As more cell and gene therapies (CGT) advance through clinical trials toward potential approval and commercialization, manufacturers are looking to automated strategies and standardized systems for scaling up production efficiently. Early process designs that came from academic labs were not focused on commercial scale-up and relied, for example, on manual handling steps and complex tubing systems for liquid transfer (1). Automated sampling has helped, but more in-line process analytical technology (PAT) and greater automation and standardization are needed (1). At the same time, the diversity of products—in the case of autologous cell therapy, a different product for each individual patient—means that any manufacturing set-up will need to be highly flexible. New systems would ideally be able to be used in a decentralized, point-of-care location, such as a hospital facility, as well as a conventional, centralized pharmaceutical manufacturing location.

Commercial production of cell therapies is currently less than 5000 doses per year, but global demand is in the millions of doses, reports Jason Arcediano, chief business officer at Multiply Labs, a robotics technology company creating systems for bio/pharmaceutical manufacturing, including an end-to-end, closed manufacturing system for CGT. Existing processes and equipment do not have the throughput needed to meet patient demand. Staffing is also a challenge. “The industry is seeing high attrition rates—over 25%—because there is a huge demand for trained teams that can run these complex processes,” says Arcediano. Complex, manual handling steps limit throughput and increase opportunities for error. “The large cell therapy manufacturers are seeking to reduce failure points, and there is general agreement that automation is the solution,” he adds.

Current manufacturing capacity is inadequate to meet patient demand, and as more new therapies are developed, the unmet need will only increase unless there is a manufacturing paradigm shift, adds John Tomtishen, vice-president of operations at Cellares, which is preparing to launch its proprietary, end-to-end automated system, the Cell Shuttle. “Automation can transform the manufacturing paradigm and help companies scale their therapies effectively to make them accessible to all patients in need,” he says.

The manual and modular approaches used in early development tend to be carried over into commercial manufacturing, but these approaches pose challenges for scalability, adds Dan Strange, CTO, Cellular Origins, a spin-out launched in January 2023 from Cambridge, UK-based technology company, TTP, with a proprietary, automated system for scalable CGT manufacturing.

Cost and drug safety are both prominent challenges for the manufacturing process, which has traditionally been highly manual and complex. “We estimate the cell therapy manufacturing process may have upwards of 40 process steps, which is not only labor intensive but creates opportunities for errors and contamination that lead to failures,” says Betty Woo, vice president of cell, gene, and advanced therapies at Thermo Fisher. “By aseptically closing and automating the manufacturing process, we’re reducing the need for the highly specialized labor required to produce these therapies, thereby eliminating touchpoints, reducing expenses, and ultimately increasing the reproducibility and predictability of the process. Automating in this way will, to some extent, also help alleviate the workforce challenges we’re seeing across the industry particularly in emerging geographies.”

The optimal workflows for CGT are still being developed and processes are not yet standardized, leading to a need for flexible instrumentation, says Woo. “As the field progresses, we expect more standardized workflows to be established, [with] improved consistency, efficiency, speed, and throughput. These improvements will likely be catalyzed by technologies that aid in further scalability—down, up, and out; in-line sensing; and autosampling,” she predicts. “Standardization reduces variability and moves us closer to a more consistent and predictable outcome, while automation provides robustness, consistency, speed, and throughput. Automation in its ideal state will drive efficiency and reduce the cost for manufacturing these life-saving therapies.”

With the current manual or semi-automated manufacturing systems, production space and the need for highly skilled operators are barriers to production capacity. Closed and automated systems address both of these bottlenecks.

“Current, manual systems require ISO 7 or 8 cleanrooms, but a fully closed system can be placed in a controlled not classified (CNC) space,” explains Tomtishen. The Cellares Cell Shuttle platform is a “factory in a box” with all unit operations inside the closed system, which is about the size of a typical cleanroom workstation. Robotics perform material transfer from one unit operation to another. The throughput of the Cell Shuttle depends on the production process, notes Tomtishen. “With a seven-day process, you could manufacture approximately 800 batches per year per Cell Shuttle. Additional Shuttles could be added to scale-out if more capacity is needed,” he explains.

All the software and hardware in the platform has been developed by Cellares over the past four years. “The software enables flexibility to develop the manufacturing workflow. The research scientist has full control over the parameters in the unit operations,” says Tomtishen. He works with users to help them develop workflows and to transfer any processes already developed on other equipment to the proprietary Cell Shuttle equipment.

Partners in Cellares’ Early Access Partnership Program include the Fred Hutchinson Cancer Research Center, PACT Pharma, and Poseida Therapeutics. The partners helped evaluate prototypes and assess product requirements, release criteria, and process workflows. Cellares is continuing to refine the technology and expects the Cell Shuttle to be market-ready in 2024, which will make it available for process development and clinical and commercial manufacturing. In the “single-platform” approach, development and all manufacturing stages take place in the same equipment, which accelerates time to market.

Multiply Labs has designed a fully automated system with robotic handling that offers a solution to the need for increasing throughput, reducing error, and allowing flexibility, Arcediano says. The system has been designed to be compatible with existing equipment through a consortium launched by Multiply Labs in 2021 to tackle the challenge of debottlenecking commercial manufacturing of CGT using robotic automation. Manufacturing development is overseen by the University of California, San Francisco (UCSF) under a sponsored research agreement. Other consortium members are contributing their expertise to bring robotic automation to their equipment within the Multiply Labs’ Robotic Cluster, including Cytiva’s bioreactors, Thermo Fisher Scientific’s incubators, and Charles River Laboratories’ rapid microbial detection platform and endotoxin testing system for automating quality control (2).

“We aim to integrate with all major equipment manufacturers, so that a CGT manufacturer can use the validated equipment and instruments they want in their process,” states Arcediano. “We’ve started with the consortium members, but we are working with other vendors already.” Using validated equipment, process, and software leads to a shorter path forward with regulatory agencies, Arcediano adds. All equipment will be integrated with the robotic handling and Multiply Labs’ in-process control system in a standardized, “plug-and-play” method of information flow that is aligned with best practices from industry organizations, such as the Alliance for Regenerative Medicine. The equipment-agnostic system can use the best currently available instruments as well as be easily updated to use next-generation instruments, Arcediano adds.

Another feature is that all data are captured digitally. Arcediano explains that the end-to-end system could be run with only one operator; the operator loads the materials and then the robots transfer material from step to step through the aseptically controlled, fully closed system. Fluid transfer is handled with a proprietary robotic click connector system that eliminates the need for tube welding.

The automated system has a much smaller footprint than traditional manual processes. Because operators do not need access to the unit operations, the modules can be stacked two to three high and five to six deep. “A ballroom facility might have 1000 to 2000 square feet, while our system requires between 200 to 300 square feet,” says Arcediano. Throughput is also higher than traditional methods. “Instead of one client [i.e., patient product] at a time, our system can prepare up to 36 products at the same time,” he adds. The system can be scaled out as needed by including more modules for greater throughput. It can also be used for product development or commercial manufacturing, eliminating tech transfer.

According to Arcediano, because the process is self-contained, it can be used in a decentralized environment, such as a hospital, which would allow application of cell therapies in other regions of the world that do not have centralized manufacturing capability.

Cellular Origins’ platform is a configurable robotic automation solution that enables scalable, cost-effective, and space-efficient cell therapy manufacture that is designed for adaptability, says Strange. The platform consists of a universal transport system for automated movement of consumables, reagents, and patient materials; a tube-management system that enables automated routing; and a sterile fluid-transfer system that welds tubes, moves fluids, and provides real-time analysis and quality control, he explains. “These tasks are carried out by a combination of autonomous mobile robots with proprietary, future-proofed end-effectors and a configurable automation console that integrates existing third-party equipment. These tasks are all integrated within a data management system to ensure full traceability of every process undertaken,” Strange explains.

“Our platform enables therapy developers to use the tools and technologies from third-party vendors and combine them into a modular factory that can quickly scale to producing 10,000 therapies per year with a fraction of the labor of existing methods. Because the platform is built upon proven familiar technologies, it can potentially be used to scale those therapies currently in late-stage development.”

Strange adds that the company is beginning to work with its first customers to develop system configurations and to deploy these systems at customer sites.

In May 2023, Cellular Origins announced a partnership with ScaleReady, which is a joint venture between Bio-Techne, Fresenius Kabi, and Wilson Wolf with a mission to simplify and standardize CGT manufacturing. The collaboration’s initial task is to standardize and automate the interconnections between ScaleReady’s manufacturing platform modules using Cellular Origins’ robotic system (3).

“Solving the liquid handling problem and proving how amenable a simple, modular, and scalable approach is to automation allows the field to see a path forward to economies of scale,” said Josh Ludwig, commercial director at ScaleReady, in a press release (3). “Through this partnership, we plan to create a viable pathway using cell therapies for larger patient indications and front-line treatments by enabling therapy developers to implement automation when it makes the most sense, and when their products require it.”

Lonza’s platform for autologous cell therapy manufacturing, the Cocoon, is a functionally closed, automated system that is currently being used to support clinical trials in Europe and North America, in both centralized and decentralized manufacturing models, says Tamara Laskowski, head of Clinical Development, Personalized Medicines at Lonza. “With a growing number of clinical-pipeline applications, we are optimistic about the future use of the Cocoon Platform in commercial applications as well,” she adds.

Several unit operations—from starting materials through to final harvest—take place inside the functionally closed, single-use cassette, in processes which are tailored for each product. The Gen2 Cocoon, introduced in April 2022, added magnetic cell separation capabilities, allowing separation of cell types of interest, such as T cells, for processes that require this additional upstream sample preparation step, says Laskowski.

Decentralized manufacturing, which is closer to the patient at the point of care, reduces logistic challenges but can present quality concerns. Automated, closed systems, however, may resolve these concerns. “Producing cell therapies in automated, closed manufacturing systems will help to reduce reliance on manual operations and variability in process performance, improve data management, and may enable the realization of a standardized level of quality across point-of-care sites,” suggests Laskowski.

Allogeneic CGTs aim to use a single source of cells to treat many patients, with manufacturing at a larger scale than autologous CGTs. Automation, however, is expected to be important for these processes as well.

“Given the large-volume productions in allogeneic processes, automated systems that support final product harvest, formulation, and fill/finish are advantageous,” says Laskowski. “Such systems can reduce risks associated with manual processing and human error, ensure proper addition of cryoprotectant, and decrease time requirements to cryopreservation and storage of the product. As additional allogeneic CGT modalities emerge, we anticipate new developments in automated platforms to support culture and expansion of adherent cells and enable multi-stage manufacturing processes involving modified culture regimens and conditions for cell differentiation.”

Spivey, C. Small Footprint Automated Closed Production Platforms for CAR-T and Other Advanced Therapies. 2023 47 (4) 28-30.

Multiply Labs. Multiply Labs Adds Thermo Fisher Scientific and Charles River Laboratories to its Robotic Manufacturing Consortium. Press Release, May 5, 2022.

Cellular Origins. Cellular Origins Partners with ScaleReady to Simplify, Standardise, and Automate Cell Therapy Manufacturing. Press Release, May 11, 2023.

Jennifer Markarian is manufacturing reporter for BioPharm International.

When referring to this article, please cite it as Markarian, J. 

Automation Aids Cell and Gene Therapy Production

Articles

Cover Story

Manufacturers face the challenge of meeting growing demand for personalized biopharmaceuticals.

Filling of syringes and vials with high-viscosity monoclonal antibodies (mAbs) or small-molecule liquid formulations is a difficult task and may present several challenges. Considerable development work may be required to develop a filling process that provides a clean fill using a small-scale or at-scale filling machine. However, performing the development work using an at-scale filling machine is neither an efficient nor cost-effective long-term option due to hectic manufacturing schedules and associated resource needs. A small-scale filling line can be used to define optimal filling process parameters, support technology transfer, and develop mitigation strategies for ongoing or potential issues with minimal disruption of normal manufacturing operation. Streamlined ways of working without requiring manufacturing-scale line time is expected to result in increased efficiency and improved portfolio progression timelines. Key learnings, case studies, and recommendations for this small-scale filler are discussed.

Submitted: Aug. 19, 2022
Accepted: April 7, 2023

, Kevin.x.hutter@gsk.com, is investigator; Kashappa Goud Desai is scientific leader; Igor Rusanov is principal investigator; and Aarti Gidh is director; all at Process Engineering and Analytics, Drug Product Development, GSK.

To whom all correspondence should be addressed.


Volume 36, No. 7
July 2023
Pages: 28–33

When referring to this article, please cite it as Hutter, K.; Desai, K. G.; Rusanov, I.; Gidh, A. Small-Scale Syringe and Vial-Filling Capability for Development of Robust Filling Processes, Smooth Tech Transfers, and to Resolve Manufacturing Issues: Summary of Key Learnings. 

Peer-Reviewed Research

Use of a small-scale filler can result in increased filling efficiency.

Small-Scale Syringe and Vial-Filling Capability for Development of Robust Filling Processes, Smooth Tech Transfers, and to Resolve Manufacturing Issues: Summary of Key Learnings

Lentiviral vectors (LVVs) offer numerous advantages for production of adoptive cell therapies. Integration of LVV genetic material into host-cell DNA enables stable and long-term expression of the therapeutic gene in the target cells, leading to longer therapeutic effects once the cells are infused into the patient. LVVs are also highly efficient at transducing a wide range of cell types and can have a large carrying capacity relative to adeno-associated virus (AAV), allowing the delivery of larger or multiple therapeutic genes. Furthermore, LVVs can be engineered to include safety features, such as self-inactivation mechanisms or inducible gene expression systems, which can aid in regulation of transgene expression or provide the ability to eliminate modified cells if necessary.

The most commonly used platform for generating LVVs in the R&D and clinical space relies on transfection of four individual plasmids. The disruption of LVV genes required for competent viral replication have been eliminated, while the structural components required to assemble virus particles have been retained. This advancement maximally reduces the potential recombination events that could lead to undesirable replication of competent lentivirus (RCL) formation in patients. While safety has been addressed, transient transfection of four plasmids to generate LVVs in this manner is challenging. Sourcing the plasmids can be difficult, and the efficiency of the reaction is influenced by numerous process parameters.

There is significant interest, therefore, in developing stable producer cell lines that simplify LVV production, afford high yields of high-purity LVVs, and measurably lower costs for manufacturing.

Producer cells are generated by introducing the gene of interest (GOI) into an LVV packaging cell line that already has stably integrated the vector-coding LVV structural proteins and a viral envelope to impart cellular tropism. This stable producer cell line then undergoes multiple rounds of clonal selection, and if a suspension system for better scalability is desirable, then subsequent serum-free suspension adaptation is required. The result is a monoclonal, highly characterized, GOI-specific stable producer cell line.

To overcome the cytotoxicity to producer cells associated with constitutive over-expression of the LVV envelope and the regulatory protein Rev required to generate viral products, two approaches have been developed. With inducible systems, the expression of viral cytotoxic proteins is regulated by the addition of an inducer or the removal of a suppressor to control initiation of LVV production at a defined timepoint and thus limit expression of the cytotoxic viral products to the time of production only, explains Luca Peterfay, process development scientist II at Charles River. Common systems turn transcription on or off using tetracycline or doxycycline (an analog of tetracycline) antibiotics (Tet-on and Tet-off systems, respectively) via the tetracycline response element (TRE).

In constitutive systems, continuous expression of all vector components occurs, with nontoxic envelope alternatives to vesicular stomatitis virus G (VSV-G) engineered into the producer cells. “This approach limits titers and transduction efficiency compared to VSV-G enveloped systems, but allows multiple vector harvests, extends production campaigns, and reduces overall LVV manufacturing costs,” Peterfay observes.

There are many reasons to use producer cells for viral-vector manufacturing, says Brian Tomkowicz, senior director of R&D for Center for Breakthrough Medicines. In addition to being designed to prevent the replication of viral vectors and eliminate the risk of generating replication-competent viruses (RCVs), producer cells are engineered to lack essential viral genes or have other safety features to minimize the chance of vector release or dissemination during production, he notes.

Simplified process flows can also be achieved with producer cells due to elimination of the multi-plasmid transient co-transfection step, observes Peterfay. As importantly, producer cells enable efficient vector production of high-quality viral vectors through incorporation of all the necessary components for viral protein expression, assembly, and packaging of the therapeutic gene, according to Tomkowicz. Furthermore, producer cells can also be grown in large-scale bioreactors, allowing the production of large quantities of viral vectors. Tomkowicz also adds that the implementation of limited perfusion-based systems for continuous production at scale will enable the scalability crucial for meeting rapidly growing demand while simultaneously lowering manufacturing costs.

Production of viral vectors with consistent quality and characteristics, meanwhile, ensures reproducibility and consistency throughout the manufacturing process, contends Tomkowicz. The use of monoclonal, highly characterized producer cells in chemically defined media (CDM) without animal-derived products increases reproducibility and reduces batch-to-batch variation, Peterfay agrees.

Not least of the benefits Tomkowicz highlights is reduced costs due to elimination of the need for large quantities of good manufacturing practice (GMP)-grade plasmids and avoidance of negative impacts of bringing foreign DNA into production cells. Eliminating the need to source large quantities of high-purity plasmids also reduces timelines, adds Peterfay. Both agree that if a constitutive system is employed, longer processing periods and multiple harvests can cut viral-vector production costs even further.

The many advantages of using producer cell lines must be balanced with the limitations of deploying this approach. The generation of the stable producer cells is an additional step prior to the initiation of production, and this step can potentially increase preclinical timelines by several months, according to Peterfay. “The generation of stable producer cells is a complex and time-consuming procedure, which entails tedious cell-line development due to the numerous clonal isolation steps followed by extensive screening procedures,” she explains.

For the constitutive approach, Peterfay also notes that due to replacement of the VSV-G with a low-cytotoxicity alternative envelope, titers may not be as high as those achieved via transient transfection, and transduction efficiency can also be lower. “The limitation in titers can be compensated by longer processing times and multiple harvests; however, due to the reduced transduction efficiency higher doses might be required to achieve the required therapeutic effect in patients,” she says.

Finally, Peterfay points out that due to the large size of the viral insert introduced into the host cell, the long-term stability of producer cells is a concern, as compromised stability will likely result in reduced productivity.

Realizing target quality attributes is essential

When developing producer cells for LVV production, the goal is to engineer cells with several important target quality attributes. A short population doubling time of less than 24 hours is ideal to reduce GMP occupancy time and operating costs, according to Peterfay. High productivity and the ability to produce high titers of LVVs is essential to ensure efficient and effective delivery of therapeutic genes to target cells, Tomkowicz adds. High cell densities should be achievable in chemically defined media and prolonged harvest times should be tolerated, notes Peterfay.

Producer cells should maintain the integrity and stability of the LVV genome during production, according to Tomkowicz. “This attribute is crucial to prevent genetic alterations or rearrangements that could impact vector functionality or safety,” he says. Peterfay also stresses the importance of genetic stability without selective pressure, as the addition of antibiotics during GMP manufacturing significantly increases costs and may pose regulatory concerns.

“It is also essential,” states Tomkowicz, “that producer cells do not generate RCLs during vector production, as they could lead to uncontrolled viral replication and potential adverse effects in patients receiving gene therapy.” In addition, he observes that producer cells should have minimal levels of impurities such as host-cell proteins and DNA contaminants and demonstrate consistent performance over multiple production runs, maintaining consistent vector yield and quality attributes.

“These properties and characteristics are important to ensure the safety, efficacy, and consistency of the LVV production process,” Tomkowicz emphasizes. “High vector yield enables cost-effective production and efficient delivery to target cells. Genetic stability and absence of RCLs reduce the risk of adverse effects. Low impurities ensure the purity and safety of the final vector product. Consistent performance enables reliable and reproducible manufacturing processes, which are critical for clinical applications,” he concludes.

Producer-cell generation for LVV production can be proprietary and company-specific, but typically involves a similar series of steps, according to both Peterfay and Tomkowicz. The process starts with vector design, which is then followed by transfection or transduction to stably integrate the vector coding elements into the packaging cell genome. Next is selection and clonal expansion of host cells that have successfully integrated the vector. Single-cell cloning is performed in adherent, stable producer cells to generate a monoclonal population. Finally, through improvements in next-generation sequencing, rapid and cost-effective mapping of producer cell lines ensures that integration events during clonal selection did not activate proto-oncogenes, thus providing added safety for patients.

Characterization and screening assays, such as vector yield determination, genomic analysis, and testing for RCL generation are then performed on the resulting cell lines to evaluate their vector production capabilities, genetic stability, absence of RCLs, and other desired properties and identify clones with highest yields, fastest growth, and high stability. If the stable producer cells are intended for use in suspension processes, suspension adaptation is then performed. The selected cell lines are then scaled up in culture and process optimization pursued to maximize vector yield.

The resulting production process is validated to ensure consistency, quality, and safety, and appropriate quality-control measures are implemented to monitor critical parameters and assess the final vector product for impurities and potency.

While the steps involved in producer cell development can be readily outlined, effective implementation is not an easy task. Maintaining long-term stability of the integrated vector can be challenging, says Tomkowicz, as it may be prone to rearrangements, deletions, or silencing over time, affecting vector production and consistency. Ensuring stable integration of the lentiviral vector into the producer cells’ genome is, therefore, crucial, but defining the number of generations where the selected clone is stable without selective pressure and maintains high productivity can be difficult, Peterfay observes.

Preventing the generation of RCLs during vector production is equally critical for safety reasons, but controlling and eliminating any potential RCL contaminants is a challenge that requires rigorous testing and screening protocols, according to Tomkowicz. Similarly, optimizing productivity and achieving consistent high-level expression of viral proteins can be challenging, as it depends on the host cell line, transfection/transduction efficiency, and the vector design itself. “Developing clonal producer cell lines with consistent vector production can be challenging due to genetic heterogeneity within the initial population of transfected/transduced cells. It may require multiple rounds of cloning and screening to identify and select stable and high-yielding clones,” he comments.

Tomkowicz adds that screening of stably integrated clones for LV packaging can be exhaustive. It is necessary to ensure the appropriate stoichiometry and high copy number of integration events to achieve optimal viral titers of intact virus particles. In addition, induction systems must be compatible with GMP manufacturing, be completely off when not desired, and have a large dynamic range upon induction.

Potential negative impacts of the transgene on the host are a secondary concern when trying to generate producer lines, according to Tomkowicz. For instance, if expression of the GOI during clonal selection impacts overall cell health another level of GOI silencing may be required.

Peterfay, meanwhile, points to the need for the constitutive LVV producer cells to overcome the limitations of using VSV-G to improve transduction efficacy in order to lower the doses required.

These challenges are magnified by the difficulties associated with implementing robust assays and screening methods to assess vector quality, absence of contaminants, and genetic stability, according to Tomkowicz. “Developing effective assays is essential to achieve thorough characterization and quality control of producer cells to ensure the safety, purity, and potency of final LVV products,” he says.

Scaling up producer cell lines from small-scale research settings to large-scale manufacturing can also pose challenges. “Adapting the production process to bioreactors, maintaining optimal culture conditions, and ensuring consistency and reproducibility at a larger scale require careful process development and optimization,” Tomkowicz states.

Ongoing efforts to address key challenges

Several technologies and approaches have been developed to help address the key challenges associated with produce cell development for LVV manufacturing. Vector engineering is a focus, according to Tomkowicz, with the goal of optimizing vector design and incorporating genetic elements that enhance stability, expression, and packaging efficiency, thereby improving vector integration and stability in producer cells. “To that end, gene editing tools such as CRISPR/Cas9 are being used to precisely engineer producer cells, facilitating targeted integration of the vector and reducing genetic heterogeneity,” he says.

The cytotoxic and cytostatic effects of some viral products in inducible systems, such as tetracycline-inducible expression systems, are limited by better controlling of gene expression through the use of inducible promoters, according to Peterfay. In constitutive or continuous packaging systems such as STAR cells, meanwhile, the cytotoxic limitations of VSV-G are reduced by nontoxic or low-toxicity envelope glycoproteins suitable for LVV pseudotyping, she says.

There is also some interest, Peterfay notes, in exploring the engineering of cell lines other than HEK293 derivatives, such as primary human amniocytes, to increase robustness and tolerance. Tomkowicz agrees: “There are efforts directed at developing novel cell lines or modifying existing ones with improved characteristics such as higher vector production capacity, enhanced stability, and reduced immunogenicity.”

Some companies are implementing more stringent screening and selection processes to identify and isolate high-yielding, genetically stable producer cell clones through methods such as fluorescence-activated cell sorting (FACS) or limiting dilution, Tomkowicz adds. To overcome labor-intensive cell engineering and cloning steps, meanwhile, Peterfay observes that high-throughput automated equipment solutions are being developed. Similarly, Tomkowicz observes that advanced analytical techniques and quality-control measures to detect and eliminate RCLs are also being developed.

Given that the use of producer cell lines for LVV production is a relatively new technology, developers of these solutions must be aware of various regulatory considerations when pursuing this route for GMP LVV manufacturing.

The main regulatory concern, according to Peterfay, is the potential for genetic drift in LVV producer cells due to the presence of the large LV insert. “Developers need to define the lifespan and the number of generations over which producer cells are stable to ensure that the product generated is consistent and has the same quality attributes from younger and older generations,” she explains. Stable producer cells also must remain stable without selection pressure, as the use of pharmaceutical-grade antibiotics is very expensive and can raise regulatory concerns, she adds.

Overall, notes Tomkowicz, the regulatory landscape regarding the use of stable producer cells for production of LVVs used in adoptive cell therapy manufacture is complex and varies across different regions. FDA (1) and the European Medicines Agency (EMA) (2), for instance, have specific guidelines and requirements for the development and manufacture of gene therapies using LVVs. Topics addressed include genetic stability, safety, quality control, process validation, GMP compliance, and specific requirements for regulatory filing, among others.

Even with the challenges facing the development of stable, effective producer cell lines for LVV manufacturing, both Peterfay and Tomkowicz believe eventual widespread adoption of this strategy to be likely, but there is uncertainty as to the timeline.

“If a stable cell line with the packaging plasmids was created that meets all the quality attributes discussed earlier, it could be patented and offered to clients to simplify their production flows and reduce timelines and costs,” Peterfay comments. Only the transgene would then require subsequent stable transfection using the stable producer cells already containing the packaging plasmids, which would substantially reduce the timelines for operation. She estimates that it will take approximately 10 years before producer cell lines for LVV manufacturing gain full trust and routine use in the biopharmaceutical industry, with the exact timing a function of both continued stable producer cell development work and regulatory/clinical expectations.

Tomkowicz does not specify any timeline, but agrees that given the rapid advances in gene therapy and the increasing recognition of LVVs as powerful tools, it is plausible to expect wider adoption of stable producer cells. He also agrees that the timeline will depend on research progress and regulatory considerations in addition to technological advances, such as advances in cell-line engineering technology, improvements in the quality of integration events such as can be achieved using transposase technology, increases in scalability and manufacturing efficiency, the development of more robust quality-control and characterization solutions, and the advent of standardized processes and guidelines. “Collaboration between academia, industry, and regulatory agencies will play a crucial role in driving the necessary developments and establishing the foundations for widespread use of stable producer cells in LVV production,” Tomkowicz concludes.

 Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), Guidance for Industry

Guideline on Quality, Non-Clinical and Clinical Aspects of Medicinal Products Containing Genetically Modified Cells 

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

When referring to this article, please cite it as Challener, C.A. State of Producer Cell Development for Lentiviral Vector Manufacturing. 

Development

Despite many development challenges, stable producer cell lines show real promise.

State of Producer Cell Development for Lentiviral Vector Manufacturing

Despite its widespread use in the chemical and biopharmaceutical industries, traditional preparative-scale liquid chromatography is considered unsustainable due to its high solvent consumption and solid waste generation. A promising green alternative to this method includes solid support-free techniques, such as hydrostatic countercurrent chromatography—also known as centrifugal partition chromatography (CPC).

In this article, the author discusses CPC’s sustainability, which is demonstrated by its solvent flexibility and recyclability. The author also investigates how CPC supports other downstream processes and extracts value from low-value waste compounds.

In contrast to conventional solid–liquid chromatographic techniques, CPC consists of liquid stationary and mobile phases. The stationary phase is held in place by a strong centrifugal force inside the column, while the mobile phase is pumped through it. The partitioning of the solutes between the two immiscible phases governs purification and affects the time spent within the stationary phase, which determines solute retention (see 

CPC, as a liquid–liquid system, offers significant cost benefits because it eliminates the need to purchase silica gel and reduces the amount of required solvents. The few types of commercially available solid silica-based packing gels limits stationary-phase options. Further, when the gel, which fills the entire column, is saturated due to adsorption and needs to be changed, the whole column of gel must be replaced, which generates a siginificant amount of waste.

Being constrained to silica gel stationary phases also limits traditional chromatography’s flexibility. When fine-tuning the separation process, chemists usually only alter the mobile phase composition instead of adjusting the stationary phase. CPC takes advantage of a wider range of possible biphasic liquid–liquid systems using the same column.

Substituting halogenated and petroleum-based solvents with green, renewable alternatives reduces the environmental impact of chromatography. Recent regulations have even banned several hazardous solvents, including n-hexane, making the use of green alternatives important.

Determining potential biphasic systems by trial and error is a time-consuming task due to the wide polarity range needed to cover the most apolar to most polar systems. To overcome this challenge, several regulatory and industry bodies have produced guides on which solvents to use based on properties such as hazardousness, toxicity, and recyclability (

Furthermore, both academic and industrial researchers have composed simple strategies to help one choose the most effective solvent system for a given application.

Ideal solvents for CPC have low viscosity, are ultraviolet (UV)-transparent, cover a wide polarity range, and have strong interfacial tension between the aqueous and organic phases. When accounting for additional post-separation fraction processing, solvents with low boiling points work best.

2-methyl-tetrahydrofuran (2-Me-THF): this solvent is a green alternative to THF in synthesis because it is nontoxic, has low miscibility in water, and is relatively stable compared to other heterocycles.

Limonene: this apolar solvent has a D-isomer, which is a major component of the essential oils in citrus fruits.

Cyclopentyl methyl ether (CPME): this solvent can directly replace dichloromethane (DCM) in the widely used methanol-DCM system.

Water: this safe, environmentally friendly solvent is usually the first choice for a green CPC application for aqueous two-phase systems (ATPS).

Another group of potential green compounds are deep eutectic solvents (DES)—more specifically, natural DES (NaDES), in which the components are of natural origin. These solvents are characterized by a large depression of freezing points, usually higher than 150 °C. DES can be acquired by simply combining two safe, cheap, renewable, and biodegradable components capable of forming a eutectic mixture. If the challenge of solute recovery and component recycling can be solved, NaDES would be a promising alternative to water-based solvent systems, providing a polarity range that facilitates the separation of less polar and hydrophobic compounds.

Sustainable separation processes reuse as much solvent material as possible, and CPC can benefit from solvent recycling through several processes such as membrane filtration, ultrafiltration, dialysis, and density-based recirculation. Research done on each method yielded the following information.

 It has been demonstrated that combining countercurrent chromatography with organic solvent nanofiltration (OSN) is a cost-effective purification method to isolate an API with a molecular weight of about 600 Da. It was found that API rejection rates are dependent on the solvents used, and, even though more than 99% efficiency was achieved with the filter membranes used, there is a need to develop membranes with 100% removal efficiency because the total API loss was 2.3% throughout the entire process. It was observed that using OSN to recycle the mobile phase solvent can provide 56% greater mass-intensity and highlights its value in optimizing CCC mass-efficiency (1).

 It has been found that this recycling method shows potential to boost solvent use efficiency. In a CPC purification run with an ATPS, dialysis was used to isolate separated phenolic compounds and recycle the sodium polyacrylate and polyethylene glycol (PEG)-rich phases of the ATPS. It was observed that when compared to CPC runs without dialysis, CPC runs with dialysis can reduce carbon footprint by as much as 36% (2).

 This method provides substantial solvent recovery results. Researchers modeled the continuous separation of PEGylated and non-PEGylated cytochrome-C (Cyt-c) using CPC. The results of the study showed that, through ultrafiltration, both the product and unreacted protein could be isolated with recovery rates of 88–100% and about 100% purity before recycling the PEG-rich ATPS with a consecutive purification run. Compared to processes without ultrafiltration, processes with ultrafiltration can reduce the carbon footprint by 67% (3).

 A method involving continuous in-line solvent recycling and density-measurement based ternary solvent system readjustment was investigated in 2020 (4). In this method, pure solvents were automatically fed into a mixer-settler unit, and the solvent system was prepared with the desired ratio based on density measurements. Here, the prepared phases were stored within buffer tanks. These tanks held the phases until they were ready to be pumped into the CPC during a chromatographic run.

After the run was done, waste solvents were separated from the impurities in the recycler skid, and then both the recovered solvents and evaporated solvents from product fractions were fed back into the mixer-settler unit. After readjusting the solvent system based on density measurements, the phases were ready for another CPC cycle. Investigation results provided 14.865 kg of crude steroid API with an average purity of 98.74% (4). In this experiment, researchers ran 153 cycles and recovered more than 90% of the methyl isobutyl ketone and acetone content for recycling and reuse purposes.

 It was studied that implementing CPC as a bioreactor is a potential solution for reducing solvent and energy resources. By pairing chemical reactions with the separation process, the manufacturing times can be shortened in applications such as equilibrium-limited hydrolysis. In this process, lower equilibrium conversions occur at high initial substrate concentrations due to the reverse reaction, making it inefficient to produce large amounts of optically pure amino acids by conventional means.

A CPC reactor can suppress the reverse reaction, but only if the reaction is faster than the reactants’ residence time and has no mass transfer resistance. With experimentally determined partition coefficients, stationary-phase retention (Sf) values, volumetric mass transfer coefficients, and kinetic parameters, one can predict peak shapes, conversion, and resolution values as a function of the enzyme and PEG concentration. Knowing these values allows for the optimization of settings for both hydrolysis conversion as well as product and reactant separation.

When compared to a traditional batch reactor, a CPC bioreactor can double throughput after optimizing CPC and system conditions. It has also been shown that this reaction method maintains performance for up to a 24-hour period before gradually diminishing. In addition, CPC reactors were found to be more hydrodynamically similar to tubular flow micro reactors because both exhibit diphasic hydrodynamics driven by pulsation.

 CPC also outperforms stirred tank reactors (STR) in converting hydrophobic substrates when using an aqueous organic two-phase system after optimizing buffer type, concentration, and system pH. When using an organic solvent, it should be compatible with the enzyme and work as an acceptable biphasic system that facilitates CPC separation. The CPC reactor doubled conversions compared to a STR, showing CPC’s capability to handle multiphase reaction systems.

When comparing STR to CPC reactors, CPC provides better mixing conditions and is more efficient when handling lower aqueous phase volumes (5). However, CPC exhibits earlier limitations than a STR when working with higher enzyme quantities, and the complex fluid dynamics within CPC constrain scalability. When better rotors and chamber geometries are developed to improve scalability and continuous processes, CPC will grow into an attractive alternative to STR.

Achieving industrial-scale sustainability

Coupling CPC with conventional separation and isolation methods was demonstrated with sustainable functionalized taxanoid production. After microbial upcycling of low-value feedstocks, CPC can be integrated into economic taxadiene isolation processes to enable consecutive chemo-enzymatic functionalization.

 extraction and purification processes such as these are traditionally performed by reversed-phase high-performance liquid chromatography (RP–HPLC). Incorporating CPC into a downstream processing strategy is a greener alternative to the traditional method because doing so combines a rapid two-step extraction procedure with a subsequent CPC purification step.

), one can avoid the admixing and adsorption issues that can occur during RP–HPLC, providing taxadiene with 95% purity and throughput of roughly 250 mg/L. 

In addition, the increased diterpene mass load of taxadiene significantly raised process efficiency, improving recovery from high-cell-density fermentation broth. This downstream CPC strategy enables biology-oriented synthesis of ad hoc-designed bioactive taxanoids at an industrial scale.

Another potential application is continuous, pilot-scale recovery of the bioactive components—hydroxytyrosol (HT), oleocanthal, oleacein, and the monoaldehydic forms of oleuropein aglycone (MFOA) and ligstroside aglycone (MFLA)—from extra virgin olive oil.

In this example, after using liquid–liquid extractors for fractionation, CPC could enrich the total phenolic fractions during the purification step. Using step-gradient elution extrusion in an n-heptane, ethyl acetate, ethanol, and water solvent system, the fractions of oleocanthal, oleacein, MFOA, and MFLA can be recovered on the gram scale with at least 80% purity. For greater purity, preparative HPLC further purifies these bioactive components to greater than 95%.

Because of the large volume of pure compounds recovered, these three industrial-scale techniques make 

 experiments a possibility for the first time.

CPC can support throughput optimization efforts by means of process intensification and switching to continuous manufacturing, instead of batch manufacturing. Improving these processes would provide greater automation and quality control, simpler scalability, boosted production throughput, and more sustainable manufacturing from start to finish.

Researchers have previously investigated the potential for CPC-based quasi-continuous purification steps to integrate with multi-step, continuous synthesis of key intermediate bioactive carbazoles. A multiple dual-mode CPC purification can follow the end of a two-step continuous-flow synthesis of the target molecule.

Using a one-phase intake of the sample solution as the input for the CPC step provides a 59% overall yield, greater than 99% purity and 2.24 g/h/L productivity. Increasing column capacity is a potential method to boost throughput and achieve industrial-scale production. The promise of industrial scalability makes CPC ideal for continuous purification in future API and intermediate manufacturing process designs (

Each year, millions of tons of agricultural, industrial, municipal, and forest waste are generated around the world, and treating this waste is a major undertaking for both industries and governments. Recycling, recovering, and reusing these materials with sustainable technologies is a more cost-effective and green alternative than traditional waste treatments.

Centering waste management strategy around CPC can reduce raw material usage in manufacturing processes and the environmental impact of waste accumulation. This approach can also help in the recovery of valuable compounds for use in nutraceuticals, cosmetics, and bio/pharmaceuticals.

A prime target for waste valorization is HT, which is found in olive processing wastewater. As one of the main phenols found in olives, HT possesses a natural antioxidative and free radical scavenging activity that makes it useful in preventing degenerative diseases. It has been shown that, after adsorption resin treatment, a pilot-scale CPC with a cyclohexane/ethyl-acetate/ethanol/water solvent system sorption resin can isolate the phenolic compounds and unlock 1.81 g/h per column volume productivity, efficiently recovering HT at a low cost.

As another example, CPC separation has been implemented to optimize and scale-up monosaccharide fractionation from hydrolyzed sugar beet pulp as an alternative to multiple resin chromatography. Sugar beet pulp mostly consists of cellulose and pectin, making it unfit as a gelling agent, and it is commonly dried and pelleted for use as an inexpensive animal feed. Due to its low cost, prevalence, and high carbohydrate content, it can be fermented into bio-ethanol or hydrolyzed into monosaccharides—which can later be fractionated with a CPC after a thermo-chemical pretreatment.

A preparative-scale CPC run with an ethanol/ammonium sulfate solvent system has been implemented to optimize monosaccharide isolation and gain a final throughput of 1.9 g/L/h when normalized by column volume. In another industrial application, integrating CPC purification into biorefinery processes allowed for purified arabinose to be converted into arabitol or other high-value compounds such as L-gluco-heptulose, a useful chemical in cancer therapy and hypoglycemia treatment.

CPC is a greener, more sustainable alternative to conventional preparatory separation methods due to its versatility and flexibility in available solvents, its wide range of possible isolation, fractionation, and purification applications, and its potential as a valuable method of waste treatment.

1. Rundquist, E.; Pink, C.; Vilminot, E.; Livingston, A. Facilitating the Use of Counter-Current Chromatography in Pharmaceutical Purification Through Use of Organic Solvent Nanofiltration. 

, 156–163.
2. Santos, J. H. P. M. P. M.; Almeida, M. R.; Martins, C. I. R. R.; et al. Separation of Phenolic Compounds by Centrifugal Partition Chromatography. 

 (8), 1906–1916.
3. Santos, J. H. P. M.; Ferreira, A. M.; Almeida, M. R.; et al. Continuous Separation of Cytochrome-c PEGylated Conjugates by Fast Centrifugal Partition Chromatography. 

 (20), 5501–5506.
4. Lorántfy, L.; Rutterschmid, D.; Örkényi, R.; et al. Continuous Industrial-Scale Centrifugal Partition Chromatography with Automatic Solvent System Handling: Concept and Instrumentation. 

 (11), 2676–2688.
5. Krause, J.; Merz, J. Comparison of Enzymatic Hydrolysis in a Centrifugal Partition Chromatograph and Stirred Tank Reactor. 

Árpád Könczöl, PhD, is lead scientific officer at RotaChrom Technologies.


Volume 36, No. 7
July 2023
Pages: 20–25

When referring to this article, please cite it as Könczöl, A. Exploring How CPC Progresses Green Chemistry Practices. 

Downstream Processing

CPC presents a green alternative to chromatography that supports sustainable operations.

Exploring How CPC Progresses Green Chemistry Practices

While the debate between stainless steel and single-use systems (SUS) will continue to rage for the foreseeable future, most would agree that there is a time and place for either approach. Naturally, however, this raises the question: when is it ideal to utilize an SUS approach? What advantages does it confer? How are they being used in the current manufacturing environment?

spoke with SUS experts Nick Johnson, director of Product Management, Biopharma, CPC; Wolfgang Walcher, chief commercial officer, Single Use Support (at the time of interview); and Helene Pora, vice-president, Technical Communication & Regulatory Strategy, Pall, to get their perspective on the current benefits of single-use systems.

Can you share your thoughts, broadly speaking, on the merits of single-use systems, consumables, etc., in manufacturing? Is there any aspect that you feel is under or over-emphasized in this discussion?

 Think about the flexibility single-use systems allowed during the pandemic. Manufacturers urgently reallocated new therapy and other capacities to commercial vaccine production. The capability to quickly switch from one kind of production to another is a huge benefit that SUS affords in everyday practice. Single-use technologies (SUT) definitely contribute to speed and efficiency by allowing multiple products to be produced in one manufacturing suite with relative ease.

SUT’s flexibility and sterility also has made it simpler to ‘fail fast’ in clinical trials, too, compared to traditional stainless steel. The impact on bioprocessing from early-stage development through commercial manufacture has been an industry game changer.

 SUS offer a level of flexibility, scalability, and cleanliness that is near impossible to achieve with glass and stainless-steel counterparts. Lines can be repurposed more easily, and there is no need for cleaning and cleaning validation. This saves a great deal of time and money while making the process safer for operators. SUS can also reduce the time to build a plant and lower overall capital expenses. And from an environmental perspective, there is a lower carbon footprint, less water, and less chemicals despite higher solid plastic waste.

Unfortunately, the amount of solid waste is frequently overstated compared to the benefits. In 2020, ISPE released a review showing that SUS is responsible for .01% of all global plastic waste, which is largely driven by consumers. This does not mean that the industry does not see the use of plastic as a problem—we are actively investing in recyclability and overall process impacts—however, it does not deserve the level of criticism it gets.

: Single-use systems are trending for a reason:

They help to improve safety, cost-efficiency, and sustainability in comparison with the use of durable stainless-steel manufacturing facilities.

With single-use assemblies, bioprocess containers, and other sterile consumables operators can transfer liquids in closed systems and as customized options.

They are adaptable to changing requirements of system setups.

Usually, the systems are designed to minimize manual interventions, thus preventing human errors which can result in quarantined or even rejected batches.

The most under-emphasized aspect is probably sustainability. Different life-cycle assessment studies have proven that single-use systems can be more sustainable than stainless-steel solutions (1). However, there is still room for improvement, and we’ll drive innovation to further improve sustainability in the pharmaceutical industry.

What are the costs of using single-use systems, consumables, etc.? What factors should be considered when deciding whether or not to employ them?

 There are different factors that come into play to influence costs of single-use systems because of the wide range of consumables on the market, which offers a wide span of prices.

The answer is strongly dependent on the application/use of single use systems. For example, in API primary packaging the alternative to single use systems is shipping hundreds of kilograms of cryo-vessels worth millions of dollars and causing tons of CO2 [carbon dioxide] emissions; alternatively, SUS, like bags, are low at cost, easy to store, and safe to ship.

For other applications, [such as] sampling or bioprocess containers, it is a question of flexibility, and people forget that stainless-steel multi-use vessels require a high amount of resources—producing, repairing and maintenance, cooling, etc. 

: Ease of use, flexibility, reliable sterility, production efficiency—these are all factors to be considered. Thanks to SUT, it’s economically feasible to manufacture therapies for diseases that affect small patient populations that have the kinds of numbers that might not justify an investment in a traditional stainless-steel operation.

Modular, purpose-built SUS can be optimized for specific processes so they’re ready when needed. The production suite can be changed over quickly to accommodate the next process. There’s no downtime between production runs because SUS don’t require cleaning or equipment requalification in between batches. The ability of SUS to eliminate the risk of cross-contamination between processes is significant. All of this contributes to more streamlined operations.

Also, SUT is the most cost-effective way for manufacturers to make a wide range of drugs from vaccines, to mAbs [monoclonal antibodies], to cell and gene therapies.

SUS are priced more affordably than traditional equipment. However, SUS require bags and connectors that will generate consumable costs over the life of each process. The cost of consumables and security of supply is very important to consider. Working with trusted suppliers that have taken steps to secure sourcing is paramount in today’s market.

It is, of course, important to consider batch size and the number of batches that will be needed per year. With size constraints usually hovering around 2000 L, any high-volume drug would require a parallel processing approach. And whether the plant will be multi-product or single product is also critical.

Though it can be argued that the ability to changeover is only valuable if it is needed, it is always a smart investment. Having the flexibility, even in a single product environment, is a good backup if anything were to go wrong (e.g., clinical failure) along the way to commercialization.

What trends have you seen occurring as it pertains to single-use manufacturing? Do you anticipate them continuing for the foreseeable future, or is the end in sight?

If you looked back a decade ago, the discussion was still, ‘will this work?’ We are so far past that now, and SUS have helped to enable an era of innovation in this industry that is unmatched. We see a diverse range of modalities, which continues to grow, and a move towards more efficacious or even curative products.

The technologies needed to support the continuous high growth projections in the industry will rely on SUS in many cases. As we’ve seen through the pandemic, SUS not only enable innovation, but they allow for the type of rapid response needed for vaccines. Undoubtedly, the COVID-19 vaccines would not have been introduced and produced so quickly without SUS.

The growth may not be as high as we have seen throughout the last two years of the pandemic, but it will continue. A new product mix with an emphasis on biologicals and smaller indications will keep that demand growing over time. We also cannot ignore that SUS will be the backbone of existing processes (pending the patient population), creating sustainable demand in those areas.

Additionally, the scope of SUS use is continuing to expand, with more critical applications than ever before like formulation and final filling.

SUS will play a vital role in pharmaceutical and biopharmaceutical manufacturing and supply chain in future. The more personal medicines and low volume high potency medication (e.g., gene therapeutics, mRNA [messenger RNA], small interfering RNA, etc.) is progressing, the more important single use system will be. Challenges [such as] recycling of single-use systems will be solved in the near future and [will], therefore, significantly improve the environmental sustainability and CO2 footprints of systems used.

: Most new and upgraded contract development and manufacturing organizations, contract manufacturing organizations, and biopharma companies are incorporating single-use bioprocessing equipment. That’s the way the industry has been moving and will continue to go.

From a big-picture standpoint, it might take five to seven years from groundbreaking to a qualified facility using stainless steel. In contrast, a SUS-based facility might require two to three years. Then, after a facility is operational, SUS allow biomanufacturers to process multiple drugs simultaneously in one space or make rapid equipment changeovers between production runs without compromising sterility. That push for efficiency and modularity within and across sites is a driver.

There’s strong interest in standardizing SUT, which begins with components purchased from SUS equipment suppliers. The same equipment, standard operating procedures, and a shared supply chain create efficiency. SUS components also will need to work in automated equipment as the industry moves away from reliance on operators toward automatic processes.

Are there any unique use cases for single-use consumables, systems, etc. that you have experienced in the industry?

: The SUS component industry is catching up a bit to create solutions specifically for small-bore tubing in small-volume work—applications [such as] sampling, seed train expansion, analytical processing, buffer/media transfers, and early cell-culture processes involving shaker flasks and rocker tables.

A growing number of companies are now engaged in small-volume (< 10 L) aseptic processes, such as early-stage drug development. You’ll also see very small volumes in cell therapy, for example, where cell availability is limited or media is expensive, and in the development of small-batch autologous therapies.

In the past, making reliable sterile connections in these smaller aseptic processes was typically handled using biosafety cabinets or tube welding. But biosafety cabinets can only handle a limited number of processes at one time. Tube welding is labor intensive, and the equipment is expensive—easily $15,000 or more per welder—and takes up valuable cleanroom space. Small companies with limited resources may not have the capital to purchase tube welders.

Until recently, tube welding was the only option for creating sterile closed systems at small volumes. Now there are single-use aseptic micro-connectors designed to sterilely connect small-bore tubing, so that’s new to the industry. Aseptic connections with new micro connectors can be completed in three steps and up to four times faster than an operator using tube welding, which can require a dozen steps or more to get a successful weld.

Also, in our line of work—connection technologies—there’s never been a genderless connector that can both connect and disconnect tubing using one product in bioprocessing until this past spring. Users appreciate genderless products because they’re more convenient. They eliminate mating complexity, reduce inventory requirements, simplify system design and reduce operator error. That genderless convenience factor was taken up a notch by creating a product that connects and disconnects aseptically without the need for ancillary equipment.

: Initially the use of SUS was focused on providing a solution for media and buffer preparation and handling. Over the last decade, we can now leverage the benefit of SUS throughout the process and even into the final filling applications. The use here is very functional—we only use exactly what we need, which is another part of how we keep the volume of waste minimized.

: There is a broad application of SUS established, so you will find all (even exotic) applications of SUS in the industry.

While the broader effects of the COVID-19 pandemic have lessened in public discourse, the supply chain interruptions it brought on remain a concern. Can you discuss the impact this has had on manufacturing? How would a mass return to the just-in-time (JIT) delivery model shift the current landscape?

The industry wasn’t prepared for such a supply chain challenge. The shortages have jeopardized business continuity of major manufacturing sites apart from COVID-19.

The shortage of SUS has therefore made biopharmaceutical manufacturers reflect on their relationship with suppliers. It has shown that it is crucial to have reliable partners, to plan ahead, and to establish reliable forecasts. Suppliers can react accordingly by building stock and procuring it early. Manufacturers and suppliers need a common understanding and common standards across the industry with respect to equipment and consumable design, as this enhances delivery speed.

A JIT delivery model in the pharmaceutical industry needs suppliers who rethink supplier relationships by offering solutions [such as] supplier agnostic solutions for flexibility or various business continuity measures to allow fast delivery times.

From a supplier perspective, it has driven us to significantly increase our manufacturing footprint and take deeper steps internally to secure our supply chain. Just about a year ago, Danaher announced an investment of $1.5 billion into the Cytiva and Pall lines of business (2). Over $300 million of that has been dedicated to strengthening the SUS line of business. Even as we start to realize some of the benefits of these investments, we remain committed to honing an agile approach to business processes standardization in parallel with our daily activities.

There will be a tradeoff for every approach. JIT will be largely dependent on the capability of biomanufacturers to produce accurate forecasts. While this can be achieved to a certain level with commercial products, it will remain very difficult for clinical manufacturing. Therefore, JIT delivery will be a combination of demand and how much some form of standardization is achieved, together with improved forecasting practices. It will still take some time to reach that level after the pandemic. The goal is really to more reliable and reproducible lead times, regardless of the model.

: From a supply-chain perspective, there’s no question that COVID-19 has disrupted everything from obtaining raw materials used to create components to the ability to ship sterilized equipment in a timely way. This isn’t exclusive to SUS, but it’s had an impact throughout the supply chain.

Supply-chain considerations are especially important when selecting suppliers of single-use assemblies. That’s because no supplier of single-use assemblies manufactures all of the products used in that assembly. For example, the manufacturer of a single-use mixer bag probably sourced the aseptic connectors, tubing, tubing clamps, T or Y connectors, filter capsules, etc., from other companies. Each of those component suppliers has a supply chain that may comprise sub-component suppliers, external assemblers, raw material suppliers, contract manufacturers, and so on. You can see that the SUS supply chain is multi-tiered and complex; the ability of the SUS manufacturer to supply product is 100% dependent upon the robustness of the entire supply chain.

Enhancing Sustainability in The Pharma industry

Cytiva. Cytiva and Pall Corporation Investing 1.5 Billion USD Over Two Years to Meet Growing Demand for Biotechnology Solutions. Press Release, July 27, 2021.

When referring to this article, please cite it as Playter, G. Weighing the Benefits of Single-Use Consumables. 

Manufacturing

Experts outline the benefits of single-use consumables relative to traditional approaches.

Weighing the Benefits of Single-Use Consumables

Analyzing extractables and leachables (E&L) is paramount to ensuring the safety of a drug product and determining any toxicological risks. Examining extractables can help identify potential risks of leachables contaminating or affecting the quality of a drug product. Extractables can be extracted through the use of solvents under certain extraction conditions or can be forced out with high heat without solvents. Extractables may be potential leachables, but not all extractables leach into drug products. Leachables migrate from the container into the drug product or directly to the patients under normal storage or use (1).

Potential sources of leachables include primary packaging components, secondary packaging components, intermediate manufacturing components, or APIs. There are also E&L that can be introduced from the component molding process and can end up becoming a leachable, such as mold release agents or lubricants (1).

Various regulatory guidelines have been published due to the potential safety risks that leachables pose. FDA published a guidance included in the 

 Title 21 that highlights guidelines on drug product containers and closures, such as:

Considering the differences between small- and large-molecule drug products can help determine analytical testing methods for E&L. 

BioPharm International, July 2023

Automation Aids Cell and Gene Therapy Production

Small-Scale Syringe and Vial-Filling Capability for Development of Robust Filling Processes, Smooth Tech Transfers, and to Resolve Manufacturing Issues: Summary of Key Learnings

State of Producer Cell Development for Lentiviral Vector Manufacturing

Exploring How CPC Progresses Green Chemistry Practices

Weighing the Benefits of Single-Use Consumables

Considerations for Analytical Testing for E&L

Drug Shortages Continue to Plague FDA and Manufacturers

The Inspection Result: Warning Letters and Form 483s

Innovation Idolatry

Drug Pricing’s Static Changes