Optimizing Electroporation for Cell Therapy Development

Cell therapy is an emerging novel treatment option for an array of diseases, ranging from genetic disorders to cancer. As the cell therapy field expands, there is a growing demand for safe and efficient approaches to deliver foreign genetic material into cells. Although electroporation offers several advantages over viral vector-based approaches for cell therapy development, the method can present challenges.  

Technology Networks had the pleasure of speaking with Thermo Fisher Scientific’s Don Paul Kovarcik, to learn more about the benefits and limitations of electroporation and key factors affecting electroporation performance. In this interview, Kovarcik also tells us about the new Xenon electroporation system and how it addresses challenges associated with electroporation, including how it preserves cell viability and facilitates scaling.

Zoe Braybrook (ZB): What are the key benefits of choosing electroporation as a transfection method for cell therapy development?  

Don Paul Kovarcik (DK): Even though a lot of the foundational work in adoptive T-cell therapy was performed using viral transduction, there still remain some limitations. One, there are added safety concerns because these viruses randomly integrate in the genome and could have unintended consequences. Two, additional testing is required such as replication competent virus. Finally, there are limits in payload size that can be delivered via viral vectors. 

In contrast, electroporation can be used to deliver a wide variety of payloads including DNA, RNA and protein including payloads larger than 10 kb. In addition, electroporation is the most commonly used method to deliver gene editing payloads. These non-viral engineering approaches could allow more specific and controllable engineering thus potentially a better therapy.

ZB: Efficiency is of high importance for cell therapy manufacturers. Can you tell us more about the performance of Xenon? How does this compare to other approaches?

DK: While there is donor-to-donor variability, the Xenon is capable of delivering up to 90% knockout and 80% viability in primary activated T cells. Ultimately, this translates to a larger number of total edited cells. One advantage of the Xenon system is that it allows developers to fine tune the electroporation parameters specific to their product or process. In contrast to program-based electroporation systems, the Xenon allows users to adjust voltage (500–2500 V), pulse width (1–30 ms), pulse number (1–10) and pulse interval (500–1000 ms). In this way, the developer has full visibility to the electroporation pulses applied to the cells.           

ZB: A limitation associated with electroporation is the substantial cell damage that can be caused by the high voltage pulses. How does Xenon address these challenges and ensure viability of cells?

DK: The ideal electroporation process is one that provides the best balance of transfection efficiency and viability. In general, one comes at the expense of the other as shown by this graph. In this experiment with primary activated T cells, voltage was the most significant variable impacting performance and while increasing voltage increased percent transfection, it also leads to a decrease in viability. 

Because the Xenon offers the flexibility for customers to set their own electroporation parameters, it gives them the best opportunity to find optimized conditions that best meet their needs.

ZB: Scaling cell therapy workflows from process development to clinical manufacturing can be a challenge. How does Xenon overcome this issue and enable seamless scaling?

DK: One of the key requirements during development was to ensure that the electroporation conditions scaled from our small-scale Neon Transfection System without the need to re-optimize on the Xenon. It was important to achieve equivalent performance regardless of what volumes our customers are working at. To achieve this, our engineering team performed an extensive characterization of the Neon’s hardware and consumables to understand how it functions. They have maintained the path length (distance between the electrodes) which was key to generating an equivalent waveform and pulse profile. They also characterized temperature and pressure generated during electroporation to understand how it impacted performance. From the very first breadboard to the final prototypes, all experiments had a Neon control as the performance benchmark. 

ZB: What are cells going through during and immediately after electroporation?

DK: An electrical charge is emitted between the top cathode (+) and bottom anode (-) electrodes in a somewhat straight line. Where the charge hits the cells, a temporary pore is opened there and an exchange of the extracellular and intracellular material occurs. The size and frequency of pores depend on the composition of the cellular membrane. For example, it is harder to create pores in rigid areas such as ones with large lipids. The resulting loss of membrane integrity and intracellular material is something the cells may or may not be able to recover from.

ZB: Does Thermo Fisher have optimized protocols for various cell types with performance data available?

DK: On the Xenon System, we have optimized electroporation of 3-day CD3/CD28 Dynabead activated PBMCs and pan T cells from fresh and frozen material with our culture and payload system at 20–100 M cells/mL (specifically 20 M, 50 M, & 100 M cells/mL). The same electroporation program works well with other culture, activation and payload systems. Because we have demonstrated scalability from the Neon Transfection System, our customers can reference over 140 optimized protocols in our database.

ZB: What are the key variables from an engineering perspective that affect electroporation performance?

DK: The engineering factors identified to have an impact on biological performance were related to consistent voltage and current being delivered to the electroporation chamber. During a MultiShot run, to ensure consistent performance from shot to shot, it was critical to have equivalent filling of each electroporation as well as to maintain pressure inside the chamber to eliminate the risk of arcing. Temperature is also a key variable. It directly affects the conductivity of the buffer and thereby the energy delivered to the cells, but also cell viability is impacted when a temperature threshold is exceeded. As a result, the system has an internal safeguard to prevent electroporation protocols that would result in unsafe temperatures. Finally, to ensure scalability from the Neon tip, we had to ensure that the distance between the electrodes of a Xenon consumable was the same as that in the Neon tip.

Don Paul Kovarcik was speaking to Zoe Braybrook, Marketing Campaign Coordinator for Technology Networks.