Automated cell isolation & activation

The CTS Detachable Dynabeads platform possesses an active release mechanism that, after the Dynabeads bind to target cells of interest, allows users to actively detach the Dynabeads from cells at any time within the process. This results in a T cell population with minimal non-target cell impurities, and an overarching process flexibility.

Processed on the Gibco™ CTS™ DynaCellect™ Magnetic Separation System, the Dynabeads firstly isolate target cells of interest. The proprietary active release mechanism then allows for active detachment of the beads from their target cells. This results in the ability to refine target cell purity, target cell yield, and desired cell phenotype, and subsequently, has benefits for downstream gene-modification and expansion.

The base of the product is a 4.5 micron paramagnetic polystyrene bead. The active release mechanism entails a biotin derivative and a variable heavy domain of heavy chain (VHH) antibody conjugated to the surface of the Dynabead itself. The VHH antibody, a 12–15 kilodalton camelid-derived short-chain antibody specific for various clusters of differentiation (CD) markers, demonstrates tunable specificity and affinity as it binds to target cell surface markers. In addition, produced in yeast, these antibodies are animal-origin free, highly stable, and exclude any risk of viral contamination or adventitious reagents.

The system process is illustrated in Figure 1CTS Detachable Dynabeads magnetic beads product concept and process.. After incubation with the CTS Detachable Dynabeads, the target cells are detached by introducing a release buffer that outcompetes the biotin derivative on the surface of the bead. After release, the Dynabeads can be subsequently removed by using the CTS DynaCellect system, which allows for the highly pure T cell population.

The CTS DynaCellect Magnetic Separation System is a closed, automated device for cell isolation and magnetic removal of Dynabeads. It enables process automation and includes fit-for-purpose single-use consumables for isolation and bead removal. The touchscreen user interface allows for customization of protocols for cell isolation, activation, depletion, and magnetic separation. This instrument can be used standalone but can also be leveraged as a plug-and-play instrument with other instruments within the workflow. This is made possible by an open platform communication unified architecture that is compatible with Emerson’s DeltaV™ software.

Improving upon current cell therapy manufacturing methods

The recently released Gibco™ CTS™ Detachable Dynabeads™ CD3/CD28 Kit allows for control over the duration of T cell activation, thus achieving desired cell phenotype, optimizing the stem cell-like properties of the final cell population, and making for a more efficacious final drug product. In addition, this kit gives researchers the ability to control and optimize activation marker profiles that they experience while obtaining desired central memory phenotypes after activation. Finally, because the CTS Detachable Dynabeads are designed for use with the CTS DynaCellect Magnetic Separation System, the process is automated and scalable.

Although this active-release kit is comparable to the passive-release CTS Dynabeads CD3/CD28 in terms of CD25 expression (Figure 2Average CD25 expression for active-and passive-release Dynabeads magnetic beads.), fold expansion (Figure 3Cell expansion for active-and passive-release Dynabeads magnetic beads.), CD4:CD8 ratios (Figure 4CD4:CD8 ratio for active-and passive-release Dynabeads magnetic beads.), and desired cell phenotype (Figure 5Memory T cell phenotype with active- and passive-detachable Dynabeads magnetic beads.), the active-release mechanism does provide improvements to the cell therapy process.

One of these improvements involves T cell purity. With the CTS Detachable Dynabeads CD3/CD28 Kit, researchers achieved target cell purity levels of >98% (Figure 6Features of the iQue advanced flow cytometry platform.). This consistency in performance is a necessity when the cellular starting material is characterized by substantial biological variability. Apheresis profiles are different from one individual to another, but also fluctuate further based on the patient’s specific indication and stage of disease. Regardless of the starting material, it is necessary to achieve consistent performance with an isolation reagent.

Another improvement revolves around maintaining healthy cells, which is required for the production of an efficacious therapeutic. As shown in Figure 7Cell viability and isolation efficiency of CTS Detachable Dynabeads CD3/CD28 beads., after actively releasing beads from the cells on days one, two and three, viability levels of >90% were observed with flow cytometry. Isolation efficiency was >90%, indicating specificity for early memory cells and revealing the robustness of the VHH antibodies.

Because the CTS Detachable Dynabeads CD3/CD28 Kit is designed for isolation and activation in a single step, the activation profile over time was also evaluated, as shown in Figure 8Activation markers for CTS Detachable Dynabeads CD3/CD28 beads.. Flow cytometry was used to assess three different samples, which were analyzed from four different donors. Expected levels of CD69 early activation were seen decreasing through days 1, 2, and 3. Inversely, CD25 mid-to-late activation levels were observed to increase within the same time span. Therefore, T cell activation markers CD69 and CD25 adhere to expected kinetics on days 1, 2, and 3.

Scaling up with automated instrumentation

As CTS Detachable Dynabeads have the ability to improve cell therapy in the context of process development, clinical trials, and commercial manufacturing, successful scale-up with the CTS DynaCellect Magnetic Separation System is vital. When scaling up with a variable starting material, researchers must achieve consistent performance with few impurities. When using an automated protocol on the CTS DynaCellect system, post-isolation purity of target T cells was 99.6% with virtually no presence of impurities such as monocytes, B cells, or NK cells. In addition, high isolation efficiency and viability were achieved during scale-up (Figure 9Isolation efficiency and viability.). Furthermore, optimal cell recovery was also achieved during the scale-up process. 106% cell recovery was demonstrated on day 3 after bead removal. The total count of more cells on day three as compared to day 0 was likely due to the fact that cells proliferate after activation begins and before incubation in expansion media. Lastly, after activation and bead removal, activation markers conformed to expected kinetics during scale-up, revealing consistently highly efficacious T cells (Figure 10Isolation efficiency and viability.).

In summary, CTS Detachable Dynabeads will provide the process flexibility, scalability, and consistent performance that cell therapy manufacturers need.

Q&A

Eugene Kang

What is the recommended ratio of Dynabeads to target T cells?

EK: When the Detachable Dynabeads CD3/CD28 Kit is being used for isolation and activation in one step, a Dynabead-to-target cell ratio of 3:1 is recommended. I say target cell because the ratio should not be calculated based on total nucleated cells. That 3:1 ratio is based on a series of titration studies performed to optimize the number of Dynabeads, the isolation efficiency, and the activation profile. If using the kit as a downstream activation reagent, a 1:1 ratio is recommended.

How do you test bead residuals in the culture post-detachment and at the end of harvest to be sure there are no beads in the therapeutic product?

EK: Because cell therapy is constantly evolving and regulatory agencies are always playing catch-up, there is not yet an official ruling on the number of residual beads that can be in the therapeutic product. However, there is guidance to be followed.

There are a number of clinical trials and commercial manufactured drugs that use CTS Dynabeads in the United States. For those clinical programs, the US FDA’s recommended guidance was a threshold of 100 beads per 3 million T cells. With these new Detachable Dynabeads on the CTS DynaCellect system, we have seen residual beads as low as two or three beads per 3 million T cells, which is obviously quite far below that 100 bead threshold.

Safety is a very important focus here. That is why we are minimizing the presence of residual Dynabeads far below recommended levels. On top of that, the Dynabeads themselves are inert during phagocytosis. Any residual beads that are left are not incorporated into the T cells and therefore, the patient.

What are the minimum and maximum requirements for sample volume and cell concentration of the starting material?

EK: Again, it is highly recommended that the CTS Detachable Dynabeads be used with the CTS DynaCellect system. This will vastly cut cost, simplify workflow, and optimize overall performance of the final drug. Having said that, the CTS DynaCellect system can handle up to 1 L of volume from a starting material, with a concentration of 10 million cells per mL. This system has also been successfully tested with a volume as low as 10 mL.

The CTS DynaCellect system can also handle continuous Dynabead removal as long as input and output bags are swapped. This is why it is suitable for both allogeneic and autologous therapies.

Does the use of CTS Dynabeads magnetic beads pose any potential risks to patient safety?

EK: As mentioned previously, not only are the number of residual Dynabeads left in the final drug minimized, but the effect of the beads on the patient is also minimized since the beads are not incorporated into the T cells.

The CTS Dynabeads are currently being used in over 200 active clinical trials as of mid-year 2023 and, in terms of safety profiles, they have continued to meet regulatory expectations.

What is the duration of activation needed to achieve the desired T cell phenotype?

EK: Every manufacturing process is different, but right now, the norm is activating within a 24–48 h period. Within that timeframe, we are seeing optimal desired phenotypes (like the central memory stem cell phenotype) after activation. However, the duration of activation will really depend on how the protocol is modified and optimized.

What is the benefit of this product having VHH antibody fragments for both CD3 and CD28 cells?

EK: With naïve T cells, there are three different activation signals to look for: CD3 for T cell receptor engagement, co-stimulatory receptors such as CD28 for activation, and also cytokine stimulation (e.g., IL2).

Instead of having separate reagents for positive cell isolation and activation, having CTS Detachable Dynabeads with both antibodies on the surface allows you to combine isolation and activation into one step, cutting down cost and the number of steps in the workflow. More specifically, the presence of both CD3 and CD28 allows for the preferential isolation of CD3 and CD28 double-positive cells. This is important because CD28 negative T cells have been shown to be associated with T cell exhaustion. By controlling that activation period, you can avoid exhausting the T cells and facing cell death.

Biography

Eugene Kang is a Senior Product Manager at Thermo Fisher Scientific, with a personal desire to establish cell therapy as a first-line treatment worldwide. He holds a Bachelor’s degree from the University of Notre Dame, a Master of Public Health from Columbia University, and a Master of Business Administration from Boston University.

Affiliation

Eugene Kang
Senior Product Manager,
Thermo Fisher Scientific

Authorship & Conflict of Interest

Contributions: The named author takes responsibility for the integrity of the work as a whole, and has given his approval for this version to be published.

Acknowledgements: None.

Disclosure and potential conflicts of interest: The author has no conflicts of interest.

Funding declaration: The author received no financial support for the research, authorship and/or publication of this article.

Article & Copyright Information

Copyright: Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.

Attribution: Copyright © 2023 Thermo Fisher Scientific. Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.

Article source: This article is based on a transcript of a webinar, which can be found here.

Webinar recorded: Sep 14, 2023; Revised manuscript received: Oct 20, 2023; Publication date: Dec 13, 2023.