February 1, 2016 (Vol. 36, No. 3)

Kaci Conaway Scientist 1 BD
James W. Brooks Ph.D. R&D Manager BD

Work Efficiently to Enhance Established Cell Lines and Base Medium

Optimization of cell culture parameters is a vital part of biotherapeutic process development, but it is not without its challenges. One such challenge is the selection of a scalable and appropriate cell culture feed that can work in combination with the established cell line and a given base medium to achieve the desired titer and growth characteristics.

The typical approach is to screen multiple commercially available feeds and identify the most appropriate feed with a specific cell line and base medium combination. However, this technique can be tedious and may not work if the base medium or cell line is altered during process development.

A more optimal approach is to use a feed that can work across multiple base media and cells in a platform process. A series of studies involving a chemically defined (CD) feed, BD CHO CD Feed (hereafter referred to as the Product), demonstrated high performance with multiple CHO cell lines in different base media in both shake flask and benchtop bioreactor cultures.

The ability to enhance monoclonal antibody (mAb) production irrespective of CHO cell line or base medium makes the Product a candidate platform feed for process development.

Materials and Methods

Three mAb-producing CHO cell lines were used: CHO Line 1 (CHO K1), CHO Line 2 (GS CHO), and CHO Line 3 (CHO dhfr-). Each cell line was maintained in its respective CD medium.

For fed-batch studies with the Product, cultures were provided with 5% of total culture volume on days 2, 4, and 6. Commercial feeds were used as per manufacturer’s recommended protocol. Cells were passaged three times in respective test media prior to fed-batch study.

Cultures were conducted in shake flasks (Corning). The ambr® 15 microbioreactor (Sartorius Stedim Biotech/TAP Biosystems) was used to model bioreactor conditions at micro scale (15–17 mL) under controlled conditions. DASGIP® bench scale bioreactors (Eppendorf) were employed for confirmation studies at one-liter working volumes.

mAb production was determined with an Octet QKe system (Pall Life Sciences). Cell viability and viable cell density (VCD) were ascertained with a ViCell® XR cell viability analyzer (Beckman Coulter).

Results

BD CHO CD Feed was developed to serve as a platform feed to be applied to cultures of different cell lines with different base media formulations. As an initial assessment of this capability, a fed-batch study was conducted to evaluate the performance of the Product across multiple CHO lines in their respective base media. Three CHO cell lines, each maintained in its respective base medium, were fed with the Product as described previously.

In each CHO cell line and medium combination, the Product significantly enhanced mAb production over control base medium alone (Figure 1). The Product also improved growth of CHO Line 1 and CHO Line 2. These results demonstrated the ability of the Product to enhance mAb production in multiple CHO cell line and base media combinations.


Figure 1. Evaluation of BD CHO CD feed across multiple CHO lines. Viable cell density (VCD) and protein production (µg/mL) of CHO Lines 1–3 in shake flask culture were measured. Each cell line was maintained and tested in cell line specific control base medium.

To further determine its versatility, the Product was assessed across multiple base media using a single CHO cell line, CHO Line 1. Prior to the fed-batch study, CHO Line 1 was passaged three times in each of five commercial and one in-house CD base media. During the fed-batch study, cultures were fed with the Product as previously described. As shown in Figure 2, feeding cultures with the Product enhanced mAb production in all base media tested. This demonstrated that, while levels of mAb production varied due to the base medium, the Product consistently enhanced CHO Line 1 mAb production across various base media.

The performance of the Product was also compared with five commercial CD feeds on a single cell line/base medium combination. CHO Line 1 was passaged three times in an in-house CD medium prior to the fed-batch study as described in the Materials and Methods section. The mAb production achieved with the Product was comparable or better than the commercial feeds evaluated (data not shown).

While the Product demonstrated impressive results in various shake flask fed-batch studies, ultimately the feed needed to demonstrate similar performance in bioreactor culture. To assess utility in bioreactor culture, the Product was evaluated in ambr microbioreactor culture using CHO Line 1 and two differentiated in-house media (BD Medium A and BD Medium B). The Product similarly enhanced growth and protein production in both base media (data not shown).


Figure 2. Evaluation of BD CHO CD feed across multiple CD media. CHO Line 1 protein production (µg/mL) was measured in batch and fed-batch shake flask culture with multiple commercial and BD in-house CD media.

To confirm microbioreactor performance and assess bioreactor scalability, BD Medium B and Product were scaled to bench top DASGIP bioreactor culture. As shown in Figure 3, addition of the Product increased mAb production almost threefold above batch culture. Moreover, the addition of the Product enhanced cell viability (data not shown) and extended overall culture longevity. Taken together, these results confirmed the enhanced performance and scalability achieved with the Product in bench top bioreactor culture.

These studies demonstrate the versatility and broad applicability of BD CHO CD Feed. When tested on multiple CHO cell lines in their respective base media, the Product enhanced growth and/or mAb production. Furthermore, the Product was found to enhance mAb production from a single CHO cell line across a number of commercial media and resulted in mAb levels comparable to or better than several commercial feeds when tested in a single base medium. Importantly, the Product’s enhancement of mAb production was scalable to bench top bioreactor cultures. While there is no truly universal feed due to unique nutritional requirements of cell lines and differing composition of base media, the Product demonstrated robust “platform” performance that was evident with different CHO cell lines and across diverse base media.


Figure 3. Evaluation of BD CHO CD feed in 1 L bench-scale bioreactor culture. Viable cell density (VCD) and production (% increase over control) of CHO Line 1 in the DASGIP bioreactor were measured.

Kaci Conaway is scientist 1 and James W. Brooks, Ph.D. (james_brooks@bd.com), is R&D manager at BD.