August 1, 2010 (Vol. 30, No. 14)

Novel Configurations Feature Control Capabilities Similar to Traditional Systems

Single-use bioreactors in various designs have been introduced onto the market over the last decade. At small scale, there are many potential solutions, including classic shake flasks in single-use design, roller bottles, T flasks, and so on.

These offer little or no control over cell growth and production. However, they do provide a simple means of small-scale screening and cell cultivation. The introduction of two-dimensional rocking bioreactors over a decade ago brought a larger scale seed cultivation vessel to the market. These systems offer gentle mixing by rocking back and forth with low shear. They also provide surface aeration that is well suited to most cell culture applications. The sensors, which are reusable and offer some control, also have some limitations in terms of handling and accuracy.

The introduction of Sartorius Stedim Biotech’s Biostat® CultiBag RM in 2006 represented a new and advanced version of the rocking bioreactor. By applying control systems similar to classical reusable bioreactors, and employing single-use sensors for pH and dissolved oxygen (DO), good feedback control loops are now available in a single-use system. These single-use sensors are patches that have been gamma irradiated inside the bag at manufacture as a closed system.

A reusable fiber optic cable is placed into the sensor sheath and passes a light of known wavelength through the bag to the patches and excites the fluorescent molecules within. The excitation response is sent back to the measuring amplifier and is converted to measurable pH and DO.

The key advantage here is the presence of a single-use sensor in a single-use bioreactor. The insertion of reusable sensors into a single-use system introduces additional handling and risk issues involving the bioreactor chamber in terms of sterility and leaks.

In recent years the disposable bioreactor market has featured stirred-tank configurations that can replace traditional stirred tank bioreactors. The control systems required to operate these novel designs usually have to be sourced from a second supplier. In some cases this can be advantageous as existing controllers from various manufacturers can be connected without much difficulty. However, when things go wrong, as often happens, two vendors are  involved in trouble shooting problems.

Overcoming Limitations

The innovative design of single-use stirred-tank bioreactors has helped overcome some of the oxygen transfer limitations seen with rocking 2-D bioreactors. Impeller-based mixing, aeration via sparger, and overlay in novel single-use designs are among the advances. These designs are available in various configurations, including top-driven motors that require insertion of a stainless steel rod into an off-center single impeller shaft to prevent vortices.

Other designs are single bottom-driven impellers without a stirrer shaft, located off center to prevent vortices. Additional innovations include box-shaped vessels with paddle impellers. These systems typically utilize reusable sensor technology or, in some cases, may rely on single-use sensors that still have to be aseptically inserted into the bioreactor chamber. The insertion of sensors into a disposable system always introduces a sterility risk to the process.

The Biostat CultiBag STR is a single-use bioreactor with a classical stirred-tank design. It features a central stirrer shaft, sparger, and dual impeller design. The design of the impellers, and location of the shaft and the impellers on the stirrer shaft, mimic a traditional bioreactor. Impeller designs include marine-type impellers and rushton-type impellers as well as typical ring spargers.

The Biostat CultiBag STR has a turn-down ratio of 25%, allowing smaller minimum volumes in fed-batch operation. Since the system is intended for single-use applications, where convenient operation is key, additional user-friendly handling features are part of the design. This includes a bag holder with clam shell opening for easy installation of the bag and a motor lift and separate platforms for the bag and controllers, which facilitates faster turnaround by allowing additional bag holders to be processed downstream while a new bag holder starts upstream.

Like the CultiBag RM, this system also utilizes single-use sensors for pH and DO control. The design was modeled on traditional bioreactors incorporating the same aspect ratios—impeller diameters and location of the shaft and impellers on the shaft—so that the universally accepted tip ratios and power input per volume criteria and KLa’s can be applied for easier transition from stainless steel to single use (Figure).

Through the introduction of traditional bioreactor-like control platforms to the CultiBag RM and STR, real process development capabilities are now possible. These control capabilities can not only supply the user with additional information from continuous data-logging capabilities but they may also help to extend batch age and increase cell growth, product titers, and even specific productivities.

Test data generated using CHO-produced mAbs compares BIOSTAT® CultiBag STR with a large historical dataset from 2,000 L stainless steel production processes. Solid lines: Single-use Biostat CultiBag STR 200 L. Dotted Lines: Classical reusable bioreactor 2,000 L.

Millie Ullah, Ph.D. ([email protected]), is product manager for disposable bioreactor technologies at Sartorius Stedim Biotech.

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