as well as microorganisms remaining. This introduces variations with chemicals, application methods, and personnel competency, each of which are additional determinants for microorganisms remaining on a surface after attempts have been made to ‘clean’ the surface. The criteria for cleanability are close to the criteria for attachment, with the Ra range that best facilitates the ease of cleaning being 0.4 to 1.5 μm.32 Since cleanability is not necessarily the same as long-term attachment, the 0.8 μm material specification remains an important purchasing requirement.

Design Considerations

As well as standard requirements for finish and roughness of the material, an important design factor is with welding. Bacteria have been observed to colonize preferentially near welds as a result of surface roughness in relation to certain equipment designs. In particular, as bacteria begin colonizing, attachment occurs most greatly on the grain boundaries of the base metal (between the weld and the base).33 The principles of hygienic design should also extend to the avoidance of macroscopic faults such as crevices and sharp corners. Research by Riedewald led to the recommendation that “scratches or faults deeper than a multitude of the particle diameter are significantly more difficult to clean than the mother plate material”.34

Different design considerations may be required for equipment within the cleanroom and materials used for water systems. Of the  two, surface roughness is more significant for materials immersed in water, under both static and turbulent flow conditions (albeit with flow velocity being a key variant),35 compared to equipment held within the cleanroom and subject to an atmospheric environment.36 Hence, 316 stainless steel will always be preferred for liquid immersion rather than 304 grade. Uniformity is also of importance as microbial attachment is aided by surface irregularities in water pipework as bacteria are protected from shear forces and hence, they can utilize more attachment points on the substratum.37

Surface Damage

Damages to surfaces will make the surface rougher and introduce abrasion and crevices that can influence the likelihood of microbial adhesion or entrapment. Here, the degree of damage is important (as indicated earlier, adhesion becomes easier across a surface as surface roughness increases).38 The morphology of damage is also a factor. For instance, one study determined where damage is linear (as with scratches), then cocci are more likely to become trapped than rod shaped bacteria, which is related to cell alignment and the binding energy within or across surface features.39 Another factor is the size of the abrasion: if the features are considerably larger than the microbial cells, then retention may be less significant (or at least more ‘cleanable’; whereas, if the features are close to microbial cell dimensions, then microbial retention is more likely to be a problem. Correcting damage is an important part of preventative maintenance for microbial communities as biofilms are more likely to cause corrosion when the stainless-steel passivation film is damaged than with an intact electropolished and suitably smooth surface.

Conclusion

Specifying surface finish and surface roughness for materials used in pharmaceutical facilities is important for durability, corrosion resistance, and microbial attachment. Roughness levels are more important than finish. It is important that pharmaceutical specifications include at least an assessment of the Ra levels and that material of Ra ≤0.8 μm is selected for water systems, product contact equipment, and equipment used where microbial contamination is a concern (such as aseptic processing). This is necessary for ensuing the appropriate hygiene criteria of a surface are in place and this consideration should form part of the contamination control strategy.

References

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