Closed, easy-to-use, scalable transfection system for cell therapy manufacturing

The Xenon Electroporation System delivers superior non-viral gene editing and transfection performance with an intuitive touch-screen interface. A closed system with a single-use electroporation chamber can help facilitate regulatory compliance and reduce clean-room costs. Scale seamlessly from research and early development on the Neon Transfection System to process development and commercial manufacturing on the Xenon platform.


Robust and efficient gene editing and transfection into hard-to-transfect cells

Transfection—introducing exogenous biological material such as DNA, RNA, or protein into eukaryotic cells—is now commonly used in a broad range of applications, including cell and gene therapy, gene editing, protein expression, mRNA vaccines, and immunotherapy. Of the major delivery or transfection methods, electroporation (a physical method) is the most widely used due to its ability to achieve high transfection efficiency, even with hard-to-transfect cells such as primary, stem, and immune cells. Electroporation can also deliver larger payloads (>7 kb) than cationic lipid-based (chemical) or viral (biological) methods, and is easier, safer, and less costly than viral transfection.

Electroporation overview

Electroporation uses an electrical pulse to create temporary pores that allow payloads to cross the cell membrane.

Complex line drawings that show electrical impulses being used to create temporary pores in cell membranes; these pores allow material to enter cells, and they close after the electrical impulses are removed. This process is called electroporation.

The Xenon Electroporation System offers reliably high transfection performance in volumes of up to 25 mL in less than 25 minutes with exceptional cell viability and recovery. In the accompanying data graphs, chimeric antigen receptor (CAR) T cells were generated from three different donors by using Cas9/gDNA to knock out the endogenous T-cell receptor (TCR) and knock in an FMC CAR. As a benchmark, lentiviral transduction would be expected to achieve 20–40% success for this process.

 

The graphs show that knock-in and knock-out performance with the Xenon system were strong. Across all three donors, successful transfection percentages (cells both knocked out and knocked in) ranged from about 22% to more than 45%, depending on the electroporation chamber used. Transfection efficiency on the Xenon instrument surpassed even the Neon Transfection System on which the process was developed. Cell viability—which must be balanced against transfection efficiency—exceeded 70% in all but one case, and was usually within 10% of the untransfected controls.

Knock-out and knock-in performance in CAR T cells

T cells from three donors were transfected with an FMC CAR construct using Cas9/gRNA on the Neon Transfection System (100 µL) or the Xenon Electroporation System with the SingleShot (1 mL) or MultiShot (9 mL) electroporation chamber, or were left untransfected (0). Cells were characterized after 72 hours as untransfected (TCRαβ+, gray), knocked out but not knocked in (TCRαβCAR, light blue), or successfully knocked out and knocked in (TCRαβCAR+, dark blue). Across all donors, successful knock-in percentages on the Xenon system ranged from 21.9% to 45.6%, exceeding even the Neon system.

Three bar graphs, representing three donors. Each bar graph includes a negative control, a sample run on the Neon system, and two samples run on the Xenon system.

For data on cell viability and preservation of CD4/CD8 T cell ratios, see Sample Data.


Closed, automated electroporation system

Xenon With MultiShot Facing Right

Together, the Xenon electroporation instrument, chamber or cartridge, buffers, and software comprise a closed cell electroporation system. The versatile Xenon instrument fits on a benchtop and is designed to scale from process development to GMP manufacturing. It accommodates a single-use electroporation chamber and features an intuitive touch-screen interface.

 

There are two single-use electroporation configurations, one a chamber to process a 1 mL sample in a single batch, the other a cartridge to electroporate 5–25 mL in a sequential, automated process, 1 mL at a time. You can also choose between two electroporation buffers, one for gene editing (designed to increase knock-out and knock-in efficiency) and one for other applications. Finally, the Xenon software runs on the touch screen, allowing you to view and modify protocols and control processing in real time.

A complex drawing that shows the four major components of the Xenon system: the instrument, liquid buffers, single-use plastic consumables, and a software package accessible by an embedded graphical user interface on the instrument.

The closed system, single-use chambers, MultiShot cartridge format, and software automation help to reduce operator error and limit sample contamination and leakage. They can also help to reduce staff training and clean room costs.

 

The single-use 1 mL SingleShot electroporation chamber can transfect 2–10 x 107 cells in one batch.

The plastic single-use consumable SingleShot Chamber holds 1 mL of cells for electroporation.

The single-use 5–25 mL MultiShot flow-through electroporation cartridge can transfect 0.1–2.5 x 109 cells in a continuous process. Its cartridge format with pre-routed tubing enables efficient and worry-free loading, while setup cues and the software interface confirm correct installation.

The plastic single-use consumable MultiShot Cartridge can process up to 25 mL of cells on the Xenon system, when the cells are loaded into an input bag that is attached to the tubing on the cartridge.

Though the system is closed, the platform is open, allowing the freedom to test and perfect transfection conditions during process development. Process developers can optimize the electroporation process by tailoring parameters like voltage and pulse width, number, and interval. You can use your own bags and sterile-weld to PVC or C-Flex™ tubing. The Xenon system can also be incorporated into a closed, automated, complete cell therapy workflow under unified software control, as described under Digital connectivity for integrated workflow.


Seamless translation from process development to commercial cell therapy manufacturing

The Xenon Electroporation System is process- and protocol-compatible with the Invitrogen Neon Transfection System, a compact, benchtop electroporation instrument that can transfect from 2 x 104 to 6 x 106 cells per reaction. After completing small-scale research and early development on the Neon system, you can transition smoothly into process development on the Xenon system using the 1 mL SingleShot chamber, and from there to GMP manufacturing using the 5–25 mL MultiShot flow-through cartridge. Your process scales efficiently from discovery to manufacturing using the same electroporation protocol without re-optimization or drop in performance.

 

Efficient scaling

Your electroporation process scales seamlessly from research and discovery on the Neon Transfection System to large-volume GMP manufacturing on the Xenon Electroporation System. In this Cas9/gRNA genome editing experiment, knock-out efficiency on the Xenon system was comparable to the Neon system for all three T cell loci studied.

A complex figure showing the research use only Neon Transfection System and the GMP-compliant  large-volume process development and manufacturing Xenon System, illustrating the similarities and differences between the two systems.

At any scaling level, you can adjust the electroporation parameters, such as voltage and pulse width, number, and interval, to meet the needs of your product and process. If you have already developed your manufacturing process, the modular Xenon system is designed to efficiently integrate into it.


GMP-manufactured consumables and buffers

Both the 1 mL SingleShot and 5–25 mL MultiShot electroporation consumables are manufactured using Good Manufacturing Practices (GMP) methods. These single-use consumables are designed to enable reliability and consistent performance and fit into your GMP-compliant facility and workflow.

GMP-manufactured buffers

Both the standard and genome editing electroporation buffers are manufactured to strict Cell Therapy Systems (CTS) standards using GMP methods.

Clear, colorless liquid in square plastic bottles with a white screw caps, for lab use

Specifically, the chambers are manufactured in facilities certified for ISO 13485 compliance, where they undergo testing for sterility, biocompatibility, extractables, and particulates. The MultiShot chamber is designed to minimize both cell loss and hold-up/dead volume, and its cartridge format helps to enable error-free loading.

 

Similarly, both the standard and genome editing electroporation buffers are CTS labeled and GMP manufactured. They meet all requirements for ancillary and raw materials for cell therapy manufacturing.


Digital connectivity for integrated workflow

The cell therapy manufacturing workflow can be complex and fragmented, comprising many different steps and products. It generally involves numerous labor-intensive manipulations, making it susceptible to errors. Several open processes also make it prone to contamination.

 

The Xenon Electroporation System can be integrated with other Thermo Fisher Scientific instruments and consumables into a complete, closed cell therapy manufacturing workflow. For example, the CTS Rotea Counterflow Centrifugation System can be used for multiple processes in your workflow, including cell separation, concentration, washing, buffer exchange, and cryopreservation. Similarly, the HyPerforma Rocker Bioreactor can be used for cell culture processes such as activation and expansion of immune and transfected cells.

Example of a connected cell therapy manufacturing system.

The Xenon system fits into a complete, closed cell therapy workflow. Left to right: CTS Rotea Counterflow Centrifugation System (cell isolation), CTS DynaMag Magnet (cell activation), Xenon Electroporation System (transfection), 2 x HyPerforma Rocker Bioreactor (expansion of transfected cells). The modules are designed for physical and digital integration.

Several pieces of cell processing equipment arranged in a line on a laboratory benchtop to show an example of a cell therapy process development workflow.

Just as physical connectivity (sterile tubing and connectors) allows the Xenon system, like the Rotea system, HyPerforma bioreactor, and other modules to be integrated into a closed workflow, digital connectivity allows them to be controlled by bioprocess controllers and software. With full Open Platform Communications–Unified Architecture (OPC-UA) compatibility, these systems can be combined into an integrated, semiautomated, closed, modular workflow for cell therapy manufacturing. The concept is demonstrated in a recent webinar, in which all instruments are controlled using the DeltaV™ Distributed Control System from Emerson.


Clinical manufacturing mode with 21 CFR part 11 support (option)

When you scale up to commercial manufacturing, we recommend switching into clinical manufacturing mode. This mode locks down your optimized protocols so that manufacturing settings can’t be inadvertently altered.

 

Clinical manufacturing mode also includes an optional software module that supports electronic record-keeping in a way that is compliant with the US Food and Drug Administration’s Electronic Records and Signatures Rule, Part 11 of Title 21 of the Code of Federal Regulations (21 CFR Part 11). This rule defines the requirements for use of electronic documents in place of paper ones. The software also includes the option to lock in a single protocol to minimize any mistakes that could be made during manufacturing.

 

Capabilities of the software include restriction of unauthorized users, password policies and expiration dates, defined user permissions and roles, tracking of all data changes through an audit log, audit reports, and on-board e-signature workflows. These are sometimes abbreviated as Security, Audit, and Electronic-signature capabilities, or SAE, and you can control them through the SAE Administrator Console.

Functionality of the SAE Administrator Console

Function

Description

System security

Controls user access to the software through user IDs, passwords, roles, and permissions. Three default user roles are provided, one with full privileges (Administrator), one with some privileges removed (Lead scientist) and the other with no privileges (Technologist). Default user roles can be edited, and additional user roles and permissions can be created.

Auditing

Tracks actions performed by users, and changes to the SAE Admin Console settings. The software automatically audits some actions silently. You can select other items for auditing and specify the audit mode. The auditing function provides reports for audited SAE Admin Console changes and actions.

Electronic signature (e-signature)

Determines the functions for which users are required to provide a username and password. You can configure e-signatures so that a user can export a signed file and print a signed report. You can also configure the e-signature event to require multiple signatures and to require users with specific permissions to sign.

 

To run your Xenon system in clinical manufacturing mode, you need a Xenon 21 CFR Software License. The license can be purchased and a field service engineer will complete the installation at your site. Per 21 CFR Part 11, your institution will also need to establish and document standard operating procedures in a compliant way.

Ordering information for Xenon clinical manufacturing mode

The clinical manufacturing mode software upgrade is a one-time purchase either standalone or as part of your instrument purchase.


Intended use of the products mentioned on this page varies. For specific intended use statements, please refer to the product label.