Characterization of Rocking Bioreactors: Residence Time Distribution

In recent years, therapeutic antibodies have played an increasingly important role in modern medicine.

Due to the high dose requirements of these antibodies, there is a growing interest in technology development aimed at improving biomanufacturing capabilities.

Most of these technologies focus on mammalian cell culture, as they are capable of post-translational modifications, which are essential for obtaining antibodies with biological functions. Most of the developments in the field of biomanufacturing are related to the improvement of bioreactor performance.

Currently, there are several types of bioreactors available for adherent or suspension cultures. Among them, single-use bioreactors have garnered increasing attention in recent years, with rocking bioreactors being one of the most intriguing options. In this system, the fluctuation (rocking) motion of the cell culture is induced, ensuring good mixing and oxygen transfer without causing shear damage to the cells. Furthermore, this bioreactor eliminates the need for any cleaning or sterilization processes, making it simple to operate and maximizing the prevention of cross-contamination.

The innovative rocking mechanism of rocking bioreactors necessitates better characterization of reactor operations. This will enable the proprietary technology associated with rocking bioreactors to be comparable to more common bioreactors, such as Stirred Tank Reactors (STRs).

One of the most important and widely used tools in this characterization is the measurement and analysis of Residence Time Distribution (RTD). In simple terms, the RTD function E(t) is used to measure the time that various components of a "fluid element" (a small volume of fluid where continuous properties such as concentration can still be determined) spend within the reactor. By comparing the RTD of a real reactor with that of ideal reactors, such as a Continuous Stirred Tank Reactor (CSTR, where the inlet tracer is perfectly mixed into the bulk of the reactor) and a Plug Flow Reactor (PFR, where there is no mixing and fluid elements exit in the same order they entered), it is possible to evaluate the mixing within the real reactor and identify the nature of any deviations from ideal behavior, which are often the source of many operational issues. The experimental schematic is shown in Figure 1.

Figure 1: Schematic Diagram of Experimental Setup

(a) Rocking and (b) Stirred Tank Bioreactor Experimental Setup

(1) Beaker with Water; (2) Inlet; (3) Outlet; (4) Sampling Beaker; (5) Peristaltic Pump.

Evaluating the Residence Time Distribution (RTD) of rocking reactors to characterize their mixing and flow characteristics, and comparing them with ideal models and commercial STRs, is a commonly used method. The RTD is typically measured using a methylene blue pulse input method, with cultures conducted at three common flow rates for mammalian cell cultures, typically low (L: 3.3 × 10-5 m3/h), medium (I: 7.9 × 10-5 m3/h), and high (H: 1.25 × 10-4 m3/h). Samples are taken periodically, and the absorbance values are measured at 660 nm.

The final results indicate that at the L flow rate, the behavior of the rocking bioreactors in continuous experiments approximates that of a STR. For the I and H flow rates, the least squares fits of the data from the rocking bioreactors show considerable deviations from the ideal models. Furthermore, the comparison of the mean residence time (tr) and the transit time (τ) between the rocking bioreactors and STRs provides a possible explanation for their non-ideal behavior. For STRs, at all tested flow rates, tr is lower than τ, indicating the presence of dead zones within the reactor (where tracer entering the culture during operation becomes trapped in inaccessible corners due to stirring and flow velocity effects). In the rocking bioreactors, the same result is observed at the H flow rate. However, for the L and I flows, deviations from ideal behavior may be the result of short-circuiting within the reactor (where tracer entering through inlet 2 exits directly through outlet 3 without mixing), as indicated by tr being higher than τ. The current study suggests that the choice of flow rate will strongly influence the behavior of rocking bioreactors. Using low flow rates appears to be a closer approximation to the ideal model (CSTR).

Figure 2: Comparison of Residence Time Distribution in Rocking Bioreactors and Stirred Tank Reactors (STRs).

L: 3.3 × 10-5 m3/h, I: 7.9 × 10-5 m3/h, H: 1.25 × 10-4 m3/h.

The CytoLinX® WB Single-Use Rocking Bioreactors offer flexibility in adjusting the optimal rotation speed and angle according to different types/modes of cell culture, inducing wave-like motion of the cell culture to ensure excellent mixing and oxygen transfer. Equipped with a built-in temperature monitoring and control probe, it provides suitable temperature conditions for cell culture. Additionally, the pH and DO cascade control modules facilitate achieving better cell culture outcomes.

CytoLinX® WB Single-Use Rocking Bioreactors

Features

• The software interface is easy to operate, stable to run, and supports audit trail;

• Supports easy switching among 10 L, 20 L, and 50 L trays. The bags and the trays can be mounted easily and rapidly without removing screws;

• Supports perfusion and automatic calibration of pump flow rate for accurate feeding and harvesting;

• Sparging, pH/DO, and perfusion modules are optional, depending on process requirements;

• Adding partial linkage design makes it more suitable for application scenarios of cell therapy customers;

• Precise measurement of gases with mass flow controllers (MFC).

Reference:

WAVE BIOREACTOR CHARACTERIZATION: RESIDENCE TIME DISTRIBUTION DETERMINATION. M. Elisa Rodrigues, A. Rita Costa, Mariana Henriques, Joana Azeredo, Rosário Oliveira IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal