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According to the PAT guidance and the scientific literature, the monitoring of the critical process parameters, process variables, and quality attributes can be performed following different methods: off-line, at-line, on-line, and in-line. Although in-line (or in-situ) and on-line are the methods of choice for real-time monitoring and process control, methods involving at-line and off-line are still options for CPPs, KPIs and even CQAs which cannot be accurately measured in the bioreactor.

What is Off-Line Measurement?

The sample is taken out of the bioreactor in sterile conditions and analyzed in the lab after physical pretreatments (e.g. filtration and dilution). The preparation and handling require clear Standard Operating Procedures (SOPs) as well as skilled personnel. If problems occur during these stages, the accuracy of the results will decrease. Together with the complexity involved in manual handling, the major disadvantage of off-line measurement is the time delay, which results in lower measurement frequency and possibly of lower product yields.

Due to these issues off-line measurements should not be considered true PAT unless there are no other measurement possibilities (e.g. HPLC for product titer or mass spectroscopy for product quality). In these examples, automated controls are not a possibility. Off-line laboratory measurements are commonly used to monitor and validate the accuracy of the in-line/ on-line process analyzers. However, factors such as temperature changes and degassing can negatively influence the accuracy of these reference measurements.



What is At-Line Measurement?

In at-line measurement, the sample is removed and analyzed in close proximity to the production process, either manually or by using automated sampling devices. Similar to off-line measurement, sterile conditions must be maintained for accurate results. At-line measurement is most common for parameters which cannot be measured accurately in-situ or on-line.

Advantages of at-line measurement include shortened time delay (relative to off-line), and the possibility for automated control; however the final results might be too slow to effectively monitor cultures with fast growth rates such as microbial cultures, according to PAT principles.



What is On-Line Measurement?

In on-line measurement, the sample is diverted from the manufacturing process with a by-pass stream and may be returned to the bioreactor. The sample is automatically measured in the by-pass by process sensors. The advantages of this method lie in the simple sterilization and the straightforward access to the sample in stationary conditions. The implementation of such a solution requires a specifically designed or modified bioreactor. The added complexity of set-up makes this method less common than in-line monitoring, yet it is one of the two methods with which constant monitoring and, therefore, control are possible in real-time.



What is In-situ (or In-Line) Measurement?

In-line or in-situ measurements occur directly in the bioreactor with a process sensor. The generated measurements are sent in real time to PLC/ SCADA systems for automated control. Process parameters such as pH, ORP (redox potential), dissolved oxygen, dissolved CO₂ (DCO₂), temperature, and conductivity are all common in-situ measurements.

In-line and on-line sensors are the optimal choice for application of PAT principles. They are required to accurately measure without manual intervention over the entire process run, which can last several weeks or even months. Therefore, the operation and maintenance of the sensor should not be underestimated to guarantee reliable, accurate measurement. Preventative measures such as calibration and cleaning should be implemented at specified intervals to avoid drift or loss of signal. The sensors need to be compatible with repeated CIP and SIP cleanings. Extended time at temperatures of 120 to 130°C should not affect the sensor‘s performance.



In conclusion, it is critical to understand the advantages and disadvantages of the various monitoring and control strategies for bioproduction, such as fermentation processes. Exploring ways to improve in-line / in-situ measurement allow process engineers to collect more data which can aid in mathematical models or model-based approaches to increase process knowledge and process optimization. Paired with modern cell culture techniques (batch, fed-batch, or perfusion), measurement of CPPs inside the process will greatly improve what can be accomplished by control systems.

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Sensor Systems for Bioprocess Monitoring