Bottlenecks in Cell and Gene Therapy: Steps That Separate Patients From Treatment
Cell separation methods influence yield, viability, and scalability in cell and gene therapies.
Cell therapies have revolutionized treatment for conditions once considered incurable, including blood cancers and genetic disorders such as sickle cell disease (SCD).
The latter, SCD, affects more than 8 million individuals worldwide; it results in sickling, or misshaping, of red blood cells, which can accumulate within and occlude blood vessels. Not only are these episodes painful, but they are also serious—sometimes life-threatening—given that the blood flow to major organs can be affected. The condition requires lifelong treatment: an assortment of tailored medications aiming to prevent cell sickling, control pain, and reduce the risk of complications.
However, hope came in recent years with the US Food and Drug Administration’s approval of the first cell-based gene therapies for SCD. These one-and-done treatments promised patients a “new life,” free from the shackles of symptom management.
With hundreds of novel cell and gene therapies in development for incurable conditions, many patients live in hope of one day receiving the same opportunity. Thousands of therapies will be tested, and some will be approved, but how many patients will receive them?
To explore the bottlenecks in scalable cell therapies, Technology Networks sat down with Dr. Sophie He, vice president of cell therapy at Bracco. She explored current industry manufacturing standards and the limitations they impose on both production and patient access.
What are the key stages of the cell therapy manufacturing pipeline, and where does the separation step fit within this?
Sophie He, PhD (SH):
The standard cell therapy manufacturing process includes washing, cell selection (or separation), activation, transduction, expansion, further washing, formulation, and fill. Separation is a critical early step because it affects not just purity and yield, but also cell viability and health. Each of these factors affects the success of manufactured cell therapy.
Cell separation in cell therapy manufacturing
This is the process of isolating the desired cells from patient samples. It removes unwanted cells, cell debris, and toxins, aiming to produce uniform batches of viable cells. Common cell separation techniques include:
- Immunomagnetic cell separation: Magnetic beads are coated with antibodies that bind the desired cells, ensuring that they are held in place during washing.
- Fluorescence-activated cell sorting: Cells pass through a laser and are sorted based on fluorescence and light-scattering patterns.
- Density gradient centrifugation: Spinning heterogeneous samples causes cells to separate into layers based on density and size.
Cell separation has historically been done separately from cell activation, prolonging the process. New methodologies combine cell selection and activation, shortening processing time and reducing the number of steps while still yielding the same results. Notably, the selection technology used can also shape downstream chemistry, manufacturing, and controls strategy, automation compatibility, and long-term manufacturing scalability.
Some manufacturing processes require an additional enrichment step before final formulation to guarantee the final product is free of undesired cell types. Therefore, cell separation is a key manufacturing process, not only to get the right starting material but also, in these cases, to ensure the final product is within specifications.
IH:
Why are populations such as hematopoietic stem cells (HSCs) particularly vulnerable during separation, compared to other cell types?
SH:
HSCs are especially vulnerable during cell separation because they are a rare population; they primarily reside in the bone marrow and account for as little as 0.01% of total nucleated cells. They are also highly sensitive to stress such as shear forces that can negatively impact their clinical effectiveness.
For rare cell populations like HSCs, the limiting factor for commercial rollout is often the generation of sufficient yield with high purity. Traditional methodologies, such as magnetic separation, can force compromises between purity and yield and require handling steps that disproportionately affect fragile populations.
Scalability is also a challenge, as methods like magnetic beads that work at a small scale don’t always translate cleanly to scaled production. This can be overcome by adopting novel technologies, such as microbubble-based approaches, which can scale effectively from laboratory to manufacturing.
IH:
What technical and biological limitations have you observed in investigating magnetic separation, the current industry standard?
SH:
Certain limitations have become normalized, such as additional column hardware, extra unit operations, limited flexibility in selection type and sequence, and the inherent risk from residual magnetic materials downstream. This can potentially impact electroporation, risking batch failure.
We’ve also seen cell stress and yield loss due to sheer forces when magnetic separation is used.
Challenges in magnetic cell separation
There are many challenges in magnetic-activated cell sorting. Dependence on antibodies opens the door to background noise, which can be contributed to by dead cells, undesired cells with similar protein expression to the target cell, and non-specific binding. If the percentage of desired cells in the starting material is low, background noise can increase. Furthermore, sorting involves mechanical manipulation, which can compromise cellular integrity.
IH:
How do these limitations influence downstream manufacturability and dose consistency for one-and-done therapies, such as SCD treatments?
SH:
SCD gene therapies have persistently struggled with yield issues, with insufficient recovery of CD34+ cells preventing the production of viable doses and limiting commercial launches.
CD34+ cells in SCD gene therapy
CD34+ is a marker for hematopoietic stem cells, which serve as the foundation for SCD gene therapies. These cells are targeted for isolation during manufacturing.
Yield can be directly related to the chosen separation methodology, so it is imperative to choose techniques with high efficiencies that leave behind minimal-to-no residuals. This helps to reduce the amplification of inefficiencies downstream, such as requiring a long expansion period due to the low number of selected cells.
In cases where a patient’s life depends on the successful manufacturing of a single treatment, failure can pose a danger to the patient, lead to tremendous expense, and result in valuable time lost.
IH:
Looking ahead, what do you believe next-generation cell separation technologies must deliver to support consistency and scalability across cell therapies?
SH:
A few things are required. Firstly, technology must be gentle and preserve the target cell population’s functionality. Given the limitations of existing technologies, a biocompatible approach that leaves no magnetic residuals could help preserve the viability and functionality of targeted cell populations.
Secondly, as an industry, we need to develop cell therapy manufacturing processes that allow us to treat more patients. This can only be achieved by streamlining the process and making it cost-effective. Having a single, scalable separation technology that can be used for positive, negative, and sequential cell selection could be key to enabling the enrichment of complex cell phenotypes. It could help researchers to fine-tune their workflows at the early process development stage and provide greater flexibility and scalability.
Finally, next-generation cell separation technologies must be compatible with closed, automated Good Manufacturing Practice workflows, aligning with industry-wide movements toward digitalization and containment.
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