Cell lysis is often seen as just another step in the upstream workflow for gene therapy production, but it’s anything but routine. This moment of breaking open cells determines not only how much vector is released, but how pure and consistent the final product will be. As gene therapy manufacturing processes scale to meet broader clinical demand, many teams discover too late that their lysis method doesn’t scale or comply.
Here, Avantor’s Beth Kroeger-Fahnestock explores the technical and operational challenges of cell lysis, from reagent selection to large-scale implementation, as well as optimizing the cell lysis step and key considerations for selecting the best reagent to ensure scalability and meet environmental and regulatory standards. As well as working within industry, Kroeger-Fahnestock has served on the ISPE task force responsible for writing the ISPE Guidance: Cleaning Validation Lifecycle – Applications, Methods, and Controls Good Practice Guide, published in 2020 and was an Adjunct Lecturer, Temple University, School of Pharmacy, RA/QA Graduate Program for several years.
Give us an introduction to the cell lysis step in gene therapy manufacture…
Cell lysis is a critical step in upstream gene therapy manufacturing, particularly for viral vectors such as AAV, where viral particles remain intracellular post-production. To release viral vectors from the producer cells during upstream processing, a lysis step is needed to rupture and break down the cell membrane, leading to the release of intracellular content. An often-underappreciated component within the upstream workflow, cell lysis may, in fact, constitute its greatest vulnerability, potentially compromising the integrity and reliability of the entire process. The primary objective of the lysis step in the viral vector workflow is to efficiently release high yields of intact viral vectors while minimizing vector damage and impurity load, which directly affects downstream processing and overall product quality.
If the lysis step is inefficient, a significant portion of the viral vector may remain within the producer cells, reducing recovery. A poorly optimized lysis process can shear viral particles under uncontrolled chemical or mechanical stress, or release an excessive amount of host cell impurities (e.g., host cell DNA, proteins, lipids), increasing the complexity and cost of downstream clarification and chromatography, and potentially compromising product quality.
The approach to cell lysis must be tailored to the vector system. For example, AAV and adenovirus require active lysis. Detergent-based chemical lysis of the cells producing AAV vectors is common and typically followed by enzymatic digestion with an endonuclease to degrade host cell and plasmid DNA. In contrast, lentiviral vectors are released into the culture medium, so the harvest typically involves clarification without the need for cell lysis.
The gene therapy field continues to grow, addressing a broader range of indications for larger patient populations. With this growth, the need to scale processes and deliver greater consistency and efficiency is essential. This imperative includes the cell lysis step; having a solution that offers effective lysis and one that ensures complete cell disruption, vector integrity, and easy removal during purification, all while meeting environmental and regulatory standards.
What makes cell lysis particularly challenging at large scale, and what are some of the most common failure points?
At large bioreactor volumes, it is essential to ensure consistent and complete lysis. Factors such as mixing efficiency, reagent distribution, and contact time are difficult to control at larger scales, potentially leading to uneven lysis, reduced vector recovery, or increased product variability. Another concern is the shear sensitivity of vectors. The lysis method must be aggressive enough to release intracellular vectors but gentle enough to preserve their structural integrity. This balance is more difficult to maintain in large-scale systems, where mechanical stress and process parameters, including temperature or pH, can fluctuate more widely.
Impurity management also becomes more critical at scale. Larger batch sizes mean greater quantities of host cell proteins. DNA and lipids are released during lysis, placing a heavier burden on downstream purification steps. Excessive impurities can foul filters, reduce chromatography efficiency, and lead to lower overall yields if not adequately controlled.
Additionally, lysis reagents used at large scale must be highly consistent, scalable, and compatible with regulatory expectations, including requirements for low endotoxin levels, animal-origin-free materials, and validated removal in the final product. Operationally, the lysis process must be easy to integrate into automated, closed systems to support aseptic manufacturing and reduce contamination risk.
Ultimately, effective large-scale lysis depends on selecting reagents and protocols that are robust, reproducible, and optimized not only for vector release, but for downstream compatibility and regulatory compliance.
Are there “rules of thumb” or design considerations you recommend for optimizing lysis without damaging viral particles?
Once the viral particles are released from the cell during the lysis process, the detergent lysis step must not have any effect on the integrity, infectivity, or yield of the released viral particles -- particularly from shear stress due to agitation. The viral vector may denature and unfold as a result of shear stress and adsorption to surfaces during the downstream process. This shear stress and resulting viral particle damage can lead to a decrease in downstream yield, and low yields can create a dosing problem. If the vector concentration in a gene therapy batch is too low, developers would have to increase the dose volume to an unreasonable level.
To avoid this, mixing speeds, temperature, and incubation time during the lysis step must be carefully controlled. Gentle agitation, combined with a well-optimized lysis solution concentration, can promote efficient lysis while minimizing physical stress on the viral particles. In addition, choosing a lysis solution that preserves capsid integrity and can be effectively removed in downstream purification is essential for both product quality and regulatory compliance. Ultimately, process development teams should balance lysis efficiency with product protection, using small-scale models to test and tune conditions before scaling up.
What types of detergents are most commonly used for viral vector lysis?
Chemical detergents are widely used to disrupt host cell membranes during the lysis step. The most commonly used detergents fall into several categories. Non-ionic detergents (e.g., Triton X-100, NP-40, Tween 20) are relatively mild, helping to preserve viral capsid integrity while effectively lysing the cell membrane. Triton X-100 has historically been a popular choice, though its use is declining because of regulatory concerns. Ionic detergents (e.g., sodium deoxycholate, SDS) are typically avoided in AAV processes because they can damage the viral capsid and reduce infectivity. Cell lysis by SDS also works only at alkaline pH, which is not an optimal condition for viral particle stability.
The choice of detergent for cell lysis is influenced by several key factors. First, the type and sensitivity of the viral vector play a major role. AAV, for example, is particularly susceptible to harsh conditions and requires a gentle yet effective lysis method, whereas adenovirus or HSV may tolerate more aggressive detergents. Process performance is also critical; the detergent must lyse cells efficiently without damaging the viral particles or reducing yield, and it must be compatible with enzymatic steps such as endonuclease treatment.
Downstream compatibility is another consideration, as some detergents can interfere with filtration or chromatography, causing fouling or product loss. Regulatory and safety requirements, such as low endotoxin levels, animal-origin-free formulations, and ease of removal, are increasingly important in GMP settings. For example, environmental regulations like the EU’s REACH have driven the industry away from using Triton X-100. Finally, scalability and robustness are essential; the selected detergent must perform reliably at large volumes.
As gene therapy production matures, there’s growing interest in next-generation detergents that offer effective lysis with improved safety, regulatory profiles, and downstream compatibility.
How do regulatory expectations around detergents shape the selection of lysis reagents?
In 2012, the European Chemicals Agency (ECHA) identified Triton X-100 (octylphenol ethoxylate), a detergent commonly used in biomanufacturing, as a "substance of very high concern" (SVHC) because of its toxicity to aquatic life. As part of the REACH framework, ECHA has placed restrictions on the use of Triton X-100, prompting its phase-out in existing processes by 2030 and prohibiting its use in new manufacturing processes within the EU.
These regulations have global implications because many biopharma companies prefer to harmonize production practices as much as possible to ensure continuity across manufacturing sites, and prepare for potential restrictions in other regions. As a result, manufacturers are increasingly shifting away from Triton X-100 to comply with European regulations and to future-proof their processes relative to evolving environmental and regulatory expectations.
In addition to environmental concerns, Triton X-100 presents several practical challenges that make it less than ideal for large-scale biomanufacturing. Its pure form is extremely viscous – comparable to cold honey – which makes it messy and difficult to handle, especially when precise dosing or automated delivery is required. The detergent also has a high foaming profile, which can limit the usable volume of bioreactors by increasing the risk of overflow during processing. Furthermore, Triton X-100 offers little protection for viral particles against shear stress, a common issue during cell lysis that can damage capsids and reduce vector yield.
In response to these challenges, suppliers are innovating new, eco-friendly alternatives.
What advice would you give to early-stage gene therapy developers as they plan their upstream and lysis strategies for the long term?
The cell lysis strategy must scale with the vector production process and meet long-term regulatory and performance requirements. Now is the time to adopt an eco-friendly alternative early in process development to avoid costly changes later in development.
Equally important is ensuring that the detergent is compatible with downstream steps, particularly enzymatic DNA digestion, which is used to remove residual plasmid and host cell DNA after lysis. The detergent must not inhibit endonuclease activity and should maintain the purity and integrity of the viral vector product throughout processing. Developers should also evaluate how variables such as pH, ionic strength, and temperature affect lysis efficiency to fine-tune their process early on.
Finally, it’s essential to consider how easily the detergent can be removed from the final product. Compatibility with downstream purification techniques, such as filtration, diafiltration, or polishing steps like anion exchange chromatography, will help ensure product quality, process consistency, and regulatory compliance.
Building these considerations into the process at an early stage can streamline scale-up, support global regulatory approvals, and ultimately enable more efficient and sustainable gene therapy manufacturing.