In the first part of this series on bioburden control, Chris Davis defined biopharmaceutical products, outlined the risks inherent in biopharmaceutical processes and examined how to prevent microbial contamination. In this concluding part, he examines the control measures that can be applied to the process, such as steam-in-place and hot-water sanitisation, and the increasing use of single-use technology. The bioburden control measures applied to the process are listed in Table 1:

Steam-in-place (SIP) Heat to 121oC and hold; Generally by direct injection of clean steam
Hot water sanitisation (HWS)   Contact all surfaces of the equipment with hot water (over 65°C) for a defined time
Chemical sanitisation   Treat the equipment with a chemical sanitant that has a validated sanitisation effect
Bioburden reduction filtration   0.2 micron absolute filtration
Single-use technology   Supplied as sterile (irradiated)  
Table 1: Bioburden control measures
Most environmental contaminants are vegetative organisms and therefore sensitive to microbicidal chemicals and/or temperatures greater than 65°C. Sodium hydroxide is commonly used as a general sanitising agent in biopharmaceuticals as it is effective, cheap and does not give rise to waste disposal issues. It does, however, need to be rinsed away before processing starts. SIP is commonly applied to process vessels, which provides a robust hygienic environment that, when combined with influent 0.2 micron filtration, allows the process stream to be held for extended periods, subject to validation of the hold time. Biopharmaceutical processing uses systems that are thermally sensitive and cannot be subject to SIP, such as chromatography matrices, ultrafiltration membranes and some filtration systems. Therefore these will be sanitised, either chemically or thermally if possible. Sanitisation can also be used for equipment that is part of a system that contains elements that cannot be subject to SIP. There are no fixed rules governing what form of sanitisation has to be used in what application and it is the responsibility of the process owner to justify what is used where. Table 2 gives a comparison of SIP and HWS. This indicates that HWS can offer significant benefits over SIP in that it can be a simpler operation requiring simpler equipment, however the levels of bioburden reduction are much lower.
Parameter SIP HWS
Microbial Inactivation Excellent Good
Regulatory Acceptance Excellent OK
System Complexity High Low
Operational Complexity High Low
Operational Time High Low
Hold Time After Sanitisation High Low
Capital Costs High Low
Operating Costs High Low
Table 2: Comparison of SIP and HWS
When assessing the methods of sanitisation for process equipment it is important to determine how long equipment will need to be held between sanitisation and processing. If equipment is subject to SIP it could, in theory, be held indefinitely, however if equipment is subject to sanitisation the hold times will be significantly less, as the bioburden levels will not have been reduced to zero and will be slowly increasing as the equipment is held. The increase in microbial numbers can be significantly reduced by ensuring the equipment is dried after sanitisation, using 0.22 micron filtered air, as vegetative bacteria need water to multiply. However drying equipment is time consuming and difficult to validate. It is good practice to minimise equipment hold times to minimise the risks to the process. [caption id="attachment_32510" align="alignright" width="300"]bioburden-control-regime CLICK TO ENLARGE Figure 1: Typical biopharmaceutical process bioburden control regime[/caption] No matter what methods of equipment preparation are used the regulatory authorities will expect operators to set maximum hold times for equipment and have the justification for those specifications. Figure 1 illustrates a typical biopharmaceutical process bioburden control regime where, typically, facilities may use SIP in areas that need to be free from bioburden (such as fermentation and cell culture), and areas that are low bioburden, HWS & chemical sanitisation.

Single-use technology

Within the industry, the use of single-use processing systems is becoming increasingly common, with some facilities being designed for almost entirely single-use equipment. Single-use systems consist of bags, similar in design to intravenous (IV) bags used in hospitals, and tubing to allow filling, sampling, additions and transfer operations. The systems, ranging in size from hundreds of millilitres to 5,000 L with custom-designed bag holders, are supplied as sterile systems and can include probes and liquid filters if required. These now include systems for all parts of the process including microbial fermentations, mammalian cell culture, depth filtration systems, solution preparation and storage, chromatography and ultrafiltration. The scale limitations of each system differ and are constantly changing as the industry develops and innovates. The advantages of single-use systems are that the level of bioburden control is high, 0.2 micron filtration into sterile bags, and there is no need to subject systems to CIP, sanitisation or SIP regimes. The advantages, in terms of operation and capital cost, have been well documented and focus on reduced capital equipment, fewer equipment preparation activities and reduced validation effort. The physical design of a facility for use with single-use systems is generally different to those designed for fixed plant, in that the areas for fixed plant become lay-down areas for the storage of the filled bags and areas where the bags are located for processing. Additional circulation space is required to allow the bags to be moved around the facility. There is however an upper limit on the scale of operations with single-use systems and many of the large capital projects currently ongoing exceed this limit.

Conclusions

Whilst adequate bioburden control is of paramount importance to the production of biopharmaceuticals this can be achieved through a variety of techniques including cleaning, SIP and hot water sanitisation and careful control of process additions. The blanket use of SIP across all areas of the process is a common approach. However, there is a discernible trend away from this and an increasing reliance on sanitisation. Whilst sanitisation can provide significant capital and plant complexity reductions the sanitisation activities need to be carefully managed, as part of a holistic contamination control strategy, in order to ensure the integrity of the process is not compromised. The increased use of single-use systems, either as part of a large fixed stainless-steel facility or as the mainstay of smaller facilities, can offer significant benefits. Author: Chris J. A. Davis CEng FIChemE, Jacobs Engineering Ireland Acknowledgements The author would like to thank Thakur Rathi (Jacobs Ireland) and Shane Breslin (Jacobs Ireland) for their help in preparing this article.