This article is based on a presentation given at a recent Engineers Ireland seminar on biopharmaceuticals in Ireland, ‘A Global Perspective: Facility Design, Trends & Positioning for Growth’. It will outline the importance of process closure in biopharmaceutical production and how to undertake a risk assessment, process closure analysis, to support both a process design and operational procedures. Before examining process closure, one must understand the importance of bioburden control in biopharmaceutical manufacture. 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. Biological molecules are a food source for microbes and the processing conditions are generally ideal for microbial growth. Thus, biological medicinal products are susceptible to microbial contamination. If a process or product becomes contaminated with microorganisms, the ensuing microbial growth may destroy the product and/or produce metabolic by-products which will not be removed by subsequent processing and could be toxic to the patient. Thus, bioburden control of biopharmaceutical processes is of paramount importance. This control 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 drug product will be sterile and impact of bioburden contamination of the process will be significant. Whilst the bulk drug substance (or active pharmaceutical ingredient (API)) 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. 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. It is therefore essential that, when processing biological materials, one minimises open processes as ‘open’ steps will require additional protection to prevent environmental contaminants. The presence of ‘open’ steps can therefore have a have major influence on layout and area classifications of the facility. Facility design and environmental controls are not a factor when the process is closed and therefore it is important to define and document where the process is closed, where it is not and how it will be qualified.

Process closure analysis

Biopharmaceutical processes are, wherever possible, designed and operated such that the process stream is never exposed to the local environment, i.e. is closed (the attributes of a closed system are well documented (e.g. ISPE Biopharm Baseline Guide)). In order to support the development and/or operation of a closed process, a process-closure analysis can be carried out. This is a risk assessment that will evaluate the process operations to assess the degree of closure associated with the process in order to confirm that, where operations are not closed, the associated room classifications provide adequate protection against the risks of adventitious environmental contamination. It should be noted that, in this instance, the analysis is only looking at the risks of environmental contamination and not including wider product risks such cross-contamination. A process-closure analysis can be carried out both to support a process design, based on process flow diagrams, and to support operations, based on standard operating procedures and installed equipment. The fundamental process is the same in that one: 1. Identifies the process step to be reviewed and the associated boundaries; 2. Identifies the inputs, outputs and manipulations associated with the process step, and any other potential risk points; 3. Identifies the process requirements in terms of bioburden specifications. These are:
0 None Typically, these will be non-product contact utilities such as jacket services
1 Controlled bioburden Typically, these are process solutions that will undergo a bioburden reduction step. They are controlled in that one should know the bioburden loading onto the reduction step to ensure it is not overloaded.
2 Low bioburden Generally, the process stream, after cell culture/fermentation, is classified as low bioburden. This includes the bulk drug substance. In reality the bioburden levels should be zero.
3 Bioburden free/ axenic/sterile These will be upstream steps such as cell culture/fermentation and the associated solutions.
4. Defines the closure rating of the step in question. This reflects the process equipment status:
1 Unexposed/single-use
2 Cleaned and subject to steaming-in-place (SIP)/sterilisation or aseptic flexible connection
3 Cleaned and subject to hot water sanitisation (HWS)
4 Cleaned and chemically sanitised
5 Briefly exposed
6 Open
The definitions of the closure ratings are as follows:
Open system A system that is not closed. The product, or product contact equipment, is exposed to the local environment and may require appropriate environmental controls to mitigate the risk of contamination from the environment.
Briefly exposed Open processes containing process and/or product components that are rendered closed by means of an appropriate closing process, within a pre-defined period of time. Examples of briefly exposed processes include open buffer or media preparations where the solution is ‘briefly’ exposed to the environment prior to closing by filtration.
Functionally closed A process that is opened, or briefly exposed, prior to coming in contact with the product or process, but is rendered ‘closed’ by a cleaning, sanitization or sterilization process that is appropriate or consistent with the process requirements; whether sterile, aseptic or low bioburden.
Closed system A process system that is designed and operated such that the product is never exposed to the surrounding environment. Additions to and draws from closed systems must be performed in a completely closed fashion. Sterile filters may be used to provide effective barriers from contaminants in the environment. A system is closed (or isolated from the environment) when the risk of contamination to the product or process cannot be mitigated by housing the operation in a bioburden-free or particulate-free environment.
5. Calculate the risk rating by multiplying the process requirements and the closure rating.
CR / PR 0 1 2 3
1 0 1 2 3
2 0 2 4 6
3 0 3 6 9
4 0 4 8 12
5 0 5 10 15
6 0 6 12 18
A risk rating of 0-5 is low, 6-10 is medium, 11-18 is high. 6. If the risk rating is low, then no further action is required. 7. If the risk rating is not low, then a further evaluation is required using the fault-tree diagram below: [caption id="attachment_38391" align="aligncenter" width="300"] CLICK TO ENLARGE: Fault-tree diagram[/caption] Whilst it is recognised that the fault-tree analysis is subjective, the results should be subject to peer review to ensure a consistent approach is taken that is supported by industry practices and the proposed operating practices. Examples of the analysis for each question include:
  • A 0.2 micron filtration step will effectively isolate the process from the environment;
  •  The SIP or sanitisation of a closed connection onto a process system before use will provide closure that is consistent with the process requirements;
  • A downstream 0.2 micron filtration step will mitigate the risks of environmental contamination from a process that is exposed in some manner;
  • The use of a laminar flow hood in a Grade C area will provide adequate protection against environmental contamination for the open filling of the bulk drug substance into bottles;
  • A negative answer to Question 4 will indicate inadequate process closure and will therefore require a re-evaluation of the system design to provide adequate closure.
An example of open processing might be the preparation of shake flasks within biological safety cabinets in a Grade C environment (standard practice within bulk biopharmaceutical production). The shake flasks need to be monoseptic (PR = 3) whilst the process is open (CR = 6). This is clearly a high-risk activity and the fault-tree analysis will conclude the existing environmental conditions provide adequate protection. If one was to examine the preparation of media or buffer solutions one would identify that the process requirements for bioburden would be controlled (PR = 2), arising from a requirement to understand the bioburden load on the subsequent bioburden reduction steps to ensure the validated level of reduction was not exceeded. The closure rating would be Briefly Exposed (CR = 2) as the material would, within a defined and relatively short time-span, be subject to a bioburden reduction step (such as filtration) that would reduce the bioburden levels to acceptable levels (low or zero). The risk assessment would therefore be low indicating no further analysis is required. Given the low-risk assessment, one could ask if a classified room would make a significant difference to the bioburden control of the process compared to an unclassified one. The microbial air action limits for Grade D are 200 colony-forming units (cfu)/m3 and whilst there are no limits for unclassified space one could, conservatively, assume an equivalent limit of 500 cfu/m3, for what is a controlled space with limited human activity. Would the potentially increased bioburden loading in the air result in a greater risk to the process in this instance? Before answering this question, one should consider the potential bioburden load in the raw materials being used in the process, as these will not have been prepared or packaged in a clean room environment. Similar considerations can be applied to chromatography column packing, where the matrix raw material is supplied with a microbial specification, which would be reduced to acceptable levels by column sanitisation after packing is complete. One could estimate the potential bioburden load of the matrix, if it were, for example, 1% of the specification, and compare this to the potential bioburden loading from the air in an unclassified space. The results may prove interesting.


Process-closure analysis is a powerful tool that can provide assurance that the design of biopharmaceutical processes will provide adequate protection against environmental contamination and the proposed operational methods will not compromise this. It can be subjective, but if applied in a logical manner that is subject to peer review, it is robust. The risk-analysis tool is becoming increasingly popular in the industry with some operating companies carrying out the analysis against the final installation, and standard operating procedures, and formally recording the results such that they can, if required, be shown to the pharmaceutical regulatory bodies. Author: Chris J. A. Davis CEng FIChemE, biopharm consultant, Jacobs Engineering Ireland