May 30, 2024

Advances in Sterilization Technologies for Overcoming Viral-Vector Manufacturing Challenges

Sterility must be assured for all biologic drug products. Terminal sterilization achieved via treatment with heat, radiation, or certain chemicals (vaporous hydrogen peroxide, vaporous peracetic acid, or ethylene oxide) is preferred, but it is only applicable to drug substances that remain stable under the harsh conditions involved. For most biologics, therefore, sterility must be assured by using aseptic processing techniques, which involve use of sterile raw materials, equipment, and processes under conditions that prevent microbial contamination and maintain sterility. Viral vectors are no exception.

Human and process-related risks
There are two main potential sources of contamination, according to Cyrill Kellerhals, COO at Andelyn Biosciences: operators and the facility design. “Proper training for operators on proper procedures and immersing them in the quality mindset is foundational,” he insists. “Investment in employee satisfaction and career development can help keep turnover low and thereby enable continuity in operational excellence,” he adds. A fit-for-purpose facility design leveraging state-of-the-art technologies such as centralized building sensors, monitoring, and control for greater connectivity and single-pass-through air, meanwhile, can mitigate both microbial and cross-contamination risks, Kellerhals notes.

The actual sterilization process for viral vectors involves sterilizing-grade final filtration followed by aseptic filling. For these steps, the risks for microbial and cross-contamination are largely associated with manual errors that lead to batch failure or batch record deviations, according to Nigel Jackson, principal engineer, bioprocess at Cytiva.

Additional challenges for the sterilizing-grade filtration step include the need to perform a pre-use post-sterilization integrity test (PUPSIT) and a post-use integrity test in situ, the potential for leak development within single-use manifolds during installation or processing, and the need to minimize hold-up volumes, Jackson comments.

In addition, viral-vector final formulations are very concentrated, and filtration of these solutions is challenging, notes Holger Laux, director of viral vector process development at CSL Behring.

Challenges to sterility can also arise during other unit operations required for production of viral vectors. “Multiple steps are involved in upstream (e.g., seed train, infection/transfection, harvest), downstream (e.g., cell lysis, chromatography, filtration, and formulation), and fill/finish (e.g., filling and freeze drying) processes, with each step bearing the risk of losing sterility,” says Karl Heller, vice-president CMC development with Ascend Advanced Therapies. Cell-stack production involving several manual manipulation steps is a prime example of operations that introduce contamination risk, says Laux.

Variability challenges
“Sterile manufacturing of live viral vectors used to be the ultimate challenge in the biopharmaceutical industry,” Heller continues. Today, however, the variable nature of viruses (DNA/RNA, enveloped/non-enveloped, size range from 20 nm to 400 nm) and their associated properties (e.g., chemical, physical, and thermal stability) remain to some extent the biggest challenges for sterile manufacturing, he believes. “All these properties must be considered and evaluated when developing upstream and downstream unit operations to obtain high-quality, stable, and sterile drug substances and drug products in high yield,” Heller remarks.

Lentiviral (LV) vectors tend to present a greater challenge for sterilizing-grade filters than adeno-associated viral (AAV) vectors, according to Jackson. “LV vectors at 0.1–0.12 µm in diameter are far closer to the pore size of the filters, and therefore, developing a robust filtration process is more difficult, especially if aggregation occurs,” he explains. The high concentration of viral vector final formulations increases the challenges to process development and scaleup, adds Laux. “Effective control of LV formulation and a strong toolbox of various sterilizing-grade filter options are required to find a solution,” Jackson notes.

AAV vectors, which are much smaller (approximately 0.02 µm), can readily undergo a 0.2-µm or even a 0.1-µm filtration to obtain a sterile product, adds Heller. For some large viral vectors, such as poxvirus vectors including vaccinia and modified vaccinia virus Ankara (MVA) with a size of approximately 350 nm, that type of filtration is simply not possible.

Whether a virus is enveloped or nonenveloped also impacts the sterile filtration process, according to Heller. “Enveloped viruses are usually [much] more susceptible to thermal or chemical influences, so no extreme conditions (e.g., low pH) can be applied, particularly during downstream processing. These limitations may increase the risk of losing sterility,” he observes.

Regardless of the indication or program, Kellerhals emphasizes that quality systems and regulations are universal and overarching, and adherence to strict quality standards is essential to ensure consistency and safety for patients.

Scaling considerations
Scaling itself does not necessarily increase the risk of losing sterility during viral-vector manufacturing, particularly for high-volume, low-titer products such as vaccines. Maintaining sterility can be more burdensome, however, for low-volume/high-titer advanced-therapy products, notes Melanie Langhauser, team lead downstream processing at Ascend Advanced Therapies.

“The challenge in scale comes in adapting the GMP [good manufacturing practice] systems to the smaller batch volumes in viral-vector processes compared to traditional products,” agrees Jackson. “Experience in delivering adaptable systems to suit specific product needs becomes vital to overcome this challenge,” he adds.

As an example, Langhauser points to the need to find appropriate single-use equipment suitable for GMP manufacturing at smaller scale. “For a 50-L bioreactor run, the intermediate downstream processing volumes can already be below 2 L after the capture steps, but single-use mixers currently on the market start at 50 L,” she explains. Chromatography systems have presented challenges as well, as most were developed for monoclonal antibody processes with starting volumes of 1000 L. Suppliers are, Langhauser observes, working to address these gaps and meet the specific needs of advanced therapy manufacturers.

As a final note on scaling viral-vector sterile filtration processes, Laux points out that at CSL, it has been observed that not the volume, but the number of virus particles per filter area is the critical factor for upscaling.

Several strategies for overcoming sterilization challenges
“Consistency and quality in any type of biological manufacturing process, including [those for] viral vectors, can be ensured by using quality-by-design and design-of-experiment techniques to address questions of optimization and reduce variability before implementing a drug substance/drug product sterile manufacturing regime,” states Kellerhals. Indeed, conducting a screening study to identify filters that perform best at small-lab scale followed by their evolution at larger-lab scale is recommended before deciding on the best options for upscaling sterile filtration processes, according to Laux.

“For LV vectors, titer losses of 30–50% during sterile filtration have been reported in several publications, often due to excessive aggregation and vector loss. Through our filter screening efforts, however, CSL has been able to develop a sterile filtration process that affords infectious titer yields above 90% at scales relevant for clinical trial and commercial manufacturing,” Laux says. The increase in titer is important because higher yields reduce the cost of goods for these expensive products. “It is therefore worth evaluating several sterile filters based on the process and formulation conditions, as it is possible to achieve both higher yields and increased patient safety,” he observes.

In addition, training of operators in aseptic techniques, use of certified and controlled manufacturing spaces (e.g., biosafety cabinets), single-pass-through air supply, continuous environmental monitoring, and placement of quality team members in manufacturing suites for additional oversight are other steps that can be taken to ensure sterility, according to Kellerhals.

Logical design and physical layout of single-use manifolds with a focus on user experience can also facilitate the design of standard operating procedures that minimize operator errors during complex processing steps such as PUPSIT and in-situ post-use integrity testing, adds Jackson. “The ultimate solution, though, is implementation of fully automated, good-manufacturing-practice-compliant systems which minimize the risk of operator error,” he contends.

The choice between the two, Jackson comments, is a cost-and-benefit risk assessment. He does note that separate pre-sterilized, single-use manifolds with sterile connectors can improve ease of installation and prevent the risk of damage during installation, and integration of effective leak testers into the production system can confirm a leak-free system at the point of use.

The role of single-use systems
While single-use equipment and materials have facilitated sterile manufacturing of viral vectors, Heller notes that these tools come with a price. “The cost differences between single- and multi-use equipment and accessories can be significant, which can be an issue especially for smaller companies in the early stages of development at smaller scales, where cleaning validation is not a top priority,” he explains.

Hold-up volumes also become increasingly important as the process scale shrinks. “For low-volume/high-titer products, every single drop of dead volume in a system can be a massive product loss,” Heller states. Minimizing hold-up volumes and maximizing yield can be achieved through effective custom design of the filtration system and manifolds to optimize them for the specific production scale, according to Jackson. “Optimizing the sterilizing-grade filtration performance through process optimization or selection of higher throughput products can lead to a smaller final filter, reduced hold-up, and higher yield,” he adds.

Finally, Langhauser highlights the increased availability today of GMP-compliant small tangential-flow filtration (TFF) systems with peristaltic pumps for performing buffer exchange at extremely low volumes with single-use tubing, especially at the end of a manufacturing process.

Optimization ongoing
While successful production of viral-vector drug substance and drug products is achievable today, there are always opportunities to optimize and improve existing processes and systems to enhance yields and ease-of-use without compromising on critical patient safety, according to Jackson.

Suppliers and manufacturers have already made progress in addressing many of the challenges to ensuring the sterility of viral-vector products, adds Langhauser. She points to the development of smaller single-use mixers for small-volume applications, single-use filters and TFF membranes at various scales that are available or under development, and irradiated single-use biocontainers with integrated mixers and pre-calibrated pH probes as useful solutions for advanced therapy manufacturing.

One of the keys to successful development of new technologies, Langhauser adds, is close collaboration between suppliers and manufacturers with regards to design and suitability testing of potential new solutions, as working together helps facilitate the introduction of new solutions that can dramatically improve efficiency, productivity, quality, and safety.

Sterile filtration will remain the standard, but with more automation
The fundamental issue with sterile viral-vector manufacturing is the need to keep out or remove undesired organisms such as bacteria or viruses from processes that are intended to generate live viral vectors in sterile formulations. “Most likely, sterile filtration for sufficiently small viruses will remain the method of choice, whereas for larger viruses, use of sterile single-use materials, stringent control of starting materials, and aseptic processing will be hard to replace,” concludes Heller. Advances are likely to occur, therefore, in automation, which, according to Jackson, will further drive down the risk of batch failure.

Kellerhals agrees. “Balancing human ingenuity and adaptability against reducing opportunities for human error is a natural outcome of the scale up process, so opportunities for contamination are very limited at the manufacturing scale. Still, human error, including shedding of contaminants from a human operator into the manufacturing environment, is something we limit today with automation and procedural rigor. Future innovations will reduce these risks even further and as close to zero as possible,” he says.

Finally, Kellerhals observes that technology and human ingenuity go hand in hand when striving for continuous improvement. He also emphasizes that “the evolution of sterility standards over time to where they are today should reassure us all that our medicines are safely manufactured for our patients.” In addition, he believes that future iterations of technology will only further this assurance, but that well-trained operators and well-honed and followed procedures will always be the foundation for aseptic manufacturing.

Keep it simple
As for many other technologies in biopharmaceutical manufacturing, there are certain basic principles that should be followed to ensure sterility, according to Heller. “The best way to protect your product from undesired organisms is to strictly obey the famous KISS principle: Keep It Simple Stupid,” he says.

That includes careful selection of equipment and starting materials with scalability and manufacturability under current GMPs taken into consideration and being guided by data, not perception.

“The fewer the unit operations in a process, the fewer the interventions—manual or automated—that will be required for it to run properly. That makes it easier to maintain sterility throughout all steps and produce sterile, high-quality viral-vector products,” Heller concludes.

Sterility in viral vector therapies is a critical and mandatory aspect in delivering overall patient safety, adds Jackson. “Driving improvements in the safety profiles of viral vectors is key to the industry expanding the application of this technology to a wider variety of diseases, which,” he states, “will enable the industry to make a difference for more varied patient populations.”

Originally published as Challener, C.A. Advances in Sterilization Technologies for Overcoming Viral-Vector Manufacturing Challenges. BioPharm International 2024 37 (5).

Tags: sterile filtration, process development, viral vectors, cell and gene therapy, biologic, biologic contamination risk