The last twenty years has seen significant investments in biopharmaceutical manufacturing in Ireland and this is set to continue with the likes of BMS, Pfizer, Eli Lilly, Regeneron, Shire and Alexion all undertaking major biopharmaceutical capital projects in Ireland. Adequate bioburden control is of paramount importance to the production of biopharmaceuticals and this is achieved through proper equipment preparation, a combination of cleaning, steaming-in-place (SIP) and hot water sanitisation and careful control of process additions. Whilst the industry has seen a move away from the blanket use of SIP across all areas of the process this is still a common approach. The removal of SIP from process steps can provide significant capital and plant complexity reductions. However 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, perhaps limited by processing scale, can offer significant benefits. This article is based on a presentation given at a recent Engineers Ireland seminar on ‘New Approaches and Technologies in Biopharmaceuticals’. Before examining bioburden control itself, however, one must define what constitutes biopharmaceutical products and hence the importance of bioburden control, in order to place it context. A biopharmaceutical is typically defined as ‘any medicinal product manufactured in, extracted from, or semi-synthesised from biological sources’. They can be composed of proteins, sugars, nucleic acids, living cells or tissues. Biopharmaceuticals can also be defined by reference to their method of manufacture (generally isolated from natural sources: microorganisms, humans, plants or animals). They include a wide range of products such as:

  • Recombinant proteins, e.g. erythropoietin (used to treat anaemia) and insulin (used to treat diabetes);
  • Monoclonal antibodies, e.g. etanercept (used to treat autoimmune diseases) and trastuzumab (used to treat breast cancer);
  • Vaccines and
  • Advanced therapy medicinal products (ATMP), which are being developed to treat a wide range of conditions.
Biopharmaceuticals are a huge market with an estimated market value of over $500 billion (€445 billion) within the next five years.

Chemical and biological pharmaceuticals

[caption id="attachment_32308" align="alignright" width="300"]figure-1-monoclonal-antibody Figure 1: Biological and chemical API[/caption] Up until the latter stages of the 20th century, most pharmaceutical products (active pharmaceutical ingredients, or API) were relatively small molecules produced by chemical synthesis. The differences between chemical and biological API (biopharmaceuticals) are outlined in Table 1 and it is these differences that require the different production methods and drive the regulatory requirements particularly with respect to bioburden control.
Feature Chemical Biological
Molecule Small, Well-characterised Large, complex Difficult to characterise
Reaction and process Well-defined chemical process Complex
Process conditions Organic solvent based, elevated temperatures and pressures Aqueous based, ambient (room/body) temperature and pressure
Product stability Good Poor
Susceptible to microbial contamination No Yes
Route of administration Oral (tablet, liquid), Injected Injected
Product sterilisation Terminal, thermal Aseptic processing, filtration
Table 1: Comparison between chemical and biological API
Biopharmaceuticals are large complex molecules whose biological activity is dependent on the three dimensional structure and therefore they have to be synthesised by biological systems. This is illustrated in Figure 1 (above), showing a monoclonal antibody and two small molecules and contrasting the differences in size and molecular complexity. Generally, biopharmaceutical processes consist of five functional steps, with each step including a number of different unit operations (as illustrated below in Figure 2):
  • Fermentation/cell culture – at the start of the process, the cells are cultivated in the fermentation (microbial cell systems) or cell culture (mammalian cell systems) stages;
  • Harvest and recovery – the cells, or cell debris (if the cells have to be broken to release the product) are separated from the product stream;
  • Purification – the product is then purified in a series of process steps, including chromatography and ultrafiltration;
  • Final purification – where the product stream is concentrated; and
  • Bulk filling – where the bulk drug substance is filled into containers for transfer and further processing into the final dosage form.

Why are biopharmaceuticals at risk?

[caption id="attachment_32305" align="alignright" width="300"]bioburden-control CLICK TO ENLARGE Figure 2: Typical biopharmaceutical process[/caption] Biological systems operate in aqueous-based environments at ambient temperatures and pressures and therefore are often unstable to solvents and extremes of pH or temperature. This defines the processing conditions for biopharmaceuticals and is why they are susceptible to microbial contamination. The biological molecules are a food source for microbes and the processing conditions are generally ideal for microbial growth. The elevated temperatures and pressures, and the absence of water in the solvent-based chemical processes, effectively protects the products from microbial contamination. In addition, the stability of chemical API means that, if required, they can be heat sterilised in the final container to produce a sterile product. Biological API need to be filter sterilised into sterile containers, thus requiring processing in a manner that maintains a sterile product. If a process or product becomes contaminated with microorganisms the ensuing microbial growth may destroy the product and/or may produce metabolic by-products that will not be removed by subsequent processing and could be toxic to the patient. Thus bioburden control of biopharmaceutical processes is of paramount importance. Bioburden control of the process is achieved by ensuring that the process equipment is clean and sanitary before processing starts, controlling the bioburden of all process contact raw materials and utilities, including in-process bioburden reduction steps (primarily filtration) where required, and then ensuring contact between the process and the environment is minimised to prevent adventitious contamination. In order to have bioburden control, one needs bioburden limits and specifications. Bioburden limits are set by the requirements of the product and the ability of the process to remove or inactivate bioburden. In most biopharmaceutical processes the final product will be sterile and impact of bioburden contamination of the process will be significant. Whilst the bulk drug substance will be defined as ‘low bioburden’ there is, in reality, zero tolerance of bioburden contamination. In terms of the process requirements for bioburden specifications the upstream process (fermentation or cell culture) will be axenic (free from living organisms other than the species required), with bioburden free media and additives, with the subsequent process steps, and associated solutions, defined as low bioburden.

Prevention of microbial contamination

In order to prevent microbial contamination of the process, the equipment must be sanitary (free from contamination) and the process protected from adventitious environmental contamination. Therefore it is necessary to:
  • Ensure the equipment is designed such that it can be easily cleaned without dismantling;
  • Ensure the process equipment is clean and sanitary before use;
  • Ensure the process stream is not exposed to the environment;
  • Ensure any process additions (gas/liquid/solid) do not compromise the bioburden specifications;
  • Include specific bioburden reduction steps where appropriate;
  • Document and validate the above.
Before processing, equipment must be cleaned and then sanitised. Cleaning is required to remove residual process material that could cause cross-contamination or could prevent effective sanitisation. In order to clean and sanitise equipment it must be designed for hygienic operations. This means smooth internal surfaces with no dead-legs or cervices that could harbour material. It is important to understand, in terms of biopharmaceutical API, the difference between sanitisation and sterilisation:
  • Sterilisation is defined as the absence of living organisms (1) and, in terms of pharmaceutical regulations, must be validated as such. Generally it is based on thermal processes that will inactivate heat-resistant bacterial spores and requires exposure to saturated steam at >121°C for at least 15 minutes.
  • Sanitisation is the reduction of microbial levels to below acceptable levels and can be carried out with either heat or chemical processes.
As bulk biopharmaceutical production is a low bioburden process, sanitisation is generally therefore the defined end point. However, as there is a zero tolerance of bioburden in the process, steaming-in-place (SIP) with sterilising conditions is commonly used as the sanitisation method of choice. In the next article, the authors examine bioburden control-measures such as steam-in-place, hot-water sanitisation, chemical sanitisation, bioburden reduction filtration and single-use technology. 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. References: (1) Rules and Guidance for Pharmaceutical Manufacturers and Distributors 2002. Sixth Edition. HMSO