The evolution of automation in biopharmaceutical development and manufacturing continues to move forward. Technologies have advanced from possibilities to realities in recent years, according to David Sheedy, head of Life Sciences Manufacturing Ireland, at Cognizant. He points to connected smart instruments, digital twins, and facilities that incorporate Industry 4.0 as examples. “Automation integration layers now make it possible to bring huge volumes of control data traffic from distributed sources across OT [operational technology] networks through a central function, making it available for further use to improve manufacturing intelligence,” Sheedy states.

According to Cory Perelman, PE, Lead I&C Engineer and ASME BPE PI Vice Chair at Arcadis, the use of single-use systems will be advanced by new technologies, such as single-use mass flow control, and traditional stainless-steel manufacturing will benefit from smart technologies. “Single-use and cell and gene therapy typically associated with many ‘islands of automation’ should be looking at a new ‘spoke-and-hub’ type architecture based on a message query telemetry transport (MQTT) data broker within a unified namespace.” He points out that some technologies are specific to one therapy while others can be applied across a range of therapies. “Ethernet-APL [advanced physical layer] is a databus with a two-wire Ethernet topology which will simplify network architectures and allow for [the] passing of a lot of useful information for all plants. For new continuous manufacturing processes, end users should focus on smart instrumentation for process downtime reduction and advanced process insights that can inform maintenance and SOPs [standard operating procedures],” Perelman states.

Ryan Thompson, senior specialist, Industry 4.0, at CRB sees open-sourced automation as a development, where data are treated as an asset to other manufacturing systems. “This [change] pushes original equipment manufacturers (OEMs) to offer connectivity to their equipment in the manufacturer’s data ecosystems instead of the typical vendor-locked approach with a separate system for each equipment or equipment type,” Thompson says.

Robotic process automation (RPA) allows processes to be automated quickly and repeatedly, which makes it a valuable tool for biopharmaceutical manufacturing, remarks Ronan Fox, PhD, senior vice president, IT R&D, at ICON. “RPA is one tool in the toolbox for automation engineers, and can be used as standalone, fully automated solutions, human-assisted, or used in conjunction with AI [artificial intelligence] and integrations (to mention a few) so that complete solutions can be provided to enable consistency, higher quality, and quicker throughput,” Fox says.

According to Edita Botonjic-Sehic, head of Process Analytics and Data Science at ReciBioPharm, there will be an increase in new process analytical technologies (PAT) that integrate assays online, inline, or at-line. “With the implementation of PAT, drug developers and manufacturers are reducing manual labor efforts through automation, eliminating the need for time-consuming manual sampling steps and removing the travel time associated with sending samples to external laboratories. This efficiency improvement enables what previously took two weeks of offline analyses to be completed in a matter of days,” says Botonjic-Sehic.

To implement automation in biopharmaceutical processes, Sheedy emphasizes that process decisions should be made early and rationally to ensure they can be automated. “Teams need to identify the steps that can be streamlined or sequenced differently to allow the overall delivery gain from the incoming automation,” says Sheedy. “Automating processes means significant change for personnel; therefore, investing in organizational change management (OCM) is critical in ensuring all stakeholders are identified, informed, and brought on the journey to minimize the impact on productivity, from go-live and beyond.”

The costs associated with implementation of automation should be looked at holistically, says Thompson, including integration of equipment and the personnel to maintain systems. “Having expertise in your technologies is an important consideration in biopharma manufacturing,” he stresses. “[It is not just a case of] looking at the cost of automation but what it will cost to have a connected factory.”

For more information on implementing automation in biopharmaceutical manufacturing, Perelman suggests the International Society for Pharmaceutical Engineers’ (ISPE’s) Pharma 4.0 guideline (1). “The Digital Maturity Model helps understand where a plant currently is on its path to digitalization. What existing systems and infrastructure (IT and OT) are in place to support digitalization is the first step. From there, it is important that the company has a clear vision and strategy as to where they want to go, and how to get there,” says Perelman. “A culture of change and digitalization across each project should be implemented as soon as possible. Often on the design firm side, we start with individual case studies, small and large, that are suitable for a given project. Then based on project budget, case studies can be selected for implementation, removed, or saved for a later date.”

When implementing automation into analytical processes, understanding the analytical instrument and the data it processes is important, but more so, is the context behind the data, explains Botonjic-Sehic. “Beyond tracking a number, a user needs to understand what part of the process gave that number and how it is related to the process performance. Properly contextualized data [are] critical for troubleshooting and success as drug developers and manufacturers transfer the process from process development (PD) to commercial scale,” she says.

A new system or operation that is being implemented in a regulated environment will require validation to ensure good manufacturing practice requirements are met. Validation ensures controls are in place for the automation of processes that are regulated, says Fox. “We must be able to prove that the automation applied is fit for the intended use, and that the execution of that automation is equivalent to the activities that would have been undertaken by trained humans,” Fox says. “The validation of the automation should match the level of validation that we would perform across any technology that is developed for a regulatory process—be that a bespoke software-based solution or an off-the-shelf purchased technology.”

For any new technology, validation strategies should be considered, notes Perelman, and new analyses should be performed. “[For example,] for cloud-based technologies, Amazon Web Services (AWS) tends to be a risk-adverse option with auditable security practices than a lesser-known player,” he specifies. “ISPE is starting to create local GAMP chapters where other regulated end users can get to together locally to discuss best practices. For new technologies, risk-based and well-documented validation approaches should be implemented in a logical way, rather than shy away from new technologies and revert to antiquated ones.”

Thompson also points to GAMP 5 on how to validate an automation system and take a risk-based approach. “Depending on the application, using a Computer Software Assurance instead of a Computer Systems Validation model may be appropriate,” he states.

In what may be a positive shift, according to Sheedy, validation is moving to streamlined procedures that enable multi-product facilities. “The 2020 lockdowns highlighted that there are better, more streamlined ways of doing things. Shifting to a digital approach to validation as standard enables the industry to work in a much more collaborative and flexible way,” Sheedy says.

When it comes to data, secure databases and historians help to streamline data access and integration, according to Botonjic-Sehic. Validation can be quickly performed by directly feeding process data into a data base. “Many processes and analytical instruments in drug development and manufacturing default to providing data in flat files, leaving the onus of building and connecting to secure databases on the users,” says Botonjic-Sehic. “Preventing bottlenecks that can arise as a result and maintaining data integrity hinges on creating an automation platform that seamlessly communicates with the entire workflow, ensuring faster, more reliable data flow from multiple systems on the operational floor. Addressing potential issues at the outset of building this architecture can help prevent the need for rework and minimize data loss.”

The development and advancement of AI and machine learning (ML) have entered the bio/pharmaceutical space. AI can process large sets of data; therefore, it may help to speed up drug development and reduce costs, according to Sheedy. “It also enables early issue identification to reduce waste, creates digital twins with greater accuracy and forecasts and executes faster supply chain decisions,” says Sheedy. “In manufacturing, the uptake has been focused on data analytics and in non-GMP applications within the manufacturing environment for now, but it is only a matter of time before AI infiltrates other aspects. As we move forward with the integration of AI, the interface between automation and AI algorithms will see increases in yield and productivity that drive business benefits and enable clients to retain competitive and make patient health improvements.”

Models for real-time monitoring and control can be created using AI and ML, according to Botonjic-Sehic. Real-world events can be simulated by in-silico models and provide proactive risk identification associated with critical quality attributes (CQAs). “Additionally, these models can determine trends that could lead to CQA deviations, allowing process development adjustments accordingly. As a result, drug developers and manufacturers can proactively minimize failure risks, reduce waste, and ensure a higher quality product,” says Botonjic-Sehic.

According to Perelman, AI and ML can optimize the work of lab scientists. “[For example, AI and ML] can pick ‘green’ solvents for synthesis best suited for the process and environmental impact (both from production of the solvent and production using the solvent).” Processes can be streamlined from pilot to scale using AI and ML, confirms Perelman.

However, regulators and industry will need to develop better validation and qualification procedures, according to Perelman. “For example, is an ML-generated process model that then feeds an ML model-predictive control allowed to change a setpoint or react to a process upset? Will an AI change on a timestamp satisfy 21 CFR [Code of Federal Regulations] Part 11 compliance? Are CPPs [critical process parameters] and tech-transfer documents largely AI-generated defensible? What kind of testing do we need to prove so? These are questions we need to ask.”

Vast datasets from biological experiments potentially may be analyzed by ML to identify patterns missed by human researchers, says Perelman. In addition, the success of drug candidates might be predicted by ML models. “Like AI, ML models can be difficult to defend to the FDA or other regulatory bodies, may contain insufficient or biased data that can lead to inaccurate models or poor predictions, and rely on expertise to create and implement the models that may be scarce in the organization.”

Data architecture may be a challenge, however, according to Thompson, because operational technology systems do not typically support data models for AI systems to scale. “Data science teams spend the bulk of their time organizing and cleaning data instead of refining models and providing insights. Spending time to define your data strategy and tools to model data will allow for AI/ML to scale,” says Thompson. “The quality of an AI/ML model improves as the data set grows. Using one piece of equipment to make inferences will deliver a lot less value than having a scaled architecture that can support the analysis of multiple pieces of equipment and take advantage of the natural experiments that occur in day-to-day production.”

“During biopharmaceutical development, particularly the clinical research phases, AI can play a key role in automating regulatory and non-regulatory processes,” says Fox. “AI, layered on other automation techniques, enables the automation of more sophisticated processes as they tend to leverage more unstructured data sources to proceed.” Fox cautions, however, that “care must also be taken to ensure that bias is minimized in the data and thus the models created. Increasing trust through demonstrable and explainable AI-powered decisions will be key to the further deployment and uptake of those AI-enabled automations.”

https://ispe.org/publications/guidance-documents/baseline-guide-vol-8-pharma-40-1st-edition

®
Vol. 37, No. 7
July/August 2024
Pages: 7–9

When referring to this article, please cite it as Haigney, S. Streamlining Bioprocesses with Automation. 

Articles

Cover Story

Automation technologies used in the development and manufacture of biopharmaceuticals continue to evolve, providing the potential for reduced costs and time.

Streamlining Bioprocesses with Automation

Viral vectors continue to grow as a major category for biomanufacturing processes based on the growing clinical programs for gene therapies. Optimizing the manufacturing process for viral vectors starts with the expression systems used. A look at the expression system used for manufacturing adeno-associated virus (AAV) vectors will highlight ways to troubleshoot problems in upstream processing.

The most common cell line used across the biopharma industry to produce AAV vectors is the human embryonic kidney 293 (HEK293) cell line, says Elie Hanania, PhD, vice-president, Process Development, Viral Vector Technologies, Avid Bioservices, and Pouria Motevalian, director of Process and Analytical Development, Viral Vector Services, Thermo Fisher Scientific.

AAV vectors are produced by transfecting three plasmids into HEK293 cells: Rep/Cap plasmid, helper plasmid, and gene of interest (GOI) plasmid. “The process is mediated by chemical reagents (transfection reagents), which can be pure lipid-based, mixed protein/lipid-based, or non-lipid based,” Hanania says.

After HEK293, the next widely used cell line to produce AAV vectors is Spodoptera frugiperda (SF) 9, says Motevalian, who also notes that there is additional cell line development work currently underway in the industry, where the focus is on novel producer cell lines.

According to Hanania, AAV production via triple transfection of HEK293 cells can take place in either adherent or suspension cultures. “The former approach may have some scale limitations as well as having animal-derived components in the culture media (although there have been advancements with commercially available serum-free and/or chemically defined culture media). The majority of AAV manufacturing focuses on expansion of HEK293 cells in suspension (using serum-free and/or chemically defined culture media) with options to scale out and scale up the process depending on the desired amount of viral vector,” he explains.

High viral vector titer and overall yield are important aspects of any viral vector production, and to support these requirements, the production process needs to be optimized. Hanania points to cell line development as one of the key process components that has a significant impact on titer and yield, stating that clone selection and testing may be a simple approach to boost productivity, while more complex approaches, such as focusing on gene editing, have also been used to further increase AAV titer and yield.

“It is important to mention that, although HEK293 cells coupled with triple plasmid transfection is the go-to approach for AAV production for the majority of programs, there are other manufacturing upstream expression systems,” Hanania adds. Alternative upstream expression systems include, but are not limited to, co-infection of Sf9 insect cells with baculoviruses that harbor the Rep/Cap and GOI sequences, and infection of HEK293 (or HeLa) cells harboring the Rep/Cap genes and GOI with replicative adenovirus providing needed helper functions. “Both these approaches can support large-scale production of AAV with favorable cost of goods (GOG) reduction compared to the traditional transfection-based process,” he observes.

The use of yeast as a potential production system (with favorable COGs) is also being investigated at the R&D stage, Hanania says.

Meanwhile, other alternatives are also being explored. Motevalian notes that cell-free or synthetic biology options are being considered and implemented in R&D settings. Widespread commercial adoption, however, remains a work in progress.

Hanania says that, aside from focusing on generating AAV for cell and gene therapy applications, there is a need to consider the overall cost of producing these viral vectors. Traditional approaches, especially at commercial scale, are extremely expensive, and this results in costly therapies that have limited accessibility.

Significant strides have been made in the field of synthetic biology that have direct impact on the biomanufacturing of large molecules for advanced therapies, Hanania notes. “This concept is still in its emerging phase when it comes to AAV manufacturing,” he says. “Aside from the use of baculoviruses and herpes simplex viruses for AAV production, synthetic biology showed great strides when considering plasmid production. The chemical synthesis of doggybone DNA is being evaluated across several therapeutic programs and clinical applications.”

The focus on safety (i.e., decreased risk of adventitious agents and other contaminants) and efficacy (i.e., potency of the AAV as the intended therapeutic molecule) are two factors driving the evaluation of these new onsets in AAV manufacturing, Hanania says.

Minimizing contamination risk is an important consideration in optimizing upstream processing. Process control strategies are needed to achieve the product’s safety profile. Currently, the industry employs a number of strategies, as applicable, to minimize process-related contamination, Motevalian says. “We have implemented stringent quality control for all raw materials, including cell culture media, plasmids, and reagents, to help ensure the reduction of contaminants,” he says.

Motevalian further explains that use of closed and automated systems for cell culture and vector production reduces the risk of contamination from the external environment, while maintaining cleanroom standards involves controlling air quality, temperature, and humidity. In addition, it is important to implement appropriate control and monitoring on critical upstream production process parameters, such as pH, which is known to influence cell growth and thus byprocess-related contaminants, such as host cell DNA and host cell protein (HCP).

“Aside from being potent, advanced gene therapy medicines need to adhere to the highest safety and quality standards. Accordingly, these need to be void of any adventitious viruses that could be introduced by source materials or during the production and purification processes,” stresses Hanania. This task is even more critical for a contract development manufacturing organization dealing with multiple product manufacturing jobs, where measures are in place to avoid any cross contamination, he adds.

“One of the most recent measures introduced in the AAV manufacturing scheme is nano filtration. Being 20-25 nanometer in size, AAV will pass through nanofilters (with a molecular cutoff of 50 nanometers) while larger viruses (e.g., adenovirus, herpes simplex virus, baculovirus, or others), if present, will be retained by such a filter,” Hanania explains.

Hanania clarifies that, although 0.2-micron sterilizing-grade filters are used at the end of the drug substance and drug product steps, these filters are intended to ensure a sterile end product “void of bacterial and microbialcontamination, but not necessarily of other viruses,” which is why a virus-reducing filter is required during the downstream purification of AAV vectors.

Other steps used to ensure the inactivation of other viruses introduced during the AAV manufacturing process focus on low pH, lysis reagents, and/or heat, Hanania further explains. “Such measures will inactivate enveloped viruses and are typically performed in addition to multistep chromatography designed to separate the target AAV from other contaminants. Of course, employing single-use materials and supplies (pre-sterilized) is becoming the norm during bioprocessing of viral vectors.”

Hanania also emphasizes that host cell DNA and HCPs, as well as other process-related contaminants, may become partially packaged in the AAV particle and cause safety issues, including immunogenic response or the introduction of potential oncogenic sequences with long-term negative impacts. “Even though initial levels of such pollutants may be low, the final product, which typically undergoes hundreds of fold concentration, will harbor concentrated levels of such contaminants with a heightened safety risk,” he states. Therefore, robust processing needs to eliminate, or at best reduce, such contaminants. Endonucleases are commonly used to reduce host cell DNA, while clarification filters, proteases, and multistep chromatography are typically used to reduce other introduced or unwanted contaminants.

Further, extensive testing of all raw and starting materials must be factored into the production process to prevent impurities from being introduced into the manufacturing process at the start. “Such testing needs to span materials (biological and chemical), supplies, and any product contacting surfaces and equipment,” says Hanania.

Meanwhile, to prevent cross contamination across multiple batches, facility design comes into play. According to Hanania, robust cleaning measures—with validation—must be applied to a facility’s design, in addition to physical segregation of the different products (i.e., designated suits). “Using vaporized hydrogen peroxide or ionized hydrogen peroxide as part of the change-over process will further reduce cross contamination risks. It is expected that controlled access is implemented to limit personnel-contributed cross contamination,” he says.

Looking to the future, the utilization of synthetic biology is anticipated to become central to AAV manufacturing because large amounts of the viral vector will be required for systemic applications, Hanania observes. “System-level genome engineering will advance the field and ensure higher productivity and yield without the constant need of scaling up the manufacturing process,” he states, adding that artificial intelligence, machine learning, conceptual computer designs, and in-silico models will support the advancement of synthetic biology and its utilization in AAV manufacturing.

Beyond the growing use of synthetic biology, better and sustainable producer cell lines are expected to become available, Hanania further notes. “Although this effort started more than a decade ago, the outcome has not been as successful as producer cell lines for retroviruses (cytotoxicity of the helper and Rep genes),” he states.

Meanwhile, the demand for large quantities of viral vectors will become a bottleneck as there is expected to be more systemic applications for gene therapies as well as developers targeting more common disorders, including cancers and neurodegenerative diseases, Hanania says. Effort will focus on optimizing production and purification processes, engineering better starting (and raw) materials, and improving manufacturing platforms. “Although template processes are highly desirable from a manufacturing standpoint, it is clear that the outcome is not optimal across the different AAV serotypes and among the various transgenes. Continuous processing with intensified culture approached may be a good foundation for scaling up targeted advanced therapeutics,” Hanania explains.

From a process standpoint, Motevalian expects that continuous manufacturing with intensified processing enabled through adoption and implementation of process analytical technologies (such as Raman) for real-time monitoring and ultimately release will become more standard. “Concerning facility design, the cell and gene therapy field is progressing towards digitized modular manufacturing facilities, providing flexibility for different production scales and types,” he states.

Hanania points to safety and quality, which will be at the forefront of approved AAV gene therapy products. As such, improved analytical assays will need to be in place. These assays will need to have better sensitivity and higher specificity to characterize these AAV gene therapy products and identify (as well as quantify) contaminants. “Batch to batch consistency is a must,” he stresses.

While high demand for AAV gene therapies may be met by scaling up, there is a limit in the current equipment and approaches to achieve adequate scale up. “It seems gene and cell therapy trajectory are following the early manufacturing pathway for antibodies and recombinant proteins. The latter focused initially on scaling to larger volumes (multiple 10,000 L bioreactors) before considering cell line development, which resulted in high productivity (> 8 g/L of monoclonal antibodies),” explains Hanania. “This negated the need to seek such giant bioreactors and made the scaling process better and more streamlined. Accordingly, scaling up for cell and gene therapy products needs to incorporate process improvements, better reagents and supplies, state-of-the-art analytics, and new designed platforms. Strides across all these have started and will continue in the next several years,” he concludes.

Feliza Mirasol is the science editor for 


Vol. 37, No. 7
July/August 2024
Pages: 18–20

When referring to this article, please cite it as Mirasol, F. Optimizing Expression Systems for AAV Vectors. 

Upstream Processing

Optimizing AAV vector manufacturing will be necessary to meet anticipated future market demand.

Optimizing Expression Systems for AAV Vectors

As the market for gene therapies matures, the industry is at a point where focus is on the best method of delivery for the genetic package at the core of a gene therapy product. Today, the two most commonly known methods for delivery are adeno-associated virus (AAV) vectors and lentiviral vectors.

AAVs, as small viruses (approximately 20–25 nm in diameter, approximately 4.8kb linear single-stranded DNA) in the parvoviridae family, are considered good vectors for gene therapy delivery based on their ability to infect both dividing and non-dividing cells, says Elie Hanania, PhD, vice-president, Process Development, Viral Vector Technologies, Avid Bioservices. Recombinant and engineered AAVs do not integrate into the host genome, but maintain sustained expression for moderate duration, he notes. 

Hanania also points out that AAV has a good safety profile (thus far) and can sustain harsh manufacturing conditions while maintaining infectivity because it is small and non-enveloped. There are several serotypes of recombinant AAV (rAAV) available, which provide options for tissue-specific delivery. Furthermore, rAAV can be produced multiple ways based on plasmid transfection or other helper virus infection, Hanania observes.

Emmanuelle Cameau, strategic technology partnership leader, Genomic Medicine, Cytiva, adds that AAV is non-pathogenic to humans, does not cause disease, and has a low immunogenicity, which are crucial factors for the safety of gene therapy patients. In addition, AAV can mediate long-term expression of the therapeutic gene, which is an important factor when treating chronic conditions that require sustained gene expression.

AAV vectors can also be engineered to carry a variety of genetic payloads, including small genes, regulatory elements, and RNA interference molecules, says Cameau, who emphasizes that it is this versatility that allows for customized gene therapy approaches tailored to specific diseases.

Lentiviruses (LVs) also offer the advantage of being able to efficiently infect both dividing and non-dividing cells, allowing them to target different tissue types, such as neurons, muscle cells, and hematopoietic stem cells, says Cameau. “They integrate their genetic material into the host genome, ensuring stable, long-term expression of the therapeutic gene. This is beneficial for conditions requiring sustained gene expression over the lifetime of the patient,” she states.

“LVs can also carry larger genetic payloads compared to many other viral vectors, such as AAV,” Cameau continues. “Like AAV, they can be engineered to include various regulatory elements, promoters, and transgenes, allowing to tailor them to specific therapeutic needs, including tissue-specific expression and controlled gene regulation.”

An added advantage is that lentiviral vectors are generally less immunogenic than other viral vectors, such as adenoviruses; this characteristic reduces the likelihood of an adverse immune response. Moreover, LVs are highly efficient with gene transfer, particularly in stem cells such as hematopoietic stem cells and other progenitor cells, which are essential for therapies targeting blood disorders and other systemic conditions, Cameau explains.

“Lentiviral vectors can also be pseudotyped with different viral envelope proteins to alter their tropism and improve targeting to specific cell types or tissues,” Cameau adds.

Hanania explains that the LV is an enveloped single-stranded RNA virus that belongs to the Retroviridae family. Because of their ability to infect non-dividing cells and integrate into the host cell genome, not only can LVs carry out sustained expression, but they can also pass the ability along to cell progenies. LVs can therefore be used for both ex-vivo gene therapy application (mainly chimeric antigen receptor T cell [CAR-T] therapy and stem cell therapy) and in-vivo gene therapy, with direct injection of the viral vector carrying the desired therapeutic payload into a patient.

“Having the capability of penetrating the nuclear envelope, LVs can infect non-dividing cells (including neurons), hence potential application for neurological-based gene therapy,” Hanania says.

Manufacturing viral vectors requires consideration of their respective challenges. There are different approaches to manufacturing rAAVs, Hanania points out. “The most common approach is triple plasmid transfection into HEK293 [human embryonic kidney 293] cells mediated by a transfection reagent,” he explains. “Alternatively, rAAV can be produced by infecting Sf9 insect cells with two baculoviruses carrying Rep/Cap and the gene of interest (GOI). Similarly, two herpes simplex viruses harboring Rep/Cap and GOI can be used.”

In addition, wild type adenovirus can be used to render the helper function required to produced rAAV when infecting engineered HeLa (or similar) cell lines, Hanania states. “Being non-enveloped, rAAV is tolerant to harsh purification conditions including low pH, high salt, and high temperature (contaminant virus inactivation if needed),” Hanania adds.

Meanwhile, LVs are classically more difficult to manufacture because they are enveloped viruses, says Cameau. “Enveloped viruses are more delicate and sensitive than non-enveloped [viruses]. Therefore, LVs are sensitive to temperature, pH, foam … which makes them really challenging to process,” she explains.

The LV manufacturing process needs to be designed such that it can mitigate the yield loss all along the way, Cameau says. In comparison, AAV is much easier to produce and purify, even at the largest scales, although empty capsids generation instead of full capsids is a particular challenge of AAV that the industry is currently looking at overcoming, she specifies.

“Third-generation (improved safety profile) LV is typically produced by transfecting HEK293T cells with four plasmids,” explains Hanania. “Being an enveloped virus, temperature, pH, and shear are detrimental and hence purification approaches are typically gentler, with one single-step chromatography and one concentration/buffer exchange step. Furthermore, being enveloped and larger in size compared to AAV, [LV] final sterile filtration is a challenge and may have an impact on titer and yield.”

Hanania further explains that the common challenge in manufacturing for both AAV (rAAV in particular) and LV is the scaling up of the transfection step, which is needed to ensure high titer and yield. However, the industry has made great strides in having commercially available serum-free, chemically defined media to support high density cell cultures. Meanwhile, new and improved transfection reagents have emerged that support more efficient transfection into cells.

“The scale factor and the time element are two focus points to sustain high titer and yield across both platforms. The overall price-tag is still quite high factoring all starting materials (GMP [good manufacturing practice]-grade plasmids, media, transfection reagents, and other associated supplies),” Hanania adds.

Establishing a manufacturing process for either LV vectors or AAV vectors means strategizing from the start to avoid pitfalls and considering the unique needs for each vector platform.

“Unlike production of monoclonal antibodies, where templated approaches are applicable, manufacturing viral vectors has unique (or non-standardized) steps among the various gene therapy programs,” emphasizes Hanania. Common factors for cell thaw and expansion can be generalized, he notes; however, beyond that, optimization is typically required to boost productivity to achieve the desired titers and yield.

This optimization approach applies to plasmid ratios, ratio of DNA to transfection reagents, time of transfection, harvest, filter clarification, lysis (when applicable), host-cell reduction, volume management prior to purification, optimized chromatography approaches, final formulation, and final sterile filtration, Hanania enumerates. These aspects are all areas with impact on final product quantity and quality.

“The above comes at a hefty price of time and cost,” Hanania states, “something to be considered an investment and not ignored as an unnecessary task. Accordingly, process development and optimization need to be factored into the overall program timeline to ensure a robust, reproducible, and CGMP [current GMP]-ready manufacturing process. Once in place, this can be used for campaign runs.”

To achieve an optimized manufacturing process, the manufacturer needs to understand the biology of the viral vector platform, its pros and cons, and the production options (scales and limitation). The manufacturer must also engage a skilled team, whether in-house or at a contract development and manufacturing organization, to help establish a good road map and determine the best approach as well as how to navigate through the process successfully.

Using non-scalable technologies from the start is one major pitfall to avoid, says Cameau. “Whether manufacturing AAV or LV, the manufacturer should ask themselves: What is the final target scale of this process? And [they should] choose from the start the right technologies that will enable that scale, with as little pain as possible,” she explains.

Cameau notes that this approach also applies to reagents and starting materials, such as the right cells (transient or stable or packaging), the right DNA quality in cases other than stable, transfection reagents, and media. “If the application is in vivo, the use of HEK293T cells should be avoided, and preferably use HEK293 cells, as there can be potential risk in using vector potentially carrying the T antigen,” she adds. “Starting with the right biological and physical tools form the beginning will avoid the manufacturer loosing precious time in redeveloping and optimizing a process, thus reducing time to market.”

As both LV and AAV have their own advantages and challenges, what other factors come into play when choosing which platform to use for a gene therapy? According to Cameau, the choice depends on what the final application is. “If the use is in-vivo modification of cells or tissue, then AAV is the safest way to go, provided the size of the GOI does not exceed 4.7 kb and that an AAV serotype can target specifically the selected type of cells or tissue,” she explains.

However, in the case where the GOI is larger than 4.7 kb, drug developers have instead been using LV or other virus types, such as retrovirus. “But the number of LV-based therapies in development for in-vivo applications is significantly minor compared to ex vivo due to the reduced safety aspect of stable integration into the human genome of LV,” says Cameau.

If the application is the transduction of stem cells ex vivo, then LV is usually the viral vector method of choice, as it can package significantly large GOIs and stable integration into the genome makes the cell modification permanent, Cameau adds.

Hanania points out that researchers tend to make a decision based on the ultimate need of the advanced therapy. “Thus far,” he notes, “ex-vivo applications have been utilizing LVs mainly because they can infect non-dividing cells (most CAR-T applications) efficiently. Furthermore, the overall amount of virus needed is not too high at this point, hence large scale (i.e., > 100 L scale) [is] not needed.”

Whereas, for in-vivo applications and tissue-specific delivery, rAAV has been the “go-to” virus, Hanania says. Diverse production options and the robustness of the virus are attractive for many advanced therapies. Meanwhile, production scales nearing the 2000 L range are becoming common for some programs, although 200–500 L is still the desired scale for most early-stage programs, he specifies. However, pay-load size is a major constraint when it comes to rAAV; therefore, Hanania says researchers may opt to choose LV for larger GOIs; although, insertional mutagenesis/oncogenesis is a concern due to LV’s tendency to integrate into the host genome.

“It is important to mention that packaging and producer cell line development have been going on for both rAAV and LV. Great success has been achieved with gamma retrovirus producer cell lines where sustained production and multi-day harvests can be performed. Dealing with cytotoxicity has been one of the major challenges in advancing these initiatives for rAAV and LV. Controlled expression and gene editing/engineering are being used to circumvent some of these issues to come up with practical and functional producer cell lines,” Hanania says.


Vol. 37, No. 7
July/August 2024
Pages: 10–13

When referring to this article, please cite it as Mirasol, F. Comparing Viral Vectors for Gene Therapy Delivery. 

Development

AAV and lentivirus both have pros and cons in their use for specific gene therapy applications.

Comparing Viral Vectors for Gene Therapy Delivery

Cell and gene therapies (CGTs) are currently in use for small patient populations to either treat a rare disease or as a single-patient, single-dose treatment, but the biopharma industry is seeking ways to apply CGTs to mass patient populations. 

® spoke to David Chang, MD, PhD, president, CEO, and co-founder of Allogene Therapeutics, to gain a better understanding of how application to a mass patient population could work.

 What are the most challenging hurdles in bringing CGTs market?

 As we move CAR-T [chimeric antigen receptor T cell] therapies into earlier lines of treatment, we’re presented with a significant opportunity, as patients are generally more responsive at these stages. However, this shift also brings new challenges, particularly in terms of delivery infrastructure. Most autologous CAR-T treatments are currently delivered in academic centers, yet most patients, especially those in earlier lines, are treated in community settings. 

These community cancer centers do not have in place the complex infrastructure and resources required to administer autologous CAR-T therapies and often do not refer patients to academic centers for treatment. In fact, about 80% of patients are treated in the community cancer center setting. We believe this disconnect between where patients are treated and where CAR-T therapies are available represents a significant access barrier.

In contrast, cemacabtagene ansegedleucel (cema-cel, an investigational allogeneic cell therapy product) is designed to bypass many of the challenges that have hindered the adoption of autologous CAR-T therapies. Use of cema-cel shouldn’t require the complex logistics that have hindered CAR-T adoption, nor should there be a reliance on referrals as the intent is for CAR-T therapies to be available in these community cancer centers. These community physicians have been waiting for a product that is off-the-shelf, easily administered, and readily available on demand to bring CAR-T into their centers, and we believe cema-cel will meet these criteria.

 What is the reality that a CGT can be applied to a mass patient population?

 We see significant potential for CAR-T in larger patient populations, but this can only be done by using an allogeneic product. The move into earlier lines of blood cancers, solid tumors, and autoimmune disease represents a significant expansion in the number of patients who could potentially benefit from these therapies. However, this also brings new challenges in terms of scalability.

Autologous CAR-T therapies have inherent limitations due to their complex logistics and bespoke manufacturing process, none of which is easily scalable. In fact, despite the commercial availability of autologous therapies since 2017, only about 10% of patients receive treatment today based on reported sales. To make CAR-T available where patients are treated, we need a different playbook.

Cema-cel [is an example of a product that] offers a potentially promising solution with a safety profile comparable to approved autologous CAR-T therapies. Made from healthy donor cells, cema-cel is designed to be produced in large batches that could treat up to 100 patients from a single manufacturing run. This ‘off-the-shelf’ approach, which also opens the door to use in community cancer centers where the majority of patients are treated, could significantly increase the scalability of CAR-Ts, bringing us one step closer to making this modality available to larger patient populations.

 Do the biggest opportunities for mass application rely on Big Pharma, smaller biotechs, or other?

 The development and commercialization of CGTs represent a dynamic and rapidly evolving landscape. Both Big Pharma and emerging biotechs play crucial roles in driving innovation. Each brings unique strengths—Big Pharma with its extensive resources, commercialization expertise, and global reach, and biotechs with their agility and singular focus.

We’ve seen how biotechs can drive innovation in CGT. For instance, recogni[tion] of the increasing difficulties [faced by] autologous CAR-Ts in penetrating community cancer centers and accessing earlier line patients led us to quickly shift Allogene’s clinical development of cema-cel to explore its use in first line (1L) consolidation for newly diagnosed large B-cell lymphoma (LBCL) patients who are likely to relapse.

This led to the design of a new clinical trial (ALPHA3) that leverages an investigational diagnostic test developed by Foresight Diagnostics that detects the presence of minimal residual disease (MRD). When administered following completion of 1L treatment for LBCL, this test has the potential to offer a highly accurate prediction of future disease relapse. We believe this test could provide the ability to identify patients who are most likely to relapse after initial treatment with chemoimmunotherapy and enable physicians to take action (e.g., administer a single dose of investigational cema-cel) to prevent relapse.

This type of pivot would likely be unheard of in a bigger organization, but because of our size we were quickly able to adjust our strategy and refocus our resources. It’s this kind of innovative thinking and agility that will be key to democratizing cell therapy.

 What partnerships or consortiums facilitate access to CGTs?

 As a clinical development company, we’re still in the early stages of exploring partnerships. We know physicians in community cancer settings are eager for an allogeneic product (such as cema-cel). We’re excited about the prospect of offering CAR-T more broadly and will be exploring ways to facilitate access in the coming years.


Vol. 37, No. 7
July/August 2024
Page: 13

When referring to this article, please cite it as Mirasol, F. The Potential for CAR-Ts in Large Patient Populations. 

The biopharma industry is seeking ways to apply cell and gene therapies to mass patient populations.

The Potential for CAR-Ts in Large Patient Populations

Article submitted: Jan. 11, 2024.
Article accepted: Feb. 6, 2024.

Biosimilars have transformed access and cost of healthcare globally. There has been an explosion of technological advances in biotechnology and computational systems in the past decade. Regulations have also evolved to meet technological advances; for example, exploring real-world evidence to inform regulatory decisions is mandated by the 21st Century Cures Act. The authors have reviewed applications of these novel technologies in the major stages of biosimilars development: process development, pharmacology, toxicology, and clinical trials, with an emphasis on recent regulatory requirements. The advances in process development include process analytical technology and multivariate analysis methods for real-time monitoring and analysis of critical quality attributes and continuous manufacturing processes, which can reduce the manufacturing footprint and increase efficiencies. The applications of artificial intelligence/machine learning in pharmacology, toxicology, and clinical trials have been reviewed. These processes have accelerated development time and decreased cost of biosimilars development. Regulatory considerations on quality aspects of biosimilars are listed as well as some of the major challenges in biosimilars development.

FDA defines biosimilars as biological products that are highly similar to and have no clinically meaningful differences from existing FDA-approved reference products in terms of safety, purity, and potency (1). Since the first approval of a biosimilar in 2015 (1), the US healthcare system has saved approximately $338 billion (2). The 21st Century Cures Act of 2016 mandates FDA to accelerate medical product development and bring new innovations and advances to patients who need them faster and more efficiently (3). The regulatory pathways for biosimilar development have evolved to meet the requirements of industry, law, markets, and most importantly, the patient (4,5).

Over the past two decades, technological advances and enhancements have improved the process of establishing biosimilarity, which has an impact on global regulatory guidance (6). Some of these technologies range from gene-editing of cell lines to continuous operation of upstream and downstream processes for enhancing operational aspects of manufacturing (4). New instrumentation technologies have enhanced the accuracy and precision of measuring primary, secondary, and tertiary structures of proteins (7,8). Process analytical technology (PAT) and multi-attribute methods (MAMs) have enabled real-time decision-making (9,10). The use of machine learning algorithms has improved predictive methods for pharmacology and toxicology. Finally, the use of adaptive clinical trials and real-world evidence methods have enhanced robustness in clinical assessments of endpoints.

Zara Bukhari is PhD scholar at the University of the Pacific, Stockton, CA. Farogh Gibraiel is senior downstream process development specialist at Polpharma Biologics S.A., Warsaw, Poland. Smritie Sheth is scientist; Chaitrali Bakhale is consultant, and Narendra Chirmule is CEO, all at SymphonyTech Biologics, Philadelphia, Pa.

®
Vol. 37, No. 7
July/August 2024
Page: 28

When referring to this article, please cite it as Bukhari, Z.; Gibraiel, F.; Sheth, S.; Bakhale, C.; Chirmule, N. Biosimilars Drug Development: Advances in Technologies from Molecule Design to Clinical Trials. 

Peer-Review Research

The authors have reviewed applications of novel technologies in the major stages of biosimilars development: process development, pharmacology, toxicology, and clinical trials, with an emphasis on recent regulatory requirements.

Biosimilars Drug Development: Advances in Technologies from Molecule Design to Clinical Trials

Single-use (SU) technologies increasingly find use today at all phases of the drug development cycle from preclinical to commercial manufacturing. As the biopharmaceutical market continues to expand and reliance on single-use manufacturing rises, demand for SU consumables—bags, containers, filters, tubing, etc.—is also increasing. The value of the global SU consumables market has already reached several billion dollars and is predicted by multiple market-research firms to continue to expand at a compound annual growth rate of 16–18% for at least the next several years (1–3). A key driver for that growth is the fact that SU consumables come pre-sterilized and ready-to-use, making setup and changeover from one product to another much quicker and easier. Staying abreast of and managing trends in sterilization of SU consumables is therefore essential for biologics producers.

Contamination poses a huge risk to the production of biologic drug substances and drug products, making sterility assurance imperative, states Christy Eatmon, global subject matter expert, sterile drug products in the Pharma Services business of Thermo Fisher Scientific. Processes used for the sterilization of SU consumables play an important role in enabling biopharmaceutical manufacturers to ensure sterility.

The main techniques include ionizing radiation (gamma-, electron-beam [e-beam]-, X-ray), gas exposure (ethylene oxide [EtO], vaporous hydrogen peroxide [VHP]), and heat/steam.

The first step in establishing an effective sterilization process for SU consumables, according to James Hathcock, senior director of regulatory and validation strategy at Cytiva, is to select a sterilization process that is compatible with the materials used in the construction of the SU components. “For instance, most bags are not compatible with heat sterilization, and SU consumables composed of some fluoroplastics (e.g., polytetrafluoroethylene and fluorinated ethylene propylene) will experience more degradation by ionizing radiation,” he says.

Controlling the sterilization process so that it delivers a sufficient minimum dose over the entire surface of the component to kill microbes and render the SU system sterile is also crucial, Hathcock observes. Typically, meeting a minimum 10-6 sterility assurance level with a dose that does not exceed an established value so as not to adversely impact or meaningfully degrade the materials is involved.

The requirements for sterilization by ionizing radiation, which includes gamma, X-ray, and e-beam, are all well defined by the ISO 11137 standard series, notes Hathcock. “These standards explain how the dose range is established, how to ensure each SU consumable/system receives the targeted dose range as part of the routine process, and how to verify the continued effectiveness of the sterilization process over time,” he says. The standards also, Hatchcock adds, provide guidance on how to migrate from one technology, such as gamma, to another, such as X-ray.

Hathcock goes on to point out that not all SU components and systems are irradiated with a sterile claim in accordance with ISO 11137. Some may be provided to a biomanufacturer without being irradiated or having been irradiated to minimize bioburden risk but without any claim of sterility. “It is therefore paramount that manufacturers be aware of the sterilization state of any SU consumables they receive,” he emphasizes. Generally, documentation is provided with each component/system.

Three key trends: capacity, complexity, and process control

The increasing use of SU technologies for biologic drug manufacturing has led to the biggest recent trend related to sterilization of SU consumables, which, Eatmon says, is to investigate other means of sterilizing SU assemblies and qualify alternative or multiple sterilization strategies for product contact surfaces other than gamma irradiation alone. Ensuring the security of sterilization supply is, agrees Hatchcock, the first and foremost important trend.

One of the drivers, according to Eatmon, is that as SU systems are increasingly implemented, the compatibility with gamma-irradiated surfaces has become a concern for some products with sensitivities to residual peroxides. Another is related to the available capacity for sterilization of SU consumables using widely established processes. “As EtO and gamma irradiation, the principal methods used today, have a limited supply capacity, concerns persist that regulation changes, newsworthy events, and supply-chain constraints could easily disrupt these services and thereby impact the flow of critical materials required for patient therapies,” Hathcock contends.

The challenge is to ensure supply exists in the face of swings in market demand and the limited ability to switch sterilization methods for devices that have already been validated using a specific sterilization modality, Hathcock observes.

The desire to streamline the process for sterility validation without compromising risk is a second important trend. Sterilization validation practices for SU consumables used in biopharmaceutical manufacturing grew out of established practices in the medical device world. There are, as a result, according to Hathcock, additional challenges when managing the often large and complex assemblies required to be tested on a quarterly basis for each SU manufacturing site.

“Evolving technologies in digital modeling and dose-delivery mapping during the irradiation process are expected to help optimize the process. In silico techniques can model how boxes of SU systems are positioned in pallets during irradiation, how many times they may need to pass in front of the irradiation field, and how realistically they could be moved from one irradiation technology to another—for instance gamma to e-beam,” Hatchcock notes.

On the regulatory side, another important trend revolves around growing expectations from regulators for biopharmaceutical manufacturers to better understand the control of irradiation processes rather than just trusting the supplier-provided certificate of quality claiming sterility for SU consumables. The driver behind this trend stems back to a Form 483 issued by FDA (4) indicating that the pharmaceutical manufacturer may not have understood or challenged the statement on a supplier certificate stating a bag was sterile and endotoxin-free, Hathcock says.

“Some manufacturers have interpreted this story as a need to perform their own sterility testing on incoming goods,” Hathcock observes. “If a manufacturer were to perform its own testing, however, an enormous and unrealistic number of incoming goods would have to be tested to achieve statistical significance. It is highly unlikely manufacturers are going to pursue this strategy. Instead, education as to how the sterilization process is managed and controlled via ISO 11137 is key,” he explains.

Gamma irradiation has been and continues to be the method of choice for many SU consumables. “In most cases, gamma irradiation provides excellent assurance of sterility and good penetration for complex or multi-layer systems, where other methods might fail,” Eatmon observes. In fact, gamma sterilization via cobalt 60 has been used for the sterilization of medical materials since the early 1960s, according to Hathcock. “Much of its broader application to SU consumables grew from similar applications with transfusion sets and filters,” he comments.

“Gamma irradiation also has the advantage of being able to sterilize a wide variety of materials in the same cycle without requiring any moisture and it is good at sterilizing both internal and external surfaces of materials, which is a benefit for processes that require a high level of microbial decontamination,” Eatmon contends.

Alternative methods are considered, of course, when products may have compatibility challenges with gamma-irradiated materials, Eatmon remarks. However, with heat-dependent methods, Eatmon notes there may be a fine line between penetrating all surfaces while maintaining the integrity of the materials. In addition, while heat sterilization has been historically used for filters, connectors, and some tubing, it is not as simple to use as sterilizing by irradiation, says Hathcock.

EtO, meanwhile, is less ideal for SU systems intended to be functionally closed, because it is more difficult to ensure all surfaces are adequately exposed, according to Hathcock. He does point out, though, that there is a long history of EtO use in the medical device industry, with rich supporting data packages.

“While there have been some incidents with EtO that have caused concerns and misperceptions, with the use of science-based approaches, these issues can be properly mitigated to ensure a strong future for EtO,” Hathcock says.

VHP is used to frequently decontaminate exterior surfaces of SU systems such as in isolators, but is not amenable for sterilization of internal fluid paths.

E-beam sterilization is another form of ionizing radiation that uses a stream of electrons instead of photons to penetrate the packaging and materials of SU consumables. “Due to their mass and charge, electrons are not as effective at penetrating the materials used to make SU consumables; and therefore, it is more difficult to deliver a controlled dose to a large, complex, SU assembly,” Hathcock comments. He adds, though, that e-beam is gaining attraction for some smaller, less-complex SU consumables given that it is a similar ionizing radiation technology to gamma irradiation yet has a lower electricity cost.

X-ray irradiation has been well-recognized as a sterilization method, with a site intended for low-dose food irradiation opening in the United States in approximately 2001, according to Hatchcock. This site was later relegated to irradiation of government mail following the 2001 anthrax scare (5). The first X-ray site for commercial sterilization of medical devices was opened in 2010 by Synergy Health in Daniken, Switzerland, and since then the accelerator technologies supporting X-ray (and e-beam) have considerably matured. “Even so, given that gamma irradiation is well established with multiple contract irradiation sites located around the world and the regulatory challenges and hurdles to switching sterilization technologies, there has been little incentive to change,” he states.

Increasing use of SU technologies has driven rising demand not only for SU consumables, but for sterilizing services. This rise in demand is occurring at a time when the supply of 60Co (used to produce gamma rays) has been challenged due to decommissioning and refurbishment of many nuclear plants where 60Co is generated from 59Co (6). Addition of new gamma irradiation sites, meanwhile, is extremely difficult (7,8).

Establishing ideal, well-understood models of supply and demand for gamma-irradiation services has been complex in recent years. In addition to the perceptions of 60Co availability, spikes in demand during the COVID-19 pandemic impacted assessments, according to Hathcock.

“As a single-use supplier, much of our supply assessment is based on the short- and long-term contract negotiations with a range of different irradiators, where we and other single-use suppliers observed a continued tightening in the gamma irradiation market. With the end of COVID-19 and internal efforts for how and where we manufacture and irradiate single-use goods, we have been able to manage the capacity challenge and are expecting the critical need to hit sometime in mid to late 2025,” Hathcock says.

Hathcock adds that the specific timelines may vary by supplier, region, and SU portfolio and will depend strongly on order forecast models as well as contracts with irradiators.“From a security-of-supply perspective, to ensure critical goods are available for patient therapies, it is certainly much better to be aware, prepared, and ahead of the curve as opposed to behind and surprised,” he concludes.

X-ray irradiation is an attractive alternative

While gamma irradiation remains the most common method for SU consumable sterilization, X-ray technology has been explored and can offer lead-time advantages, according to Eatmon. She cautions, however, that as is often the case with new technologies, adoption can be a lengthy process due to additional validations required and the regulatory impact of making changes to existing processes.

X-ray irradiation is an attractive alternative because, like gamma irradiation, it is photon-based and thus kills microbes by the same mechanism. In addition, the technique has been used for the sterilization of medical devices for over a decade. The key difference between X-ray and gamma irradiation is in how the photons are generated. Even so, ISO 11137-1:2006 Sterilization of Health Care Products—Radiation—Part 1 treats the requirement for ionizing radiation including gamma, X-ray, and e-beam technologies equally (9).

The potential for supply challenges related to gamma irradiation impacts multiple regions. In addition, because the irradiation sterilization technology must be well-supported to address regulatory requirements for sterility, there is no way, says Hathcock, any one supplier or pharmaceutical manufacturer can tackle the issue alone.

“The industry therefore worked together through industry trade groups including the Bio-Process Systems Alliance (BPSA) and BioPhorum on a cohesive strategy for moving forward with X-ray that works for component manufacturers, integrators, pharmaceutical manufacturers, and regulators,” Hathcock remarks.

BPSA published background and risk-based testing strategies for SU systems in 2020 (10) and a second paper in 2023 sharing data on a variety of components from multiple suppliers and test labs all indicating an equivalent impact of X-ray as compared to gamma (11). BioPhorum, meanwhile, published in 2023 a risk-evaluation white paper illustrating a practical approach for evaluating the risks and supporting data from the perspective of the pharmaceutical manufacturer (12).

“It is likely,” Hathcock states, “that the SU bioprocess industry will move forward with X-ray irradiation together given the interconnectedness of SU component supply chains. Components such as sensors or tubing may be evaluated by the component manufacturer for X-ray compatibility, then provided to an integrator who assembles these components with others, and then manages the irradiation process before shipping to the end user.”

The medical device industry, according to Hathcock, also recognizes the value of working and publishing data supporting X-ray sterilization. As an example of a successful transition from gamma to X-ray sterilization, he points to the move made by Schott in 2012–2013 to implement X-ray sterilization of syringes (13).

As with all steps in the sterile drug manufacturing process, the sterilization of SU components must be validated. “To develop a new sterilization technique or even modify existing processes, a robust validation must be completed, including consideration of worst-case scenarios and challenging of all parameters,” Eatmon says. She does emphasize, though, that with proper validation packages, regulatory agencies should find these processes acceptable.

Building confidence that regulators are aware of X-ray and will support the BPSA and BioPhorum-aligned risk evaluation strategies has been a key focus for industry work groups, according to Hathcock. These groups have shared their experience with FDA’s Center for Drug Evaluation and Research Emerging Technology Team (ETT) and Center for Biologics Evaluation and Research Advanced Technologies Team (CATT), as well as the European Medicine Agency’s Quality Innovation Group (QIG) and Japan’s Pharmaceutical and Medical Devices Agency.

Many of the agencies have sent representatives to industry conferences and workshops on X-ray sterilization and hence awareness is expected to be increasing. FDA representatives also plan to visit some of the X-ray and gamma irradiation sites in mid-2024 as part of their experiential learning program.

“All the agencies have been receptive to the transition from gamma to a dual-technology approach, in which SU systems can be gamma or X-ray treated, but the experience has varied from agency to agency,” Hathcock says.

One issue for some of the agencies relates to the use of the word “gamma” in existing filings. “While some health authorities are not focused on the use of the term given that both X-ray and gamma irradiation are identical in the fundamental photon technology used for sterilization, dose ranges employed, and impacts to the materials, others consider a move from gamma to X-ray sterilization as a potential technical discrepancy between what one practices and what is in the filing if gamma irradiation is specified in the filing but X-ray irradiation is used in practice,” Hathcock explains. This one hindrance, therefore, is not related to the scientific underpinnings of the technologies, but to the use of the term “gamma” instead of “ionizing irradiation” as described by ISO 11137.

Going forward, the move to supplement gamma irradiation with other alternative technologies, most notably X-ray irradiation, will continue to be the main trend impacting the sterilization of single-use consumables, according to Eatmon. All the efforts made by the industry are pursued with the goal of ensuring there is an effective supply of sterilization capacity to support patient therapies, Hathcock adds.

“Education and communication about X-ray irradiation in combination with the adoption of dual irradiation technology approaches for sterilization of SU assemblies will be crucial to achieving this goal,” Hathcock contends. “Such a strategy,” he believes, “will allow the biopharmaceutical industry to continue to use the existing, well-established, and extensive gamma-irradiation capacity as an ongoing key part of the solution while also enabling operational flexibility to use mature accelerator-based forms of irradiation technology to meet the growing market demand for increased sterilization capacity.”

The Business Research Company. Single-Use Consumables Global Market Report 2024. January 2024. https://www.thebusinessresearchcompany.com/report/single-use-consumables-global-market-report

Precedence Research. Single-Use Consumables Market. Precedenceresearch.com. 

https://www.precedenceresearch.com/single-use-consumables-market

MarketsandMarkets, Single-Use Consumables Market Worth $4.7 Billion by 2027. Press Release, https://www.marketsandmarkets.com/PressReleases/single-use-consumable-market.asp

FDA. Form 483 to Biogen Idec Inc., Aug. 2, 2013. 

United States General Accounting Office. Diffuse Security Threats: Technologies for Mail Sanitization Exist, but Challenges Remain. Report to Congressional Requesters, April 2002. 

https://www.gao.gov/assets/gao-02-365.pdf

Comben, M. Viewpoint: The Critical Production of Cobalt-60 in Nuclear Reactors. World Nuclear News, March 28, 2023. 

https://world-nuclear-news.org/Articles/The-critical-production-of-cobalt-60-in-nuclear-re

Canadian Nuclear Safety Commission. Security of Nuclear Substances: Sealed Sources REGDOC-2.12.3. Ottowa, 2013.

Chou, J.; Skornicki, M;. and Cohen, J. Unintended Consequences of the Potential Phase-Out of Gamma Irradiation, F1000Research 2018 7:346.

ISO. 11137-1:2006, Sterilization of Health Care Products–Radiation–Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices.

BPSA. X-Ray Sterilization of Single-Use BioProcess Equipment, Part 1: Industry Need, Requirements & Risk Evaluation. Technical Guide, May 17, 2021. 

https://www2.bpsalliance.org/forms/store/ProductFormPublic/x-ray-sterilization-of-single-use-bioprocess-equipment-part-1-industry-need-requirements-risk-evaluation

BPSA. X-Ray Sterilization of Single-Use BioProcess Equipment, Part II: Representative Qualification Data. Technical Guide, July 11, 2023. 

https://www2.bpsalliance.org/forms/store/ProductFormPublic/x-ray-sterilization-of-single-use-bioprocess-equipment-part-2

BioPhorum. Guidance for Risk Evaluation of X-ray Irradiation of Single-use Systems (BioPhorum, Sept. 22, 2023). 

https://www.biophorum.com/download/guidance-for-risk-evaluation-of-x-ray-irradiation-of-single-use-systems/

Jaeger, B. “Gamma to X-Ray: Experience and Approach at SCHOTT,” Presentation to BioPhorum members, April 28, 2021.

Cynthia A. Challener, PhD, is a contributing editor to 


Vol. 37, No. 7
July/August 2024
Pages: 22-27

When referring to this article, please cite it as Challener, C. A. Sterilization Trends for Single-Use Consumables. 

Manufacturing

Gamma irradiation is being supplemented with alternative technologies.

Sterilization Trends for Single-Use Consumables

Contract organizations offer sponsor companies flexibility by providing capacity, expertise, knowledge, and cost savings. Contract manufacturing organizations (CMOs), contract development and manufacturing organizations (CDMOs), and other contract organizations can also offer smaller bio/pharma companies greater opportunities, by providing the resources smaller biopharma companies may not have. According to Robert O’Brien, director operations—Packaging & Logistics at Almac Pharma Services, sponsor companies should consider if contracting activities to a CMO is more cost effective than performing those activities in-house. O’Brien points to direct access to specialists and equipment, a level of control of quality standards, and requirements for scale up as factors to consider when deciding whether to outsource or not. “Outsourcing enables the sponsor company to concentrate on their core competencies, product innovation, and strategic initiatives,” O’Brien says.

When making the decision to outsource a particular process to a contract partner, there are several things sponsor companies should consider. Tech transfer is an important step when outsourcing to a CDMO and should be evaluated when choosing to outsource processes, specifies Peter Ercoli, chief operating officer, Biologics, at BIOVECTRA. Ineffective tech transfers can lead to persistent complications, adds Ercoli. “Conversely, sponsors should ensure their processes are completely defined before initiating a tech transfer, unless they want the CDMO to define it for them. In the case where sponsors are leaning on the CDMO to define their tech transfer process, both parties must be clear on this understanding from the outset,” he says.

Not following regulatory requirements can also complicate the situation when working with CDMOs, according to Ercoli. “The last thing a sponsor wants is to successfully complete the tech transfer and scale-up phases only to fail a pre-approval inspection. Such a failure can be catastrophic, potentially setting back the project by months or even years. While other challenges can be managed with guidance or collaboration, the readiness of facilities and personnel for regulatory inspections is non-negotiable and must be thoroughly vetted,” he says.

The benefits of an outsourcing partner extend to most, if not all, aspects of development and manufacturing, and analytical testing is no different. “CDMOs amass a great breadth and depth of knowledge through their exposure to a wide range of product types including small molecules, peptides, biologics, conjugates, highly potent, and controlled substances,” says Susan Hackett, associate director—Technical Operations, Almac Sciences. “Additionally, not all companies have the appropriate infrastructure to accommodate analytical testing; therefore, outsourcing these operations to an established CDMO helps to save time and cost.”

Outsourcing finished product testing is beneficial for sponsors, because it can provide unbiased results, according to Shailesh Vengurlekar, senior VP Quality & Regulatory Affairs, at LGM Pharma. Sponsor companies should consider the quality of the work and the data provided by a contractor when selected a testing partner. Turnaround time, infrastructure, cost, and communication are also key, according to Vengurlekar.

“Sponsors typically benefit most when outsourcing complex testing services that they may not have the capability (equipment, personnel expertise, time, etc.) to perform in-house,” says Vengurlekar. “[Whereas], it is also beneficial to sometimes outsource simple and routine testing involving a lot of the sponsor’s time and labor. The time can be better utilized by the Sponsors to develop their product database as well as on timely submissions to meet company goals.”

Contract organizations offer sponsors specialized equipment, capacity, and expertise for testing, manufacturing, and packaging bio/pharmaceutical products.

BioPharm International, July August 2024

Streamlining Bioprocesses with Automation

Optimizing Expression Systems for AAV Vectors

Comparing Viral Vectors for Gene Therapy Delivery

The Potential for CAR-Ts in Large Patient Populations

Biosimilars Drug Development: Advances in Technologies from Molecule Design to Clinical Trials

Sterilization Trends for Single-Use Consumables

Contracting Out Frees Sponsors Up

Igniting Pharma’s Potential?

Going Green: Updating Your Filings