In September with the launch of the new WAVE 25 Bioreactor, The Cell Culture Dish and GE Healthcare Life Sciences launched a contest looking for the most innovative ways WAVE Bioreactors had been used to advance technology. Of all the different technologies reviewed, four were selected as favorites to share with our readers. Of the four, one was selected as our winner because it stood out as not only a favorite technology, but also as an excellent fit for a technology that would be of particular interest to the Cell Culture Dish community.

Favorites:

A simplified, direct transfection using polyethylenimine for large-scale and high throughput applications.

This procedure was described in the study, “A simplified polyethylenimine-mediated transfection process for large-scale and high throughput applications,” published in the Methods. In the paper, Raymond, et. al describe a method for direct transfection using the 5 liter WAVE bioreactor for large scale production.

As demand for recombinant proteins in earlier stages of drug development has increased there has been considerable interest in developing methods for generating large amounts of protein without the time consuming process of creating a stable cell line. Transient transfection has become an attractive option by shortening the time it takes to generate protein from several months to only a few weeks.

The authors use HEK293 cells in this study. HEK293 cells and derivatives are most commonly used for transient expression because they have some key advantages including: that they allow for proper folding and relevant post-translational modification; they are easy to grow; and they are highly transfectable. Polyethylenimine (PEI) is most commonly used as a transfection agent because it works well and is cost effective. The use of PEI as a transfection agent involves the formation of nanoparticles (polyplexes) and the quality of these polyplexes determines transfection efficiency and productivity. The most common method for using PEI in transfection is indirect where the formation of polyplexes occurs outside of the culture and then is added to the cells. This study seeks to compare the traditional indirect method with a direct method where DNA and PEI are added directly to the cell culture and polyplexes are formed in situ. One aim of the study is to determine whether this method using suspension culture is directly transferrable from 6 well plates to shaker flasks and then to 5 liter WAVE Bioreactors for large-scale production.

To test large-scale production, the 5 liter WAVE Bioreactor was used with the chimeric B72.3 antibody. To begin the study, 3.5 liters of culture media was transferred into a 10 liter WAVE Cellbag. Cells grown in shaker flasks were then transferred into the WAVE Cellbag for a final volume of 4.8 liters and an initial cell density of 0.40-0.45 x 10⁶ cells/mL. Indirect transfection was tested using 200 mL of prepared polyplexes that were incubated for 15 minutes then added to the cells and followed by a rinse of the transfer line. For direct transfection, 100mL of DNA was added to cells followed by a rinse of the transfer line, then 5 minutes later, 100 mL of LPEI was added followed by a rinse of the transfer line. Results showed that for both, maximum antibody titers were reached on day 6 and in the direct transfection 116 mg/L titer was achieved and 103 mg/L using the indirect method. This proved that the direct transfection method described provided a viable and simple alternative to the traditional approach for large-scale production. This direct method provided several advantages including: minimizing the number of steps, eliminating the need for an incubation period for polyplex formation, and a robust platform for high-throughput and large-scale antibody production.

Large-scale expansion of lymphocytes for Adoptive Cell Transfer Therapy

This innovative protocol was explained in “Clinical Scale Rapid Expansion of Lymphocytes for Adoptive Cell Transfer Therapy in the WAVE Bioreactor” published in the Journal of Translational Medicine. In the study, authors, Somerville, et. al, investigated the use of the WAVE Bioreactor to expand lymphocytes to clinical scale.

In looking at treatment options for metastatic melanoma, one therapy being investigated is adoptive cell transfer (ACT) therapy. In the paper, the authors describe the use of autologous tumor infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) modified to express specific T-cell receptors targeting tumor antigens and the promising results. Adopted transfer of TIL combined with a lymphodepleting regimen can result in responses in 48-70% of patients with 40% achieving durable complete response, which is much better than the current standard treatment options.

With the promising results of this therapy, there is a need to be able to manufacture cells for reinfusion into patients. Traditionally this has been achieved using static culture by culturing cells two weeks in T175 flasks and then transferring cells to 3 liter gas permeable bags. One challenge with this method is that it requires a semi-open system and as a result very highly skilled personnel to reduce risk of contamination.

In light of the demand for these cells and the challenges with the current system, the WAVE Bioreactor was used with continuous media exchange and was compared with standard static culture production. Both systems began with culture initiated in T175 shake flasks then on day 7 the cultures were either transferred to a 3 liter static bag or a 10 liter Cellbag attached to the WAVE Bioreactor 2/10 system. The results demonstrated that use of the WAVE Bioreactor protocol resulted in a comparable number of cells and both systems were able to rapidly expand to clinically relevant scale. The WAVE Bioreactor system provided a more stable culture environment made possible by prevention of the accumulation of waste and real time monitoring of culture conditions. Major advantages of using the WAVE bioreactor included a small footprint, the ability to take samples in real time for monitoring cells and culture conditions. In addition, the ability to grow cells to high densities in reduced volume with the WAVE Bioreactor resulted in expedited downstream processing and allowed for the harvesting of multiple products per day using standard blood bank cell processing equipment. Thus the WAVE bioreactor provided a suitable alternative to static culture, provided a closed system, and a reduction in labor.

For this particular application there was a higher percentage of CD4+ cells and a lower percentage of CD8+ cells found in the WAVE Bioreactor system, so specific Cell Therapy applications need to be examined on a case-by-case basis depending on the specific cell type needed. Adjustments and optimization may need to be conducted to achieve desired results with various cell types.

Development of a Platform for Scalable and Robust Manufacturing of Lentiviral Vectors

This novel protocol was presented in “Characterization of Lentiviral Vector Production Using Microwell Suspension Cultures of HEK293T-Derived Producer Cells,” and published in Human Gene Therapy Methods. Authors of the study, Guy, et.al, describe the development of an scalable manufacturing process for the production of lentiviral vectors (LV) through transient transfection of HEK 293T cells grown in suspension in a 2 liter WAVE bioreactor system. In the paper the authors discuss the product ProSavin, a Gene Therapy for Parkinson’s disease that is currently in Phase I/II clinical trials. Material for Phase I/II was manufactured using transient transfection in adherent HEK 293T cells. Adherent culture was effective for producing a small amount of material for initial trials (30 patients) it was found to be too labor intensive and difficult to scale up for Phase III studies and ultimately product commercialization.

In an effort to develop a platform for large-scale production, authors developed two stable producer cell lines and one of those cell lines (PS46.2) was adapted from adherent to suspension culture. Authors then employed the use of statistical design of experiments techniques and a microwell-based experimental platform to analyze nine factors and their effect on titer to determine the production factors most critical for further optimization. They discovered three that had an effect – post induction period, dox concentration and liquid fill volume.

After further characterization and optimization of these factors they developed a protocol for larger-scale manufacturing using the WAVE Bioreactor 20/50 system. For the experiment a 2 liter Cellbag was innoculated with 0.5 liters of PS46.2 cells at a density of 1.2 x 10⁶ viable cells/ml. They then used the optimized microwell conditions to set harvest timing and fill volumes. To determine rocking rate, rocking angle, air flow rate and starting CO₂ concentration they relied on a review of current literature and manufacturers recommendations. Results showed that the cell growth and vector production kinetics were similar to the microwell experiment. Max titer was within 2-3 fold of maximum titer reached in microwells. Lower titer was expected and authors hypothesize that “better control and understanding of the changes in the engineering environment experienced by cells upon scale-up into wave-mixed systems will be crucial for enhancing titers at large-scale and improving scalability between the microwell platform and wave bioreactor.” The authors were able to prove the validity of using the WAVE Bioreactor for large-scale production of lentiviral vectors with the use of suspension adapted producer cells and also the use of the microwell system for developing scale-up processes. This is important work as authors state that in the paper that to the best of their knowledge the use of a wave bioreactor system for the culture of suspension-adapted stable lentiviral vector producer cells has not yet been evaluated. Protocols for large-scale manufacturing of gene therapies is increasingly important as, according to the paper, there are 49 gene therapies in clinical trials worldwide.

Special Winner

Perfusion-based High Density Cell Banking

In the study, “Development and Implementation of a Perfusion-based High Cell Density Banking Process,” published in Biotechnology Progress, the Cell Culture Development group at Biogen Idec shared their expertise in using the WAVE Bioreactor to create a perfusion-based high cell density cell banking process.

The current process for preparing cells for transfer into large-scale biopharmaceutical manufacturing bioreactors is to expand the cells using seed train culture. This involves many time consuming steps beginning with a small sample of cells taken from the working cell bank. Cells then undergo multiple expansion steps until there is enough cells and culture volume for transfer to a large-scale bioreactor. The novel process that Biogen Idec describes in the study simplifies this process and reduces process time considerably.

In the study, the team describes their process of using perfusion culture to create a high-density cell banking system that increased the number of viable cells and volume in each banked vial. The perfusion bioreactor they selected for the task was the WAVE Bioreactor system with disposable 20-liter Cellbags fit with an internal floating filter. The internal floating filter retains the cells in the bioreactor and filters only the media thus eliminating the need for an external cell separator and flow loop. This configuration was selected because it simplified the use of perfusion culture in the cell-banking lab. Perfusion conditions were then optimized for high cell density culture, including perfusion rate, rocking speed and aeration. Results demonstrated peak cell densities of greater than 20 x 10⁶ cells/ml and maintenance of cell densities greater than or equal to 90%.

Creation of the high-density cell bank enabled direct inoculation of the high-density cell bank vials into the WAVE Bioreactor, which eliminated the need for shake flask expansion. The paper states “Elimination of multiple culture expansion steps in the upstream seed train resulted in reduction of 9 days of manufacturing plant time, and also improved operational success in seed expansion steps.” Biogen Idec was then able to repeat their success in seven independent cell lines all producing recombinant therapeutics and results have been verified in GMP clinical manufacturing campaigns.