Eize de Boer

October 1, 2008

8 Min Read

The recent product recalls of PedvaxHib and Comvax vaccine batches are a reminder that the control of vaccine manufacturing processes is of the highest importance. This rings especially true because the target population for these two products is children under the age of five.

The Hib vaccine guards against meningitis and other serious infections caused by the bacterium Haemophilus influenzae. In this particular recall, type b vaccine was tested and determined to be free of contaminating microorganisms before being released to the market. However, postrelease surveillance of the sterilization methods used in the manufacturing process showed that the presence of Bacillus cereus bacteria in the batch could not be ruled out.

A Quality Risk Management Approach

Earlier this year the European authority published new guidance for the control of aseptic processing and for quality risk assessment. Because these two new documents affect the manufacturing of biopharmaceuticals, the guidance for the preparation of active pharmaceutical substances was subsequently amended to include a quality risk assessment approach.

A quality risk assessment approach adds significant weight to the knowledge and experience vaccines manufacturers need to have about their products and processes to protect the patient. Assessment of the associated risks needs to be formally laid down in clear and unambiguous writing that can be retrieved during formal authority inspections.



A Significant Change: The prevention of (microbiological) contamination is referred to in these newly published guidance documents. The original guidance on active substance preparation covered the manufacturing “only up to the point immediately prior to the active substance being rendered sterile.” With this new guidance, it is now clear that bulk vaccines manufacturers must assess, control, communicate, and review, both proactively and retrospectively, the quality risks associated with the active substance. This new guidance for the sterile status of the bulk manufacture of vaccines is unprecedented.

Compliance with existing regulations is important when considering the new requirements for the secondary manufacture of vaccines, particularly in relation to the overall qualification of the filling process. The new guidance on aseptic processing (annex 1 of the EU GMP guide) contains a detailed description of how to minimize microbial contamination. Validation of the filling operation is now clearly defined with precise statistics on the number of containers that are allowed to show positive growth during a fill simulation test, with action limits set at multiples of one out of each 10,000 containers filled. Usually such fill simulations are carried out three times consecutively during initial validation runs and twice each year for regular operation.


The main elements involved in vaccine manufacturing risk management are:

1. Establishment of a manufacturing process description: Typical unit operations of vaccine bulk manufacturing are cell culturing and harvesting methods; inoculation of cell cultures and fermentation; and downstream processes such as filtration, centrifugation, diafiltration, chromatography, formulation, filling, freeze drying, and sterilization.

2. Identification of the process parameters that affect product quality and their corresponding tolerances: This includes the equipment and utilities in use and the analytical methods to control final product specifications. This can be done by preparing detailed process flow diagrams and checklists, for example.

3. Risk analysis of manufacturing steps: An assessment of the risk associated with each individual step in the manufacturing process, commonly defined as a quantitative measurement of the probability of occurrence of harm and the severity of that harm. Examples of general recognized risk analysis methods include the failure mode effect analysis (FMEA) and the failure mode, effects, and criticality analysis (FMECA). An example of such a risk analysis, as done for the contamination of harvested bioreactor content, is illustrated by the following sequence of risk analysis:

  • Manufacturing step: harvesting of bulk vaccine

  • Potential sources for out-of-specification/contaminated product, (not an exhaustive list of sources):

    • Bioreactor cleaning deficiency

    • Deficient air filtration

    • Adding nonsterile nutrients

    • Deficient sterilization of harvesting port

  • Assessment of impact on quality for each individual source resulting in a weighed outcome based upon criticality to establish corrective actions, such as

    • Cleaning validation required; cleaning SOPs required

    • Monitoring of air filter from batch to batch

    • SOP for sterility assurance nutrients

    • SOPs for sterilization of harvesting port; integrity monitoring of harvesting port before harvesting

4. Risk control to reduce the risk to an acceptable level: Usually the process validation program is laid down in a validation master plan, which constitutes the overall steps taken during the process validation planning and execution. The same is true for other validation programs; the process validation master plan outlines the process validation protocol and report for each manufacturing step.

5. Acceptance of risk and communication: The process validation to reduce the quality risks associated with the manufacturing of vaccines must be formally accepted and communicated. Clearly there will be residual risks that might not be entirely eliminated or, based on a benefit-cost analysis, remain present. Such residual risks need to be formally accepted by and communicated to the different stakeholders, such as the (quality) management of the organization, regulators, and sometimes the patient healthcare sector.

Additionally, the new EU regulation for aseptic processing includes special emphasis on the requirements for bioburden levels before the final sterilization for terminally sterilized products or before the filtration step for aseptically processed products. The manufacturer is required to first state the acceptable bioburden limits as driven by the efficiency of the final sterilization or filtration and to measure the levels of bioburden of each individual vaccine lot during these final steps in the manufacturing process.

The new guidance concerning quality risk assessment further illustrate how important it is that the manufacturer has significant knowledge of and experience with its pharmaceutical vaccine operations and demonstrates that through meaningful process validation. Process validation evaluates the variations that commonly arise when working with living organisms and assesses the impact of such variability on product quality. Process validation is generally performed before the launch of a new product to the market. It requires a retrospective approach, however, when existing products are to be considered. Evaluating existing products often presents significant challenges to the committee in charge of designing and executing process validation and demonstrating regulatory compliance to the authorities.

For example, vaccines which, in many cases were licensed long ago have been developed without the scrutiny of the present validation programs. This makes it harder to define meaningful criteria to successfully identify the acceptably narry process boundaries and parameters. Also regulatory requirements are changing and not always compativle with the processing of vaccine that were licensed in the past.

Aseptic Processing Is Key

Because vaccines all contain a portion of the disease generating organism (or a related organism), they are intrinsically unstable and are classified as biological agents. Typical sterilization methods are not practical because they would also destroy the therapeutic effect of the vaccine. Therefore, vaccines are normally produced in biopharmaceutical facilities where aseptic processing is standard in the manufacturing process.

In aseptic processing, products and final containers are treated separately until the vaccine is placed in the container (vial or syringe) and closed. Both the container and closure are normally subjected to a separate sterilization process, such as dry or moist heat, and then brought to the fill line. The vaccine is usually processed through clean room areas operating at a low microbial burden, or bioburden, to reduce the risk of contamination.

Aseptic processing has always received a lot of attention from designers and regulators in the biopharmaceutical sector because of the high risk of microbiological contamination that can affect patient health. Usually aseptic processing is done in critical areas or controlled areas, depending on the risks associated with certain steps in the manufacturing.

Critical areas are where sterile product and sterilized materials such as vials and closures are exposed to the environment. Critical area clean rooms must be constructed as Class 100 areas (i.e., not containing >100 particles of ≥0.5 µm in diameter per ft3 of air). Air must be supplied through HEPA terminal filters and air particulate matter must be measured during processing at the point of operation (such as the filling process).

Controlled areas are where nonsterile product, in-process materials, and vials and closures are exposed to the environment. These rooms must operate at Class 10,000 or 100,000 (maximum of 10,000 and 100,000 particles respectively of ≥0.5 µm per ft3) during periods of activity. Normally, Class 10,000 or 100,000 are the rooms surrounding Class 100 clean rooms from which product and personnel are transferred.

This air particulate matter classification is based on the ISO 14644 standard for “Clean Rooms and Associated Controlled Environments.” Both the US (since 2001) and EU (since 2002) authorities have adopted this classification to define the specifications for clean rooms and other clean air devices (e.g., aseptic working benches). The EU grades A/B, C, and D correspond to US classifications of 100; 10,000; and 100,000 respectively.

Combining the principles of aseptic processing with a quality risk assessment approach, as called for by the new regulatory guidance, will make for an important contribution to the safety and quality of today’s vaccines.

A Solid Foundation

Drug manufacturing will always entail some degree of risk. However, a systematic approach to quality management offers a solid foundation to mitigate the risks. A high level of experience and skills are required to fully understand the products and processes in the manufacturing and testing of vaccines and to ensure that the right approaches are taken to control associated risks.

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