Adapting to new sterility challenges

Traditionally, cleanrooms and aseptic zones within pharmaceutical manufacturing have been decontaminated manually, using suspension test-validated disinfectant agents. However, the agents are not generally validated in situ in line with industry standards. Since a log reduction in bioburden cannot be accurately calculated when using manual decontamination methods (due to the difficulty in validation processes), alternatives are now being employed, such as hydrogen peroxide vapour (HPV)

The new microbiological monitoring systems of today can offer a robust level of detection

As companies look to develop large molecule products using targeted delivery methods, different quality control measures are needed. Tim Flanagan, Bioquell, looks at how bio-decontamination and monitoring processes are being adapted to meet this change.

It is imperative that medicinal products are manufactured within a clean and sterile environment. The risk of biological contamination during the manufacturing process needs to be reduced to a minimum. This ensures patient safety, and maintains production efficiency by eliminating the time-consuming and costly process of investigating the causes of contamination and the controlled destruction of contaminated batches.

There are clear and complementary regulatory requirements and references to ISO 14644 standards for classification and monitoring of controlled environments in the manufacture of sterile medicinal and therapeutic products. Regulatory requirements in the US are detailed in the FDA Guidance for Industry in Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice, United States Pharmacopeia (USP) including Microbiological Evaluation of Clean Rooms and Other Controlled Environments USP <1116>. In Europe the EU Good Manufacturing Practice – EU GMP annex 1 applies.

Sterility tests are required during the production validation process, as well as for routine quality control and detecting major contamination in a batch. Sterility testing can be an extremely difficult process and open to errors. Protocols must be designed and executed so as to eliminate false results. With the increasing number of medicinal and therapeutic products that have a biological profile and therefore cannot be terminally sterilised, there is also a greater requirement for aseptic processing where both biological contamination control and sterility testing play a part in batch release.

control and monitoring

Classified in terms of its microbiological profile, the environment needs to be maintained in a state that presents an acceptable low risk of biological contamination – something that will differ from facility to facility, depending on the application. This level of acceptable risk following cleaning/sterilisation is specified in terms of log reduction in bioburden, and is typically 6-log for critical areas/surfaces. 6-log provides a 99.9999% reduction in bioburden on critical surfaces, i.e. within the controlled environment where manufacturing will occur. The surrounding environment is less critical and can be maintained at a lower log reduction.

Traditionally, cleanrooms and aseptic zones within pharmaceutical manufacturing have been decontaminated manually, using suspension test-validated disinfectant agents. However, this method can be problematic as the agents are not generally validated in situ in line with industry standards, making it difficult to verify any log reductions in bioburden. Furthermore, there is no standard practice for the removal of the reagent residue. If not removed correctly, surfaces can be left ‘wetted’ and this may leave a fine coating of aggressive sporicidal agents, which can result in damage to the surfaces themselves.

There is no standard practice for the removal of the reagent residue. If not removed correctly, surfaces can be left ‘wetted’ and this may leave a fine coating of aggressive sporicidal agents, which can result in damage to the surfaces themselves

Sampling to identify bioburden contamination levels can also pose several challenges. For example, the sample may not necessarily provide a true representation of the conditions across the entire environment. Results of zero CFU (colony forming units) may not mean that there is an absence of contamination, but simply that the bioburden was below the level of detection at the monitoring location when sampled. Contamination is never likely to be homogenous across an entire environment.

As research continues to advance in the area of microbiological monitoring technologies, today’s new systems can offer a higher level of detection. However, this can present further challenges within the testing process and certified regulations in the clean environment. There is an increasing awareness that a significant percentage of the bioburden may be viable but non-culturable organisms.

evolving methods

Improvements in the monitoring of a cleanroom environment need to be coupled with enhancements in bio-decontamination control. Since a log reduction in bioburden cannot be accurately calculated when using manual decontamination methods (due to the difficulty in validation processes), alternatives are now being employed, such as hydrogen peroxide vapour (HPV).

HPV – produced by vaporising aqueous hydrogen peroxide – can safely eliminate microbial contamination within a contained environment. Specially designed vapour generators in combination with high velocity gas distribution nozzles and fans ensure a uniform distribution of HPV throughout an enclosure, which leads to an even coating of the active agent on all surfaces.

The HPV process has been validated with Geobacillus stearothermophilus as the biological indicator, to demonstrate a 6-log sporicidal reduction of bioburden within the cleanroom/enclosure (Figure 1). When HPV is introduced to the environment there is, as expected, an initial rapid increase in HPV concentration and at the saturation point, the rapid onset of bio-decontamination is triggered. This gaseous vapour phase decontamination process using hydrogen peroxide under specified conditions has been recognised as a method of achieving surface sterilisation by international regulators.

Figure 1: Hydrogen peroxide vapour (HPV) technology

One of the key elements to this process – which makes it a safer alternative to detergent agents – is that it is not ‘wet’. Vaporised hydrogen peroxide molecules are delivered only to surfaces past the saturation point, at a thickness of 2–6µm, and their controlled removal leaves the surface completely free from residue. The contact time of the active HPV and the residue-free nature following aeration enable its use with a variety of materials and within areas containing sensitive electronics. The 6-log sporicidal and broad spectrum efficacy of HPV, with starting agents at 30%–35% w/w concentration and controlled delivery/removal of the disinfection agent, also eliminates the requirement for any rotation of different disinfectants.

sterility testing

As the pharmaceutical industry is evolving, drugs are being developed to be more targeted to elicit a specific physiological response at a given site or tissue type. Because of this shift, the number of small molecule entities being produced is decreasing, while alternative large molecule products using targeted delivery methods are being developed. Therefore, different quality control measures need to be implemented.

Quality and bio-contamination control can be designed into the process using quality by design (QbD) principles, supported by risk assessed control measures. Sterility assurance can be effectively measured through advanced, continuous particle monitoring to detect any deviation from predefined specifications. Using QbD principles significantly contributes towards bio-contamination free results in routine environmental monitoring and sterility testing. As a final check for identifying major contamination within the product, sterility testing forms the last stage of this process.

Previous methods of bulk sterility testing using large, flexible-film, half-suit isolators are therefore being replaced by more versatile, scalable and adaptable systems

The sterility test process needs to be flexible yet robust to enable the processing and preparation of different sample types, closure types and batch sizes. Previous methods of bulk sterility testing using large, flexible-film, half-suit isolators are therefore being replaced by more versatile, scalable and adaptable systems. These use rapid transfer technology to achieve high throughput processing in barrier separation gloved isolators. This move towards modular systems facilitates more continuous and rapid testing of various sample types, with a high degree of biological contamination control and risk management.

With barrier separation technology, such as isolators and closed barrier workstations, users are able to ensure a sterile environment is maintained, via the application of HPV technology. Within a single Bioquell QUBE module, a rapid 20-minute HPV bio-decontamination cycle allows fast and efficient turnaround of samples. In addition, this closed system provides a physical separation between the operator and the workstation: this further reduces the risk of contamination from the operator, while providing more effective biological decontamination risk management of the testing zone.

Within a Bioquell QUBE module, a rapid HPV bio-decontamination cycle allows fast and efficient turnaround of samples

The major challenge when trying to maintain the sterility test work zone occurs when entering the test materials/samples into the aseptic environment. This needs to be completed without compromising the aseptic conditions themselves, or the sterility of the products. One such way of doing this is to implement modular sterility test systems.

There are now innovative systems coming to the fore that can provide the advanced level of flexibility required to accommodate varying sample types. Modular systems can be effectively tailored to meet the demands of each facility. For example, the Bioquell QUBE system enables different sample types to be handled on varying scales of production capacity, depending on the system configuration or material transfer method used. Generally speaking, there are four types of processing operation:

Batch processing – All sterility test material together with the separation barrier are gassed in unison, using HPV in one bio-decontamination cycle. The sterile products are supported on either point-of-contact support racks or hangers. They may form part of the load pattern or enter after the gassing cycle via a closed aseptic transfer. This, however, is not an optimal process as the gassed load takes up space and restricts movement for the sterility testing of the batch, lengthening processing times.

Gassed isolator work zone with aseptic hold and rapid gassing transfer of all test materials and product samples – A gassing transfer chamber is loaded with test materials, which are transferred upon cycle completion to an interconnected sterility test process isolator that has been previously gassed.

Gassed isolator work zone with aseptic hold and two types of material transfer disinfection processes – Test materials are bio-decontaminated in a gassing transfer chamber and, upon cycle completion, they are transferred to an interconnected sterility test process isolator that has been previously gassed. The variant in transfer disinfection is for product samples that cannot be exposed to HPV and as such, the system has a type D transfer hatch that can be used to enter product test samples into a manually decontaminated environment.

Gassed work zone designed for aseptic sterility testing and containment against operator exposure to hazardous biological or toxic samples – Sterility test materials will enter the process zone by a rapid gassing transfer process. Test product samples coming from a contaminated process zone require containment in recovery, transfer and subsequent entry into the sterility test isolator zone. A rapid transfer port container with aseptic transfer between the production process zone and the contained sterility zone would be used. All waste materials need to be treated as toxic and securely removed from the test area.

Having the flexibility to choose any one of these processing operations in a single system provides a cost-effective method of performing the different types of sterility testing. Modular sterility testing systems are therefore essential to provide an efficient pharmaceutical manufacturing protocol, regardless of sample type, while maintaining final product efficacy.

In conclusion, sterility is a critical component of the drug manufacturing process. There are implications for patient safety that could be detrimental to health should contaminated products make it through for public use. Furthermore, there are consequences relating to production and process inefficiencies with cost implications if batches were to be contaminated.

HPV eliminates many of the issues associated with manual disinfection procedures, providing a validated and efficient method that leads to a reduction in both the cost due to root cause analyses and the risk of false positives

HPV ensures complete bio-decontamination. As a scientifically robust process, HPV provides significant efficiency savings, allowing critical components of filling lines to be gassed-in-place and cleanroom facilities to be returned to a state of microbiological control promptly following facility shutdown, re-qualification, or recalibration. HPV also eliminates many of the issues associated with manual disinfection procedures, providing a validated and efficient method that leads to a reduction in both the cost due to root cause analyses and the risk of false positives.

Newly developed sterility testing systems such as the Bioquell QUBE employ integrated HPV bio-decontamination technology, along with a flexible modular design. This allows the system to be readily reconfigured to fit the manufacturing process and sample type, or the individual requirements of each facility. The modular sterility test barrier separation design enables the in-process transfer of different test samples, with varying batch process throughput. By enabling a variety of different configuration options, this can accommodate difficult-to-decontaminate samples, or simple-to-decontaminate test sample closures that may be affected by gaseous disinfection. As a result, significant process flexibility is provided and various process compatibilities can be managed.

Risk management is substantially improved where the controlled barrier separation technology equipment is fully integrated into the operational process. Effective, robust bio-decontamination and ‘real time’ monitoring technology will have a significant impact on the reduction in bio-contamination events.

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