Options for Cell Cultivation

Presented by Tina Lütke-Eversloh

Tina Lütke-Eversloh is a senior process manager at Rentschler Biotechnologie, Germany.

Rentschler Biotechnologie GmbH is a contract development and manufacturing organization specialized in the production of therapeutic proteins, listing more than 280 projects to its credit. Lűtke-Eversloh presented a comprehensive coverage of a range of bioprocessing platforms offered by the company, detailing the pros and cons of available choices.
To optimize protein manufacturing, it is necessary to generate high cell densities in culture. Her discussion covered the company’s experience with various alternatives for the cultivation of Chinese hamster ovary (CHO) cells — the cell line of choice for most bioprocessing protocols, including simple batch, fedbatch, chemostat use, and perfusion.
Simple batch is a one-time operation in which the culture reaches a plateau and then is terminated. A fed-batch system involves periodic addition of nutrients, and the chemostat also includes removal of cells and media. Superior performance can be obtained by continuous perfusion of fresh culture medium and cell retention. Two options are the alternating tangential-flow system (ATF®) and tangential-flow filtration (TFF), also known as cross flow filtration. The latter platform involves the recirculation of the retentate across the surface of the membrane. In ATF®-based perfusion bioprocessing, medium is pumped back and forth across the membrane in a wave action to prevent clogging. It is believed to be robust, without critical movements, thus minimizing shear stress.
This robust process yielded easy and reproducible scale-up, which is also applicable for ultrafiltration. These positive features have made the ATF® system the most widely used perfusion process. Rentschler’s experience comprises ATF-based perfusion bioprocessing of up to 1,000 L under CGMP conditions.

An alternative to ATF® perfusion is continuous centrifugation (Centritech®- based perfusion bioprocessing), in which cells and media are cycled through a continuous centrifugation device, removing dead cells through low g forces. Scaling up from 5 L to 250 L, the cell concentration and time to reach the plateau phase of growth were identical; however, product concentration and viable cell counts decreased in the larger vessels.
Lűtke-Eversloh compared the two platforms used independently. The ATF® system displays superior technical robustness, but bleeding losses are higher. The Centritech® technology does not completely remove cells from harvest, as is the case with ATF® perfusion. Moreover, shear stress is high with the Centritech® bioprocessing, leaving fragments of cells that will must be removed during subsequent filtration.
The Centritech® platform is available as a single-use technology, which is not the case for the ATF® perfusion, although such devices are planned. Another singleuse separation device is offered by CARR as the UniFuge® separation system, with its advantages of eliminating the cleaning and sterilization requirements and increased risk of contamination that accompany reusable devices. To date, Rentschler has applied continuous bioprocesses primarily to three types of molecules: proteins that are difficult to express and yield only low titers; molecules that either negatively affect producing cells or that are unstable and quickly degraded (such as blood coagulation factors); and various biosimilars requiring certain critical quality attributes, which are easier to maintain during a steady-state phase than during exponential or stationary cell growth.
With a range of options (including combinations of platforms), it is clear that for those companies designing a bioprocessing program, it will be necessary to take into account individual needs case by case. It may not be possible to design a “one size fits all” recommendation. Cell types, media, feeding regimes, and so on all can play a role in the decision-making process.