Functional Evaluation of Dissolved Oxygen Sensors When Installed Invasively vs. Non-Invasively in the BIOne Single-Use Bioreactor

Justin Cesmat, Greg Kauff man and David Olson- Distek, Inc.

Abstract

One of today’s primary process improvement targets within the biotechnology industry is increasing product yield. To increase yields, strict quality standards during the manufacturing process must be maintained. Quality by Design (QbD) principles provide guidance on process development including integration of control strategies and process analytical technologies (PATs) that help to ensure critical process parameters (CPPs) are maintained within strict control limits. One common CPP is dissolved oxygen (DO), which has been shown to directly impact product yield.

Stir tank reactors (STRs) such as the Distek BIOne Single-Use Bioreactor (SUB) are the most commonly used reactor system for bioprocess scale-up and production. These systems continuously monitor and control DO concentrations at defined setpoints using either a polarographic or optical DO sensors and gas sparge of air and/or oxygen. The BIOne SUB allows for direct measurement of DO without invasive sensor installation using an intuitive design that aims to reduce contamination risk and increase available processing time.

In this study, the performance of both polarographic and optical DO sensors were evaluated when installed both invasively and within the non-invasive downtube of the BIOne SUB. Results from this study demonstrated that no significant difference in DO sensing capability exists for either the polarographic or optical sensor when installed invasively or non-invasively.

Introduction

As the biotechnology industry continues to grow and become more competitive, further emphasis is being placed on significantly increasing production yields while still maintaining high product quality standards. These process improvements are often sought through enhanced characterization and optimization. To be effective, a thorough understanding of the relationship between critical material attributes (CMAs) and CPPs on final product critical quality attributes (CQAs) is required.

QbD principles are often employed to ensure consistent and reproducible manufacture of high-quality biologic products.¹ QbD emphasizes that the yield and quality of the final product depends heavily on the manufacturing process itself. Its objectives include establishing performance-based quality specifications, increasing process capability with reduced variability, increasing product development, and manufacturing efficiencies, and enhancing root cause analysis.²

The elements of QbD include defining the quality characteristics of the product and identifying CQAs, understanding the impacts of process parameters and raw materials on the final product, and establishing a control strategy to maximize consistency in the manufacturing process.³ Successful control strategies often require the integration of process analytical technologies (PAT). These tools provide a system for designing, analyzing, and controlling manufacturing through real-time monitoring of critical quality and performance attributes of raw and in-process materials and processes.⁴

Inadequate control strategies can lead to extracellular conditions unfavorable for culture growth. If severe, these conditions may result in quality excursions that could negatively impact the safety or efficacy of the final product and lead to batch rejection.⁵ Without real-time monitoring and control of process parameters using PATs, out-of-specification (OOS) conditions are far more likely to occur and go undetected. Detection of these excursions would manifest as CQA deviations during final product quality control testing, long after the batch has been completed.

STR systems are often the vessel of choice in the biotechnology industry when targeting increased product yields, as they can easily scale up to very large production volumes. They also can maintain a high degree of process control when designed and implemented properly. Because of the high cost and time investment associated with operating these systems, robust control strategies are recommended to ensure cultures perform within specification limits.⁶

One of the most commonly used control strategies in STRs is closed-loop feedback control. This type of control is highly suitable for dynamic systems using online PAT sensors such as pH or DO.⁷ Closed-loop controllers continuously take PAT sensor process readings and evaluate them against the setpoint value; the difference known as error. The controller then drives system inputs, such as oxygen sparge to control DO, to reduce the error between process reading and setpoint.⁸

DO Control within STR Systems

Due to its necessity in aerobic cellular metabolic activity, bioreactor DO (pO2 ) is often recognized as a CPP across many bioprocesses.^9,10 Molecular oxygen is required for the oxidative phosphorylation metabolic pathways (OXPHOS) within aerobic cultures, which is critical for peak titer production.¹¹ When DO levels are not controlled properly, undesired process effects like increased cell aggregation, reduced viability, and decreased titer may occur.¹²

Suspension cultures within STR systems can achieve very high cell densities, upwards of 30 x 106 cells/mL in mammalian CHO cell culture and 190 g/L dry weight in microbial E. coli cultures.^13,14 At these densities, oxygen uptake rate (OUR) of the culture can rapidly deplete DO within the medium and result in process parameter excursions.¹⁵ Therefore, to maintain DO levels at setpoint, the oxygen transfer rate (OTR) into the system must be equal to or greater than the OUR.

Within STR systems, DO concentration is traditionally monitored using a sensor submerged in the culture medium and is controlled through closed-loop feedback control. When oxygen consumption drives the sensor reading below setpoint, the controller modulates system inputs to increase OTR and maintain DO within control limits. These inputs vary depending on process application and typically include addition of headspace or subsurface gassing with air and/or oxygen along with increasing agitation rate.¹⁶

Two types of DO sensor technologies are primarily used in bioprocessing: polarographic and optical. Polarographic DO sensors are electrochemical devices that rely on diffusion of oxygen through a membrane to generate an electrical current. The current is linearly proportional to the concentration or activity of oxygen in the medium.¹⁷ Optical DO sensors rely on fluorescent intensity quenching (reduction) of a light source to determine oxygen concentration. Higher concentrations of molecular oxygen will result in increased fluorescent quenching.¹⁸

In recent years, the biotechnology industry has begun shifting away from polarographic sensors, as their detection process consumes oxygen, and sensors need to be polarized, calibrated, and maintained regularly.¹⁹ Optical dissolved sensors have slightly slower detection speeds, but are less noisy, more accurate and require far less maintenance than polarographic sensors.²⁰

The Distek BIOne 1250 controller utilizes closed-loop feedback control to maintain DO at setpoint and can effectively incorporate either polarographic or optical sensors. The BIOne Single-Use Bioreactor (SUB) contains a non-invasive DO downtube with a silicone semi-permeable membrane. This downtube enables DO reading without the need for sensor autoclaving and invasive installation within the vessel, reducing prep time and minimizing contamination risk.

In this work, we aimed to prove that the performance of both polarographic and optical DO sensors is equivalent when installed both invasively and within the non-invasive downtube of the BIOne SUB. To perform this evaluation, a model medium was degassed using subsurface nitrogen sparge. Once an oxygen-free environment was established, an oxygen saturation curve was generated by sparging air at a constant rate into the medium. Results demonstrate that use of the non-invasive downtube for DO measurement in the BIOne SUB does not affect the accuracy or performance of either polarographic or optical sensors.

Materials and Methods

A BIOne SUB model 2022-1002 manufactured by Distek was selected as the STR model for this study. This model has a 2-L working volume, single right-handed pitch blade impeller, a flute sparger with 7 x 1.5 mm holes in a linear arrangement and comes standard with a non-invasive DO downtube. The headplate also includes two PG13.5 ports for invasive PAT installation. The bioreactor was operated using the BIOne 1250 Dual-Vessel Controller by Distek. (model number 2022-8122).

During the evaluation, a model medium of 1.0X Phosphate Buffer Saline (PBS) solution was batched to a working volume of 1400 mL with a 37°C temperature setpoint and a constant agitation rate of 300 rpm (P/V = 35 W/m³ ) clockwise providing upward axial flow. The agitation rate was selected as it represented a typical power input for high density mammalian cell cultures.²¹

One optical DO sensor (Mettler Toledo InPro 6860i) and one polarographic DO sensor (Hamilton OxyFerm FDA) were used to compare performance between invasive installation and non-invasive installation within the SUB. Using the BIOne 1250, a two-point calibration was done in air for both sensors prior to the study.

With DO sensors installed in the SUB as shown in Figure 1, the model medium was degassed using subsurface nitrogen sparge to a DO concentration of 0% on each sensor. Following degassing, constant air sparge of 140 sccm (0.1 vvm) was supplied until the medium reached DO saturation. A total of three replicates each were performed with the sensors installed both invasively and non-invasively.

Following the start of air sparge, DO measurements were logged every 30 seconds for both sensors using the BIOne 1250 controller. Data from each test replicate were normalized from 0% to 100% using the formula shown in Equation 1.

The normalized data were analyzed using a nonlinear asymmetric sigmoidal curve regression (Graphpad Prism, Version 9.0). Using these data, the 50% DO saturation time was determined for each experimental trial.

Results and Discussion

Regression data for the optical and polarographic DO sensor saturation testing overlayed with the 50% saturation condition are shown in Figure 2. Also shown are the results of the multiple unpaired t-test analysis of 50% saturation time for each sensor type when installed invasively vs non-invasively. The unpaired t-test results demonstrate that the difference in 50% saturation time for both the optical and polarographic sensor when installed invasively vs non-invasively is not statistically significant (α = 0.05).

It must be noted that the 50% DO saturation time was not compared between the optical and polarographic sensors. Due to differences in sensor manufacturer, sensing technology, detection levels, and response time, it is expected that the DO saturation profiles between the two sensor types will vary, even when installed within the same system. Therefore, the scope of this study was limited to a comparison between invasive vs non-invasive installation of each sensor type within the BIOne SUB.

Conclusions

The use of PATs for monitoring and control of bioprocesses is considered one of the key elements of QbD. These PATs must be able to read accurately and reliably for process control strategies to function properly and maintain CPPs within setpoint limits. Use of these PATs should not introduce additional process risks such as contaminations that could risk batch rejection.

Distek has successfully introduced a non-invasive DO downtube into the SUB line which prevents the need for risky process contacting installation of a DO sensor. As demonstrated in this work, use of either an optical or polarographic DO sensor within the non-invasive downtube provides comparable sensing capability to invasive installation of the same sensors. These types of intuitive designs found on the BIOne SUBs make them ideal for process characterization and optimization work, as they increase available processing time while minimizing risk to the product.

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