Impact of DMSO And Freezing Technique in Developing a High Cell Density Cryopreservation Process for Upstream Bioprocessing

Developing a seed train for upstream bioprocessing involves optimizing many conditions – the medium, the feed, the starting inoculum density, etc. Integrating high cell density cryopreservation (HCDC) in the seed train also means considering parameters like the choice of cryoprotectant and freezing technique.

In HCDC, cells are frozen at high cell density and at larger volumes compared to the traditional expansion process. The ultimate goal in using HCDC is to intensify the upstream process to improve efficiency and productivity, reduce time, and contamination risks (see other benefits of HCDC here).

Here, we evaluated how dimethyl sulfoxide and freezing techniques impact viable cell density and titer production in an HCDC intensified seed train. While the HCDC process should be customized based on the cell line being used, the following offers a foundation for developing cell line-specific conditions.

DMSO Use in Cryopreservation: HCDC vs. Traditional Cell Banking

DMSO is typically added to the media used for freezing cells at a concentration ranging from 5–10% to protect them from the harsh conditions.

Cell banking typically uses 2 mL vials that are filled via pipette in a relatively quick process. This means that the cells have limited exposure to the DMSO prior to being frozen. With HCDC, volumes in the range of 60 to 250 mL are used for cell freezing and as such, the filling process takes longer to complete. The cells are therefore exposed to the DMSO for a longer period, potentially increasing the risk of damage by the DMSO itself.

To inoculate the subsequent bioreactor vessel, the entire volume of the HCDC bag is thawed and added, including the DMSO. The small volumes used in conventional banking do not typically cause a problem as the dilution factor is very high. Additionally, small vials can be centrifuged to remove medium containing DMSO followed by resuspension of cells in fresh medium. This process minimizes the presence of DMSO in the upstream process that may influence cell health and growth, as well as drug quality attributes. With HCDC, there is no opportunity to centrifuge the cells as they are frozen in bags and the volume added to the bioreactor results in a much higher dilution factor.

Impact of DMSO Concentration on HCDC

We performed a series of studies to identify the optimal parameters for DMSO use in an HCDC process. The following parameters were evaluated:

• DMSO concentrations from 0 to 15 percent
• Exposure times of cells suspended in the DMSO cryopreservation medium from no additional incubation added up to two hours additional incubation time added
• Temperatures during which the cells were in contact with DMSO
• A variety of cell lines CHO-K1, DG44, CHO-S and CHOZN^®, our proprietary cell line platform

Effect of DMSO Concentration and Incubation Time on the CHO-S Cell Line

We observed how these parameters impacted viable cell density, viability, and final IgG production of different CHO cell lines when exposed to cell culture medium containing DMSO (Figure 1). The CHO-S clone used in this study showed the highest sensitivity and deviation compared to other clones (CHO-K1, CHO-DG44, CHOZN^®; data not shown). Overall, the most robust conditions were observed with a DMSO concentration of 7.5 percent among all cell lines and even for the more sensitive CHO-S clone used, the impact of freezing technique on cell density was minimal.

Figure 1. Impact of DMSO concentration and incubation time on viable cell density, viability, and IgG concentration.

DMSO Concentration in the Bioreactor

Using one cell line, we inoculated tubes with a cell suspension containing DMSO concentrations from 0 to 10 percent. Viability decreased over time at concentrations above 1 percent when cells are in contact with DMSO (Figure 2A).

In terms of the toxicity threshold of DMSO, we found that the final DMSO concentration in the bioreactor should be approximately 0.5%. At 7.5 percent DMSO, a 150 mL cryobag added to a 2.2 liter bioreactor equates to the desired 1:15 dilution. As the bioreactor size that can be inoculated with a 150 mL bag exceeds the 2.2 liters, the final DMSO concentration in the culture vessel is negligible.

Figure 2. Impact of DMSO concentration (A) and the toxicity threshold (B) on percent viability and viable cell density. Precooling the cell suspension prior to filling the bag may be beneficial as the cell suspension coming out of the bioreactor is at 37 °C, which may increase the DMSO impact for sensitive cells.

Freezing Technique for Cryopreservation: HCDC vs. Traditional Cell Banking

Standard protocols for freezing cells recommend freezing rates of about -1 to -2 °C/min to prevent cell damage. While this approach is easy to execute with small vials of cells, it is impractical for large numbers of larger volume cryobags. As such, the possible impact of uncontrolled freezing, and whether it is necessary to control the process, was evaluated.

Freezing Methods

We used two cell lines (CHO-S and CHOZN^®) and froze them using three different techniques: (1) placing into a –80 °C freezer directly, (2) freezing in a controlled rate freezer, and (3) placing in a CoolCell^® container (Corning). The freezing technique had no impact on either cell line for any of the critical parameters measured (Figure 3).

Although there was no impact on the two cell lines, it is important to evaluate the freezing technique for individual cell lines.

Figure 3. Impact of different freezing techniques on viable cell density, cell viability and IgG production from CHO-S and CHOZN^® cell lines frozen using different techniques.

Conclusion

Use of HCDC is a proven strategy for intensifying the upstream workflow. Here, we established guidelines for the recommended concentration of DMSO and freezing process for the HCDC process. While each cell line may respond to the HCDC process in a slightly different manner, these guidelines offer a foundation for the development of cell line-specific conditions.