CIP Failures Due to Biofilm Formation: Strategies for Prevention and Remediation

Introduction

A Clean-in-Place (CIP) system aims to remove all undesirable materials from product contact surfaces to a level such that residues remaining are of minimal risk to the safety or quality of the product. While the general focus is on the removal of product residues or chemical residues, there is often a microbial control element involved. This includes the avoidance of the formation of biofilms. The term ‘CIP’ partly reflects the process, yet a CIP process is as much about disinfection as it is about cleaning. The cleaning and disinfecting aspects of CIP are complementary processes; neither alone can achieve the production of high-quality and safe products.¹

To achieve microbial control, the CIP cycle needs to remove product residues (these can serve as nutrients for any bacteria remaining on a product-contact surface) and destroy or physically remove any bacteria. It is also important to maintain conditions in process equipment during non-use periods, which prevents the growth of microorganisms.

This article is a follow-up to “The Problem of Biofilms and Pharmaceutical Water Systems” (American Pharmaceutical Review, 2017) and it looks at the objects of the CIP process and outlines measures to prevent biofilm formation and to address biofilms in the event they develop.

Clean-In-Place

CIP has been used in the biopharmaceutical (as well as the food) industry since the 1950s and has been commonplace since the 1980s, providing the advantage of automation and the ability to be qualified through replicate cycles in comparison with manual cleaning. Through CIP a variety of pipes, vessels, equipment, filters, and different fittings can be cleaned and sanitized without the need for their disassembly.

A standard CIP cycle consists of:²

• Pre-rinsing with pharmaceutical-grade water (this can be purified water or water for injections, depending on the criticality). This water serves to remove disassociated and loosely attached residues. It is important to use softened water since ‘hard water’ containing calcium and magnesium salts can affect the properties of the cleaning chemicals.
• The use of a caustic agent (most commonly sodium hydroxide), which is flushed through. This caustic acts as the primary cleaning step. Sodium hydroxide is effective at decomposing lipids and proteins at ambient temperatures. Alternatives include sodium orthosilicate, sodium metasilicate, trisodium phosphate, sodium carbonate, and sodium hydrogen carbonate.
• The caustic agent is often re-circulated.
• This is followed by a second rinse using pharmaceutical-grade water.
• An optional acid solution wash can be conducted as a secondary cleaning step. The use of acid depends on the presence of mineral or protein deposits.
• If an acid is used, there will be a third flush with pharmaceutical-grade water to remove all traces of the cleaning solutions.
• An optional disinfectant solution wash can be conducted. The types of disinfectants used in CIP systems include chlorine (such as chlorine dioxide or sodium hypochlorite), quaternary ammonium compounds (such as ammonium didecyldimethyl chloride), amphoteric, electrolyzed oxidizing water, iodophors, glutaraldehyde, biguanides, ozone, peracetic acid, and hydrogen peroxide. The use of disinfectants both reduces the number of surface-attached viable microorganisms and prevents microbial growth during inter-production periods.³
• Selecting the correct disinfectant and combination is important. Microorganisms in biofilm communities are more resistant to disinfectants compared with organisms in other natural states.⁴
• Care must be taken that the disinfectant does not cause structural damage to the equipment (such as surface pitting or rouging). Many disinfectants require copious rinsing to remove any remaining residues.
• A final hot water circulation is typically performed.
• The equipment is then dried using compressed air (which has normally passed through a sterile filter to avoid recontamination). The drying step is critical since any remaining moisture provides an environment that can encourage microbial growth.

Sometimes, to minimize water, chemical, and waste-treatment costs, cleaning solutions are recirculated.

The CIP standard efficacy can be measured routinely by performing a conductivity test, where results established above a threshold during validation can signify the remaining presence of cleaning residues. During cycle validation, an additional chemical test for total organic carbon is performed to ensure that no product residues remain (or other organic substances). For microbial control, both swabs and rinse samples are necessary.

CIP Design

For a CIP unit to work effectively, good design and build is of paramount importance. Superior design issues include:^5,6

• Ensuring that supply and return piping is separate from process piping.
• Ensuring all connections between cleaning solution circuits and product circuits are constructed to prevent the mixing of product and cleaning agents during processing.
• Designed to permit CIP solutions to be pumped through process piping at a rate to ensure turbulent flow. The flow should be designed to have increased shear forces beyond product flow rates).
• The CIP unit recirculation tank and all interconnecting piping, pumps, and valves should be fully self-cleaning and drainable.
• At the end of any completed program, the system should be as clean as the equipment to which it was connected.

In terms of equipment to be cleaned by CIP, it should be designed to optimize the cleaning process:^7,8

• Equipment should be designed and constructed so that it can be cleaned before each use.
• The surface material should be optimal for the cleaning chemicals and cleaning process (e.g., stainless steel is typically easier to clean than aluminum).
• All surfaces must have neither ridges nor crevices which could harbor organic material.
• Surfaces should be polished and be of a suitable surface smoothness. Cleaning and disinfection efficacy is influenced by the physicochemical properties (topography and roughness) of contaminated product-contact surfaces.
• Assemblies must be designed in such a way as to reduce projections, edges, and recesses to a minimum. These should preferably be made by welding or continuous bonding. Screws, screwheads, and rivets may not be used except where technically unavoidable.
• All product-contact surfaces must be easily cleaned and disinfected, where possible after removing easily dismantled parts.
• The inner surfaces of equipment must have curves of a radius sufficient to allow thorough cleaning.
• Liquid deriving from products as well as cleaning, disinfecting, and rinsing fluids should be able to be discharged from process machinery without impediment.
• Machinery must be designed and constructed to prevent any liquids or living organisms, or from any organic matter accumulating in areas that cannot be cleaned.
• Machinery must be designed and constructed so that no ancillary substances (e.g., lubricants) can come into contact with the product.

Key Parameters

The most important parameters are the concentration of the cleaning chemicals; the contact time; the efficacy of the automated process (such as the turbulent flow rate of the rinsing water); and the temperature of the cycle.⁹ Each of these creates a variable that needs to be established during qualification and one that needs to be controlled upon completion of the validation.

The balance of these four parameters is also important, as established by the German chemist Dr Herbert Sinner during the 1950s. Sinner developed a model for the effective cleaning of materials through washing, represented as a circle with four sectors:^10,11

• Chemistry – selecting the appropriate chemicals is important, especially when attempting to remove water-insoluble soil. With cleaning chemicals, alkaline detergents have a saponification capacity and acid detergents dissolve mineral deposits.¹²
• Mechanical – this can refer to water flow; any mechanical scrubbing; contact pressure; and the frequency of movement. Increases in water velocity, such as >1.6 meters per second, can result in significant biofilm detachment; hence the degree of turbulence is an important measure of system suitability.
• Temperature – certain soils are easier to loosen at higher temperatures. Higher temperatures can also assist in reducing the contact time.
• Time – the longer the contact time, the easier the soil is to loosen and the easier it becomes for the cleaning chemicals to break the physicochemical bonds holding the soil to the surface.

Hence a Sinner circle might look like as per Figure 1 (in this case, with each of the four variables being of equal value):

Operational Problems

When used according to the established validated parameters CIP is very efficient. The main concerns are often with aging equipment and the impact on the established parameters. Further complications can arise when equipment is used outside the intended use, such as the introduction of products of greater concentration or with the use of entirely various products compared with those for the initial qualification.

Another problem, and the focus of this article, is where a biofilm develops.

Biofilms

Biofilms are problematic to pharmaceutical water systems and to equipment that comes into contact with water. If a biofilm develops then an out-of-control situation is likely to emerge. Such a situation can often only be detected from point-of-use samples since several excursions are normally required to shift from an isolated event toward the probability of a biofilm. By this time, it may have become established and hence harder to treat.

Biofilms are natural communities of bacteria, and they are quite common in nature. Biofilms are bacterial populations that have attached and then adhered to a surface, as well as to each other. Attachment is the product of forces - electrostatic interactions and London-van der Waals forces. Adherence is the second step that leads to irreversible attachment. This takes longer (often days or months), and it occurs through several species within the community secreting a polysaccharide coating, termed glycocalyx (or exopolysaccharide), which is ‘slime-like’ and very ‘sticky’.¹³ The function of the coating is to encourage the attachment of other bacteria; trap nutrients; and provide a degree of protection.¹⁴ The organisms within the biofilm undergo physiological changes and undertake different advanced forms of communication (quorum sensing), signaling changes to the environment to each other. Quorum sensing controls bacterial group behaviors in complex and dynamically changing environments. In addition to cross-species communication, bacteria can also share genetic information including switching genes that confer antimicrobial resistance.

The requirements for biofilm formation reads like a simple recipe: a given concentration of planktonic bacteria, plus water, plus a surface, plus available nutrients (including a carbon source). The nutrient levels need only be relatively low.¹⁵ Ironically, the extremely low levels of nutrients within pharmaceutical-grade water probably help to direct bacteria towards developing a biofilm community as part of a survival strategy.

Biofilm formation undergoes several steps. Initially, the attracted bacteria are held in a state of reversible attachment. This, over time (minutes rather than hours,) becomes an irreversible attachment. Within a few days micro-colonies form, leading to an enlarged mass and the establishment of a mature biofilm community.¹⁶ In general, the morphological group Gram-negative bacteria form biofilms more readily; one reason is due to appendages on the bacterial cell that allow such bacteria to attach to surfaces more easily.¹⁷ These are the types of bacteria that are more often associated with water systems. A second reason for promoting bacteria is the surface charge which can attract organisms towards the surface.

Why Might Biofilms Form?

Design weaknesses are a common reason for enabling the conditions for biofilm formation, especially where chemicals cannot penetrate or where stagnant water remains. Other design issues relate to the use of non-sanitary valves and the type of material used for the pipework; the finishing of the pipework; the diameter of the pipe; the velocity of the circulating water or the presence of dead-legs (bends in the pipe where the water velocity slows).

Yet in a well-maintained item of equipment with a validated cleaning cycle, how might biofilms form? Wear and tear are an obvious reason. To this can be added service and calibration failures and as mentioned above, the extension of a cycle designed for one specific product for a different product without verifying whether the adopted cycle is appropriate.

Biofilm formation is also associated with pharmaceutical manufacturing plant shutdowns. Here the equipment may become contaminated if it is inadequately stored, and an entrenched biofilm might prove too stubborn for the standard cleaning cycle. A related reason is due to periods of very low water use, which affects the velocity of the water flow.

Another risk emerges following repairs and maintenance, such as the need to cut into the pipework stemming from an item of equipment or with the water supply. Cutting in is necessary to replace pipework and sometimes to change a valve.

These factors create conditions that can make a biofilm more or less likely to develop. However, the presence of bacteria – including specific species – is required, and a low level of nutrients. Time is an important dimension since sufficient time is required for bacteria to deposit into a surface and transition from a reversible to an irreversible state. Quorum sensing plays a significant role in the development of biofilms. This is to the extent there is often a clear connection between quorum sensing and biofilm formation. Quorum-sensing bacteria produce and release chemical signal molecules such as autoinducers (for example, many Gram-negative bacteria release an autoinducer called LuxI/LuxR). Another autoinducer called AI-2 allows for inter-species communication – perhaps a form of “universal language, enabling cross-species communication.¹⁸ These molecules tend to increase in their concentration as a function of cell density, thereby increasing in importance the larger the biofilm community becomes.¹⁹ Importantly, quorum sensing enhances resistance via altering biofilm development, bacterial secretory systems, and bacterial efflux pumps.²⁰

Preventing Biofilm Formation

Poorly designed storage and distribution systems create opportunities for re-colonization and ultimately product contamination. However, biofilm formation can be prevented from the outset with good control design principles. Pipes are more likely than any other part of the water system to develop contamination if they are poorly designed (or improperly maintained).²¹

With pipes and other parts of the CIP system, some important design elements include.²²

1. Ensuring smooth internal surfaces in tanks and pipework. This is because microorganisms adhere less well to smooth surfaces compared with roughened surfaces. This additionally makes corrosion resistance and regular steps to avoid rouging important design features (such as electropolishing of stainless steel).
2. Having continuous movement of the water in tanks and rapid flow in pipework. Flow rate is necessary for minimizing the opportunities for microorganisms to adhere to surfaces (and hence form biofilms). Where shear forces are sufficient, microorganisms adhere poorly to surfaces. In terms of general guidance, typical flow velocities in the range of 1-2 m/s tend to be suitable.
3. As well as continuous movement, areas where water flow can slow down or stagnant pockets of water can develop need to be avoided. This includes so-termed ‘dead legs’, which are pipe branches that are too long to cause turbulence to occur with the water flow. The length of branch pipes should always be minimized.
4. Valves must also be meticulously designed so they do not allow water to remain. This can occur with some user points. This is overcome with the use of zero dead-leg valves, together with sloping drainage.
5. Where leakage occurs, this must be quickly addressed, since this can allow bacteria to ingress from the external environment.
6. Use of filters on air vents for water storage tanks; this also helps to minimize the ingress of microorganisms.
7. The use of coated surfaces on pipes and in tanks, where appropriate (so as not to pose a risk of leaching toxic substances) can help to address biofouling.

It is also important that after maintenance work is performed on the CIP unit, full system sanitization should be performed.

With equipment to be processed by CIP, the good practices associated with cleaning verification should be observed: having limits on dirty hold times and ensuring that cleaned equipment is dried following processing and stored in a secure area away from dirty equipment and water aerosols.

Controlling Biofilms

Where biofilm communities are present, these can be controlled by maintaining water at an elevated temperature (the temperature required will be somewhere between 65 and 80°C). This option is only available if the system has been designed as a very hot water system. One example of this is through Steam in Place (SIP). Steaming is used in the pharmaceutical industry to further reduce the microbiological content in the manufacturing equipment. SIP either sanitizes or sterilizes equipment depending on contact time and temperature. Before effective SIP is achieved, an effective CIP needs to be run to ensure surfaces are clean to avoid any problem with heat penetration.

Alternatively, ozone can be used to maintain an environment above 10 parts per billion ozone, with the ozone removed at critical junctures for health and safety reasons. Outside of these conditions, the biofilm may flourish or, if the design is inadequate, the alternate conditions may allow biofilms to form.

Eliminating Biofilms

Even when good design and maintenance approaches are used, biofilm formation can still occur. For such occurrences, attempts are required to eliminate the biofilm community. Three methods can be considered for CIP systems to remove microbial contamination. These are heat, chemical additives, and physical activity.²³ With heat, either increasing the water temperature to 65°C to 80°C or ‘steaming’ by elevating the temperature within the region of 121°C and ensuring the temperature is held for a sufficient contact time (typically 10-15 minutes), as with a conventional Steam-in-Place cycle.

With chemical treatment, there are a range of potential chemicals with the choice being partly dependent upon material compatibility. As indicated above, detergents (either alkali or alkali followed by an acid) can be effective at removing a biofilm from the surface as well as addressing chemical or product residues. The application of disinfectants, such as chlorine, chlorine dioxide, hydrogen peroxide, or peracetic acid can address surviving microorganisms. The concentration of the chemical will vary depending upon the agent; with chlorine, chlorination can be achieved with minimum levels of 0.2mg/liter of free chlorine.

Physical activity in combination with heat and chemical treatments can enhance the process, such as agitating through mechanical means to enhance the properties of turbulent flowing water. It can be possible, in some cases, to add objects like a rubber ball into pipework to increase the agitation effect.

There are other novel methods, although these are not necessarily easily compatible with conventional CIP. These include ultraviolet light and bioelectricity. With ultraviolet radiation, this is effective at a wavelength of 254nm for a re-circulating system where water flows over a multiple-lamp system. Bioelectric effects require the use of two electrodes and direct current.²⁴

Ultimately, if these treatments do not prove effective, the final recourse is to disassemble equipment and replace it with new pieces or to discard the equipment altogether.²⁵

Summary

This article has examined the application of CIP within the pharmaceutical sector, looking at some of the specifics of the technology and the qualification of the system. The article has proceeded to consider approaches for biofilm prevention and remediation. Where biofilms are present, sometimes multiple strategies are required (given one selected treatment may not be effective against all types of biofilm).

In general terms, CIP systems can be effective in tackling biofilms when soil-covered biofilms are treated with a detergent,t and after the soil has been solubilized a hot water rinse should be applied. After this, the application of a sanitizer should kill any exposed attached microorganisms, and these can be removed through a second hot water rinse. Such an approach can help keep biofilm development to a minimum.

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Author Details 

Tim Sandle- Head of GxP Compliance, Kedrion BioPharma, UK

Publication Details

This article appeared in American Pharmaceutical Review:

Vol. 28, No. 2

March 2025

Pages: 20-25

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