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European Biopharmaceutical Review

Real-Time Approach

Biopharmaceutical production and manufacturing is mainly based on empirical approaches for process design, where product quality is ensured by the so-called ‘Quality by Testing’ concept. This approach is due to the complexity of the cellular system and the limited process observability. Process understanding and rational, knowledge-based process design are the keys to progress further in the field, and they represent central goals of the US Food and Drug Administration (FDA) Process Analytical Technology (PAT) and QbD (Quality by Design) initiatives (1,2). In consequence, advanced on-line monitoring of these processes – the basis for realtime process control – is becoming increasingly relevant.

On-Line Monitoring

Detailed information about the advantages and disadvantages of individual state-of-the-art on-line bioprocess monitoring systems has been comprehensively reviewed (3). Even though there is impressive progress in this field, real-time access on physiology-relevant process variables is still limited, and there is an ongoing effort to improve bioprocess-monitoring capabilities.

Besides the development of new in situ sensors, the measurement of compounds in the off-gas is a promising non-invasive approach to easily access additional process information. The bioreactor off-gas was already the focus of scientists’ interests to determine the O2 and CO2 concentration when calculating respiratory-related parameters or applying electronic noses (4,5). The cells in a bioreactor produce a broad range of metabolites as byproducts of growth and production (6).

Some of these metabolites are secreted to the fermentation broth, and those with sufficient volatility end up in the off-gas of the fermenter. Consequently, real-time monitoring of these volatile organic compounds (VOCs) will provide information that is directly linked to the metabolic activity of the cells. Expected concentrations in the parts per billion by volume (ppbv) range, as well as a large variety in terms of the chemical composition of VOCs, require trace gas analysers that can sensitively detect different compounds in parallel, and preferably in real-time. Proton transfer reaction mass spectrometry (PTR-MS) is often the technology of choice, since it matches these requirements (7).

To facilitate artefact-free transfer of trace gas concentrations to the analyser, a sampling setup which couples a PTR-MS to the off-gas stream of a bioreactor has been developed. This system was used successfully to monitor concentrations of emitted volatile metabolites on-line and in real-time, in different production processes, with Escherichia coli and Chinese hamster ovary (CHO) cells.

Trace Gas Analyser

The employed analyser – a highsensitivity PTR-MS – is capable of real-time analysis and quantification of VOCs down to 0.001ppbv without sample preparation. It can analyse a large variety of organic and inorganic trace gases like alcohols, aldehydes, ketones and esters but it is insensitive for common constituents of air, for example N2, O2, Ar or CO2. The PTRMS technology is a ‘soft’ chemical ionisation technique and induces no or low fragmentation of the analyte. This is especially useful for the analysis of complex samples without preseparation. The data acquired by a PTR-MS are quantitative and can be assigned to specific compounds.

Artefact-free transfer of trace concentrations to the analyser can be challenging. Condensation of the humid sample gas can lead to compound loss. Moreover, surface adsorption of compounds can have a huge impact at such low concentrations. Thus, for the analysis of trace gas concentrations in fermenter off-gas, a specialised sampling interface was developed (8).

The interface is temperature-controlled to avoid condensation, and chemically inert materials were used. For small fermenters with a comparably low aeration flow, the standard sterile filter had to be replaced by a heated ceramic filter cartridge to reduce surface adsorption of volatile trace compounds. The high flow in industrial fermenters enables a faster equilibration time and an adaptation of the filter elements is not required. Moreover, the sampling interface provides the multiplexed measurement of several fermenters simultaneously, a built-in calibration and optional dilution of the sample gas.

When the concentration of a compound exceeds the upper limit of linearity of a PTR-MS – around 10,000ppbv – the quantification of all compounds can be affected. While emitted volatile metabolites are at much lower concentrations, we observed high ammonia concentrations in E. coli fermentations, where NH3 is commonly used to regulate the pH value. A defined dilution of the sample gas with clean air resolves this issue.

E. Coli-Based Bioprocesses

A successful implementation of PTR-MS monitoring of E. coli-based fermentation processes has been demonstrated (8). Subsequently, a comprehensive study with variation of three important process parameters – temperature, induction level and growth rate – was conducted. Depending on the process settings, we observed up to 50 VOCs with notable concentrations and distinct variations over time, with some preliminary results shown in Figure 1. Trends of three different VOCs monitored under different process conditions show significant differences for each individual VOC.

Mammalian Cell Culture-Based Bioprocesses

PTR-MS perfectly matches the requirements for on-line monitoring of biopharmaceutical production processes. Monitoring mammalian cell culture is a relatively new application area for the PTR-MS technology, and consequently not much literature is available to date. In the context of biopharmaceutical process development, the PTR-MS measurement setup (see Figure 2, page 54) was applied for the first time in a study conducted at the Bioprocess Development Department of the Sandoz GmbH site in Schaftenau, Austria. The objective of this study was to determine the suitability of PTR-MS for monitoring of cell culture cultivations and to assess the biological relevance of the PTR-MS data. In order to distinguish between compounds of non-cellular, cellular origin cell-free experiments and fed-batch cultivations (with and without disturbances) with a recombinant CHO, cell lines were conducted. As a result, a series of compounds, formerly unrelated to cell culture cultivations, could be detected.

Several compounds showed a certain trend over the cultivations – however, further studies will be needed to fully exploit the mechanistic relation between these volatiles and metabolic pathways. The PTR-MS acquired complex datasets and is a valuable resource for statistical modelling to predict non-directly measurable parameters. Evaluated with partial least square regression, important cell culture parameters traditionally measured off-line – such as the product concentration – could be estimated in real-time. The PTR-MS technique has proven suitable to monitor cell culture processes. In addition, the measurement of volatiles in the bioreactor off-gas can contribute to a deeper process knowledge and offers new possibilities to control bioprocesses.

The Future

Bioprocess design will definitely move forward from empirical approaches to rational knowledge-based concepts that are already established in other industries. The FDA’s PAT and QbD initiatives offer an important step in this direction, and advanced process monitoring strategies represent the key to process understanding. In view of these facts, the implementation of new process sensors and analysers in pharmaceutical production processes is of high interest.

PTR-MS is a real-time process analyser, perfectly matching the requirements for on-line monitoring of biopharmaceutical production processes. The fast analysis time allows the multiplexed monitoring of a series of bioreactors in parallel. Moreover, the approach is non-invasive – except for the heating of the gas-transfer line, for example, no changes to the process or the fermenter are required, which greatly supports the implementation of this approach in a Good Manufacturing Practice (GMP) controlled environment. This is of great importance, as existing production plants can be upgraded with manageable efforts.

Implementing a detailed analysis of the fermenter off-gas via PTR-MS is a promising approach for several reasons. The results obtained for E. coli and CHO fermentation processes are highly repeatable – when process conditions are constant – but show significant variations as a response to changes in process parameters. Based on preliminary analysis and interpretation of these data, highly relevant information can be derived for both organism and process combinations that are investigated. Changes of some individual VOCs correspond to specific conditions in the process, some of which up to now could only be detected via complex, time-delayed off-line methods.

Conclusion

To further exploit the potential of PTR-MS-based process monitoring of the fermenter off-gas, a multitude of tasks are waiting ahead. The large number of VOCs with distinct trends over the course of a fermentation promises access to a broad range of highly relevant process information, many of which still have to be investigated further. Already available datasets must be analysed. An in-depth understanding of the underlying metabolic processes can lead to new concepts for bioprocesses. The characterisation of other production cell lines is an important issue, and it is proposed that in the near future, PTR-MS process monitoring will expand to other processes.

Acknowledgement
This work was supported by the Federal Ministry of Traffic, Innovation and Technology, the Federal Ministry of Economy, Family and Youth, the Styrian Business Promotion Agency, and the Standortagentur Tirol and ZIT-Technology Agency of the City of Vienna through the COMETFunding Programme, managed by the Austrian Research Promotion Agency.

References
1. Wu H et al, Quality-by-Design (QbD): an integrated process analytical technology (PAT) approach for a dynamic pharmaceutical coprecipitation process characterization and process design space development, Int J Pharm 405: pp63-78, 2011
2. PAT – a framework for innovative pharmaceutical development, manufacturing, and quality assurance, Guidance for Industry, FDA, 2004
3. Schügerl K, Progress in monitoring, modeling and control of bioprocesses during the last 20 years, Journal of Biotechnology 85: pp149-173, 2001
4. Aehle M et al, Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process, Cytotechnology 62: pp413-422, 2010
5. Bachinger T and Mandenius CF, Searching for process information in the aroma of cell cultures, Trends Biotechnol 18: pp494-500, 2000
6. Hussain NH et al, Recent advances in biology: intercellular communication and quorum sensing in microorganisms, Sci Prog 81(1): pp69-80, 1998
7. Lindinger W et al, Proton-transferreaction mass spectrometry (PTR-MS): on-line monitoring of volatile organic compounds at pptv levels, Chemical Society Reviews 27: pp347-354, 1998
8. Luchner M et al, Implementation of proton transfer reaction-mass spectrometry (PTR-MS) for advanced bioprocess monitoring, Biotechnol Bioeng, 2012

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About the authors

After receiving his PhD in Food and Biotechnology, Markus Luchner worked as a postdoctoral scholar in the Austrian Centre of Industrial Biotechnology (ACIB) in the field of bioprocess engineering and on-line process monitoring. Since 2012 he has been the Head of the Bioindustrial Pilot Plant at the University of Natural Resources and Life Sciences (BOKU) in Vienna, Austria, and is responsible for training and scientific projects.

Having been awarded his MSc in Bioprocess Analytics, Timo Schmidberger started his career at Sandoz, Austria as a Scientist. He was involved in development, optimisation and characterisation of industrial cell culture processes. In 2011 Timo started his PhD thesis at Sandoz in cooperation with BOKU, focusing on the implementation of PAT tools.

After achieving his PhD in Food and Biotechnology, Gerald Striedner was a postdoctoral scholar and Senior Scientist at ACIB. In 2009 he became the Head of the working group ‘Microbial Fermentation’ at the Department of Biotechnology at BOKU. Gerald is the key researcher and project leader in the areas ‘Cell Design and Engineering’ and ‘Bioprocess Engineering’ at ACIB, and he is involved in several European Union national projects.
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Markus Luchner
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Timo Schmidberger
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Gerald Striedner
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