Shear relief minimising shear stress in cgt production

17 December 2025

Cell and gene therapies (CGTs) are a rapidly developing area of technology, and therapies using chimeric antigen receptor (CAR) T-cells, stem cell and other gene-edited cell products are transforming the pharmaceutical landscape. These new therapies essentially exploit cells as living drugs, capable of proliferating and generating a sustained, targeted immune response to treat disease sites in the patient’s body.

CAR T-cell therapy involves engineering a patient’s T-cells to expand in number and generate sustained immune responses, targeting tumours and diseased tissue. Similarly, stem cell therapies use multipotent or pluripotent cells that proliferate and help regenerate damaged tissues. CAR T-cell therapies, such as tisagenlecleucel and axicabtagene ciloleucel, engineer a patient’s T-cells to attack tumours. Viral gene therapies, such as RGX-314 for neovascular age-related macular degeneration, show potential for durable disease control with a single administration. Despite these advances, CGTs face critical challenges during manufacturing, transport and delivery, where maintaining cell viability, phenotype and function remains essential.

Despite advances in CGT delivery and manufacturing, an often-overlooked challenge comes from the mechanical stress experienced by cells during handling. Therapeutic cells are highly mechanosensitive, and exposure to shear forces from fluid flow, tubing, bioreactors, microfluidic devices or injectors can reduce viability, alter phenotype and compromise functional performance, potentially impacting efficacy and safety.

Why shear stress matters for CGTs?

In fluid-mechanical terms, shear stress (τ) arises when layers of fluid move relative to one another or to adjacent surfaces and can be expressed as the product of fluid viscosity (μ) and shear rate (γ̇); that is, τ = μ × γ̇. In cellular systems, exposure to shear can have profound biological consequences, including morphological deformation, cytoskeletal disruption, membrane perturbation, altered gene expression and reduced viability. Even modest shear rates can produce measurable drift in membrane-bound proteins,1,2 indicating that fluid forces acting near membranes can mechanically perturb membrane-bound entities and thereby impose mechanical stress on the cell surface.

Figure 1: This schematic outlines some major workflow stages in CGT manufacture: harvest and transduction, expansion, separation and final formulation for patient delivery. When unmanaged, shear stress can fluctuate sharply across CGT manufacturing at any of these stages.

During the manufacture and delivery of CGTs, therapeutic cells are exposed to a variety of fluid-mechanical environments that can impose shear stress at multiple stages of processing (Figure 1), potentially impacting viability, phenotype and functional potency. In the initial cell harvest and processing stage, cells are typically collected, subjected to centrifugation and resuspended in media. Here, shear arises during pipetting, aspiration and passage through narrow tubing, with high flow rates generating localised high shear that can deform membranes or disrupt fragile cytoskeletal structures.

During transduction or gene-editing in bioreactors, viral vectors or gene-editing complexes are introduced under mixing conditions; here, even gentle agitation can create gradients of fluid velocity, exposing cells to non-uniform shear. During expansion in stirred-tank or perfusion bioreactors, cells are exposed to varying shear stresses generated by impellers, perfusion flows and sparging (i.e. when oxygen is bubbled through media). These shear stresses can alter cytoskeletal integrity, trigger apoptosis or induce subtle phenotypic drift.

The separation and washing steps, commonly conducted via tubing, pumps and filtration devices, similarly produce localised shear, particularly at constrictions, bends or membrane surfaces, where fluid velocity gradients can be high. Finally, during formulation and delivery, cells are transferred into infusion media; pass through tubing, connectors and injectors; and, ultimately, are administered via catheters or syringes – the high pressures at needle tips or microfluidic sorting channels can produce transient but intense shear spikes.

Collectively, these mechanical exposures can reduce cell viability, alter functional markers and induce apoptotic or stress signalling, potentially reducing the therapeutic potency of the final product, increasing batch-to-b

atch variability and raising regulatory concerns under GMP and International Council for Harmonisation guidelines.

Methods of shear stress mitigation

As CGTs scale to more complex processes, systems and formulations, their exposure to shear stress rises, increasing risks to product quality. Larger bioreactors, higher throughput microfluidic devices and more complex formulations all increase the frequency of shear stress exposure, compounding potential damage on cultured cells (Figure 2). This growing mechanical challenge underscores the need for robust mitigation strategies. Strategies such as optimised flow geometries, controlled infusion rates and protective formulations can help to preserve the functional characteristics of these living therapeutic agents throughout manufacturing and delivery workflows.

Figure 1: Illustration of the level of control possible over particle morphology using spray-drying process design.4 Image from A. B. D. Nandiyanto and K. Okuyama⁴

When optimised, spray drying can reduce reconstitution times from several minutes to mere seconds.(5) This has the potential to significantly improve usability and adherence, even with existing device technologies, by removing some of the difficulties users face.

Because it is perceived as a harsher process than freeze drying, spray drying has traditionally been used for small-molecule drugs. However, it has been shown to be suitable for biologics, including complex biomolecules, such as immunoglobins and viral capsid proteins, due to evaporative cooling and the use of protective excipients, such as trehalose.

Aside from unfamiliarity, adoption for injectable formulations has been limited by practical challenges, including the availability and maturity of aseptic spray drying and powder handling, high cost of physical losses at small scale (up to 50%), and the limited number of CDMOs available with experience in this area.

Today, some of these barriers, such as aseptic handling and powder filling, are gradually diminishing, increasing the accessibility of this technology. However, as with lyophilisation, spray drying requires the development of an appropriate formulation – a process that can be time-consuming and demands specialised expertise.

Alternative Reconstitution Device Technologies

A different approach to simplifying and standardising the process of reconstitution is to design around the issues. Several novel drug-device combination platforms aim to achieve this by automating some or all preparation steps. These systems reduce user burden, lower the risk of use errors and improve overall usability compared with more manual methods.

Windgap Medical’s Large Volume Dual Cartridge platform employs a side-by-side configuration of two standard single-chamber cartridges – one containing the diluent and the other the lyophilised drug product. Reconstitution occurs through cyclic fluid transfer between the cartridges, ensuring thorough mixing prior to administration. Two variants are offered – a touch-activated design and a compressed-gas-driven version, the latter capable of delivering large volumes and handling highly viscous formulations.

Eveon’s Intuity® Ject MX also uses cartridges as primary packaging and achieves reconstitution via cyclic fluid transfer between two containers. However, it replaces manual or gas-driven actuation with an electromechanical mixing mechanism. This automation standardises the mixing process and allows for connectivity features that enable data capture and adherence monitoring. Additionally, Eveon offered the Intuity® Mix, a piston-pump-based, non-portable, fully automated system intended for clinical or pharmacy use. (Note from ONdrugDelivery: As of April 2024, Eveon is in formal insolvency proceedings in France.)

Enable Injections’ EnFuse® system follows a different approach. While currently marketed versions are designed for liquid formulations, such as Empaveli® (pegcetacoplan, Apellis Pharmaceuticals, Waltham, MA, US), a variant exists that performs reconstitution immediately before pump filling. This method bypasses the challenges associated with in situ lyophilisation and container compatibility by connecting directly to a standard lyophilised powder vial. EnFuse uses an internal mechanical pumping system for fluid transfer and mixing, enabling it to handle a wide range of formulation viscosities.

Some systems address specific aspects of vial usability by automating diluent transfer but without automating mixing, transfer or delivery. Their vial-based format is generally bulkier than cartridge-based platforms and offers fewer opportunities for delivery device integration. However, it provides flexibility in dosing and route of administration, making these systems well suited to formulations with straightforward dissolution profiles or varied dosing regimens, while being less appropriate for tackling challenges such as high viscosity.

Baxter’s BaxJect III®, marketed for use with ADYNOVATE® (PEGylated antihaemophilic factor, Takeda Pharmaceuticals), uses two standard vials: one for diluent and one for lyophilised powder, connected via a sterile transfer device. Reconstitution is driven by a pressure differential between the vials, without an active mixing mechanism. The user must manually withdraw the prepared solution into a syringe through a side port.

DuoJect’s (Bromont, Canada) INTERVIAL™ family of devices, including PENPREP EVO™, approved for use with SAIZEN® (somatropin, Merck), combines the diluent and lyophilised powder within a single device. Mixing is manual, and the user then draws up the dose after reconstitution is complete.

Pfizer’s Act-O-Vial® offers a simple mechanical method to simplify reconstitution. It integrates the diluent and lyophilised drug in a single vial assembly separated by a rubber stopper. Pressing the stopper releases the diluent into the powder chamber, allowing mixing before withdrawal for injection.

Innovation in this space benefits most from partnerships that bridge formulation science, human factors expertise and engineering design into a single integrated development. By combining deep understanding of the drug product with insight into user needs and awareness of enabling technologies, it is possible to streamline reconstitution, reduce complexity and unlock the next generation of combination products. With unmet market need still evident, this remains a fertile area for innovation and a compelling opportunity for growth.

Conclusion

Lyophilisation remains the preferred solution for stabilising sensitive biologics, offering substantial benefits in shelf-life extension, storage flexibility and global distribution. However, the shift from traditional vial formats to integrated, patient-friendly delivery systems is an area rich with innovation. Dual-chamber primary packaging and in-device reconstitution platforms promise simpler preparation and fewer use errors, yet they bring their own user and technical challenges, such as larger size, potentially confusing use steps, freeze drying within constrained geometries, ensuring rapid and complete dissolution and maintaining long-term stability.

Emerging alternatives, such as spray drying, present new opportunities to tailor powder characteristics and enhance reconstitution efficiency. Still, historical barriers, including the complexity of aseptic powder filling and high material losses at small scale, have limited broader adoption. Advances in device technology are also driving progress, with platforms that aim to automate some or all steps of the reconstitution and delivery process.

Ongoing innovation in both drying and delivery technologies is fuelled by the need to improve patient convenience, adherence and therapeutic outcomes. The widespread adoption of on-device reconstitution will depend on carefully balancing formulation stability, manufacturing feasibility and human factors. The potential reward is significant – expanding access to complex biologics in formats that are both highly effective and intuitive to use across diverse markets and therapeutic areas. Achieving this will require multidisciplinary collaboration, less-siloed development approaches and strategic partnerships that unite expertise in formulation, engineering, and user-centric design.

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Written by Kristien De Clercq and Dr Alex Vasiev and featured in On Drug Delivery.

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