Basic Principles of Bioprocess Monitoring and Viability Determination

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Laura Sophie Rösner	--	1713	2023-02-08 13:27:48

This entry is adapted from the peer-reviewed paper 10.3390/bioengineering9120762

Bioprocesses involve multiple steps, including upstream processing, downstream processing and product formulation. Each of these steps must be monitored and regulated precisely, which requires suitable sensors that meet specific demands. Both the process itself and the process monitoring can be arranged differently. Likewise, this applies to the determination of viability, with each method possessing advantages and disadvantages.

sensors spectroscopy viability cell culture bioprocess monitoring soft sensor

1. Bioreactor Modes of Operation and Monitoring Techniques

Often, productivity, i.e., the amount of a product that can be obtained per volume and per time, is considered almost more important than yield in industrial bioprocesses ^[1]. In order to achieve optimum productivity, the bioprocess and the type of reactor must be well matched and the process has to be precisely regulated and controlled. Depending on the design and operation of the reactor, different reactor types can be distinguished. The stirred tank reactor (STR) is one of the most widely used reactors, and can be operated not only as a batch reactor but also continuously as a continuous stirred tank reactor (CSTR) ^[1].

In principle, a bioreactor can be operated in three different ways: batch, fed-batch and continuous ^[2].

In a batch process, a known initial concentration of cells and substrates is used without adding further media during the process ^[3]^[4]. Only after the cultivation is complete are the products removed and purified. Hence, no input or output of liquids occurs and the liquid volume in the vessel can be considered constant ^[2].

Especially in industrial production, fed-batch processes are very common. Such processes are usually the method of choice when higher initial substrate concentrations are not applicable due to inhibitory (catabolite repression) or even toxic effects. Fresh medium or substrate is added intermittently or continuously during fed-batch processes. Thus, a free volume must remain in the reactor for the added medium at the beginning of the cultivation. By using a fed-batch process, the frequency of downtime that occurs in the batch processes can be reduced, which often makes fed-batch processes a more economical alternative compared to batch processes ^[4].

In continuous processes, the substrate is continuously fed via the feed stream and products are removed via the product stream. Depending on the method of maintaining the steady state, a distinction can be made between chemostats and turbidostats ^[2]. To increase the productivity of a CSTR, cell retention can be implemented. Reactors using cell retention are referred to as perfusion bioreactors ^[1]. Another reactor type, although only occasionally used in biotechnology, e.g., when valuable gaseous substrates are involved, is the plug flow reactor (PFR) ^[1].

In particular, fed-batch and continuous processes require precise regulation to ensure optimal process control. The classification of sensor types for process control is based on their position in the process and the method and frequency with which they provide information on the process. A basic distinction is made between on-line, at-line and off-line sensors.

During off-line analysis, individual samples are taken from the reactor and examined in an (external) analytical laboratory ^[4]. The resulting time delay often impedes efficient process control, since the window for intervention in the process has usually elapsed by the time the result is available ^[4]. Additionally, whether sampling is performed automatically (at-line) or manually (off-line) is often associated with a high risk of contamination. Hence, on-line sensors are preferable ^[5]^[6]^[7].

As mentioned above, at-line sensors require regular (automatic) sampling by means of a suitable sampling device. This procedure is often used for analysis via chromatographic methods or mass spectrometry (MS). Biosensors are usually operated at-line in flow injection analysis (FIA) systems as they must remain outside the sterility barrier ^[8]. Although this method involves a time delay, it has the advantage that samples can be adjusted to the optimal assay conditions.

On-line sensors, in contrast, are directly in contact with the bioprocess and are either located directly inside the reactor (invasive) or separated from the reactor by the reactor wall (non-invasive) ^[4]. They can also be operated in bypass mode, with a stream continuously diverted from the reactor and measured in flow-through mode. On-line sensors can deliver results directly from the reactor environment in real time without the need for manual interaction ^[6]^[8]^[9].

2. Sensor Requirements

Devices considered for on-line-monitoring are required to possess certain attributes which qualify them for their use in bioprocess monitoring and control.

In order to endure the harsh conditions during sterilization, the sensor, or at least the optical window for the sensor, must be robust enough and needs to maintain its calibrated state ^[6]^[9]. In addition, the sensor should not interfere with the sterile barrier ^[9]. The measurement accuracy of the sensor is determined by various interactions with the bioreactor and the cultured species. If increased cell debris occurs, as is the case in the cell death phase, spectroscopic analysis of the culture broth can become more complicated. In addition, the measurement can be affected by gas bubbles, solid particles, stirring and very high cell densities. For these reasons, a consistently good signal-to-noise ratio is crucial for applicability of the sensor throughout the entire cultivation process, regardless of changes in chemical and physical process parameters.

Viability sensors must also fulfill general sensor criteria, including high specificity and high selectivity. While a sensor’s selectivity represents its ability to measure a target analyte in presence of other compounds, sensitivity refers to the change in the output signal as a result of a change in analyte concentration ^[10]. Other criteria such as stability, linearity, robustness and repeatability must also be fulfilled ^[5].

Sensor requirements also depend on the cultured species, the type of medium and reactor, as well as the achieved cell density. While the cultivation of mammalian cells requires very sensitive and specific sensors with low detection limits, microbial production processes with high occurring cell densities, high viscosity and high gassing rates impose very high demands on the robustness of the sensors used for monitoring ^[11].

Especially in the field of single-use (SU) bioreactors, there is a particularly great need for the research and development of viability sensors ^[12]. Invasive probes which are sterilized within the reactor are usually not well suited to single-use application as they cannot always reliably guarantee the integrity of the sterile barrier. Non-invasive spectroscopic methods, on the other hand, can be easily implemented in SU bioreactors, which also prevents the risk of cross-contamination ^[13]. However, one challenge which must be overcome for sensor application in SU bioreactors is the permeability of the plastic reactor material for electromagnetic waves, to enable measurement in the reactor bag without loss of intensity or interference effects. In addition, the development of plug-and-play devices for sensor integration in SU devices is of particular interest ^[14]. One reason for this effort is that standardized ports such as the Ingold port are not available for SU applications ^[10].

3. Off-Line Methods for Viability Determination

Even though on-line sensors offer promising possibilities for the determination of viability, they have not been widely used to date. Off-line methods are still the method of choice and are stipulated in many standard operating procedures (SOPs) for quality management.

The choice of the appropriate test method depends on the cultured organism. The standard method for the assessment of bacterial viability is the colony count method. However, this method requires several days of incubation for colony formation and is limited to culturable bacteria that grow on agar plates. Furthermore, it can be difficult to obtain reproducible results due to the high sensitivity of the test to changes in the culture conditions and human counting errors ^[15].

A more robust off-line method than traditional cell counting is cell viability assays. The most prominent amongst these assays are dye exclusion assays, colorimetric assays, luminometric assays and flow cytometric assays ^[16]. Nevertheless, a disadvantage of these viability assays is the requirement of several (time-consuming) steps for preparation and analysis and the use of detection devices such as (fluorescence) microplate readers, (fluorescence) microscopes or flow cytometers.

According to not only commercial GMP manufacturing but also to research and process development, the gold standard for the simultaneous determination of total cell density and viable cell density is at-line analysis via live/dead staining ^[17]. These so-called dye exclusion assays, e.g., trypan blue, propidium iodide or 7-aminoactinomycin D (7-AAD), are based on the membrane integrity, and the dyes can enter the cells when cell death occurs ^[18]. The counterstaining of living cells can be performed using calcein acetoxymethyl (calcein AM) ^[16].

A variety of tetrazolium compounds can also be used to detect viable cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is positively charged and can penetrate viable eukaryotic cells ^[19]. MTT is reduced to a purple formazan product by cells with an active metabolism ^[20]. This reduction can be quantified by measuring absorbance at 570 nm after one to four hours of incubation. It was one of the first viability assays suitable for high-throughput screening. The assay has to be considered as an endpoint assay, as MTT has a cytotoxic effect and can be influenced by reducing compounds in the medium such as ascorbic acid or coenzyme A ^[19].

The resazurin reduction assay is comparable to the MTT-assay. Similar to MTT, resazurin also acts as a redox indicator. Viable, metabolically active cells can reduce resazurin, leading to the pink, fluorescent resorufin. However, the MTT-assay has limitations as well, since media compounds may interfere with the fluorescence of resorufin which, itself, is cytotoxic ^[19]^[20].

In contrast, adenosine triphosphate (ATP)-based assays function differently. Here, the addition of the assay reagent leads to immediate rupture of the cell membrane; therefore, no incubation is required ^[19]. This assay utilizes the enzyme firefly luciferase, which converts luciferin, resulting in a long-lasting luminescent signal ^[20]. It provides a very rapid, sensitive method to determine viability and takes advantage of the fact that cell death is accompanied by loss of membrane integrity. However, a transmembrane proton gradient is mandatory for ATP synthesis. Consequently, ATP synthesis becomes impossible after cell death and any remaining ATP in the cytoplasm is consumed by endogenous ATPases ^[19].

Flow cytometry as a method of quantitative single-cell analysis can be used for viability determination as well. With this method, cells can be characterized within a liquid flow with the aid of lasers, depending on their size, granularity or ability to carry specific fluorescent molecules. Since dying cells are often smaller than viable cells, changes in viability can be observed via forward- and side-scatter analysis. Common cytotoxicity and viability staining methods can also be applied to cytometry ^[16].

References

Villadsen, J. Bioreaction Engineering Principles; Springer: Berlin/Heidelberg, Germany, 2011; ISBN 978-1-4419-9688-6.
Doran, P.M. Bioprocess Engineering Principles; Elsevier: London, UK, 2004; ISBN 0-12-220855-2.
Simpson, R.; Sastry, S.K. Chemical and Bioprocess Engineering: Fundamental Concepts for First-Year Students; Springer: New York, NY, USA, 2013; ISBN 978-1-4614-9126-2.
Chmiel, H. (Ed.) Bioprozesstechnik, 3rd ed.; Spektrum Akademischer Verlag: Heidelberg, Germany, 2011; ISBN 9783827424778.
Claßen, J.; Aupert, F.; Reardon, K.F.; Solle, D.; Scheper, T. Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application. Anal. Bioanal. Chem. 2017, 409, 651–666.
Beutel, S.; Henkel, S. In situ sensor techniques in modern bioprocess monitoring. Appl. Microbiol. Biotechnol. 2011, 91, 1493–1505.
Reyes, S.J.; Durocher, Y.; Pham, P.L.; Henry, O. Modern Sensor Tools and Techniques for Monitoring, Controlling, and Improving Cell Culture Processes. Processes 2022, 10, 189.
Mandenius, C.-F.; Titchener-Hooker, N.J. (Eds.) . Measurement, Monitoring, Modelling and Control of Bioprocesses; Springer: Berlin/Heidelberg, Germany, 2013; ISBN 978-3-642-36838-7.
Sonnleitner, B. (Ed.) Bioanalysis and Biosensors for Bioprocess Monitoring; Springer: Berlin/Heidelberg, Germany, 2001; ISBN 9783540487739.
Steinwedel, T.; Dahlmann, K.; Solle, D.; Scheper, T.; Reardon, K.F.; Lammers, F. Sensors for Disposable Bioreactor Systems. In Single-Use Technology in Biopharmaceutical Manufacture; Eibl, R., Eibl, D., Eds.; Wiley: Hoboken, NJ, USA, 2019; pp. 69–82. ISBN 9781119477839.
Teixeira, A.P.; Oliveira, R.; Alves, P.M.; Carrondo, M.J.T. Advances in on-line monitoring and control of mammalian cell cultures: Supporting the PAT initiative. Biotechnol. Adv. 2009, 27, 726–732.
Kuhnke, L.M.; Rehfeld, J.S.; Ude, C.; Beutel, S. Study on the development and integration of 3D-printed optics in small-scale productions of single-use cultivation vessels. Eng. Life Sci. 2022, 22, 440–452.
Samaras, J.J.; Micheletti, M.; Ding, W. Transformation of Biopharmaceutical Manufacturing through Single-Use Technologies: Current State, Remaining Challenges, and Future Development. Annu. Rev. Chem. Biomol. Eng. 2022, 13, 73–97.
Busse, C.; Biechele, P.; de Vries, I.; Reardon, K.F.; Solle, D.; Scheper, T. Sensors for disposable bioreactors. Eng. Life Sci. 2017, 17, 940–952.
Mauerhofer, L.-M.; Pappenreiter, P.; Paulik, C.; Seifert, A.H.; Bernacchi, S.; Rittmann, S.K.-M.R. Methods for quantification of growth and productivity in anaerobic microbiology and biotechnology. Folia Microbiol. 2019, 64, 321–360.
Kamiloglu, S.; Sari, G.; Ozdal, T.; Capanoglu, E. Guidelines for cell viability assays. Food Front. 2020, 1, 332–349.
Pörtner, R. (Ed.) Cell Culture Engineering and Technology; Springer International Publishing: Cham, Switzerland, 2021; ISBN 978-3-030-79870-3.
Al-Madani, H.; Du, H.; Yao, J.; Peng, H.; Yao, C.; Jiang, B.; Wu, A.; Yang, F. Living Sample Viability Measurement Methods from Traditional Assays to Nanomotion. Biosensors 2022, 12, 453.
Riss, T.L.; Moravec, R.A.; Niles, A.L.; Duellman, S.; Benink, H.A.; Worzella, T.J.; Minor, L. Assay Guidance Manual: Cell Viability Assays; National Library of Medicine: Bethesda, MD, USA, 2004.
Braissant, O.; Astasov-Frauenhoffer, M.; Waltimo, T.; Bonkat, G. A Review of Methods to Determine Viability, Vitality, and Metabolic Rates in Microbiology. Front. Microbiol. 2020, 11, 547458.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Biotechnology & Applied Microbiology

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Franziska Walter

View Times: 972

Update Date: 08 Feb 2023

Table of Contents

Video Upload Options

Confirm

1. Bioreactor Modes of Operation and Monitoring Techniques

2. Sensor Requirements

3. Off-Line Methods for Viability Determination

References