Benefiting from Single-Use Tech Downstream

Single-use technologies (SUTs) offer many advantages in biologics manufacturing, both for upstream and downstream processing. They provide flexibility to quickly scale out, scale up, or scale down to accommodate rapid changes in production volume requirements; reduced downtime needed for cleaning, sterilization, and maintenance; and customization for specific production processes. Initially, more efforts were focused on the development of single-use bioreactors, which translated to single-use bags for various downstream applications. Filters were the next ideal targets, whereas today there are disposable solutions for most downstream unit operations, with continued innovation and introduction of new solutions to meet the processing needs for different steps, different modalities, and different development phases.

Widening adoption for some downstream operations

Single-use technologies are becoming more widely adopted into downstream, whether it be single-use systems or single-use purification technologies, according to Christopher Nieder, director of viral-vector downstream process development at SK pharmteco. Generally, though, use of disposable technologies occurs on a case-by-case basis and are determined after considering economic factors.

With the increased focus on flexibility in the industry, speed to clinic, and preference for closed systems processing, the application of single-use systems has increased across all modalities, adds Mardhani Aparajithan, associate director of manufacturing science and technology (MSAT) at SK pharmteco. “However,” she notes, “specific process requirements and manufacturing considerations (i.e., viral clearance validation, membrane integrity, scalability) should be considered for each modality before implementation.”

The operations seeing the widest use of SUTs include those involving filtration, because, observes Marc Noverraz, process technology consultant for viral-based therapeutics at Sartorius, filtration media, by definition, are not reused like other consumables, such as chromatography resins, and the equipment setup is of low complexity. Fluid mixing and storage (such as for buffer preparation) is also commonly performed using SUTs at small- to medium-scale.

Applications that have seen lower levels of SUT adoption include those for which disposable systems have not yet been able to match the performance of stainless-steel. That includes centrifugation and high-performance liquid chromatography, which must require that equipment withstand high accelerations or pressures, according to Noverraz.

There are few single-use continuous centrifuge options on the market specifically designed for mammalian cell culture, and they are not very efficient for microbial cell separation, agrees Tibor Nagy, director of bioprocess strategy and development at FUJIFILM Diosynth Biotechnologies. “Solutions are just starting to infiltrate large SUT monoclonal antibody (mAb) processes due to the fact that depth filtration as a harvest step is not feasible or economical at the 4000 L to 6000-L scale,” he says. “SUTs for ultracentrifugation,” Nagy adds, “which is generally performed on the small scale in viral-vector manufacturing, has technical challenges as well.” Adam Goldstein, senior director of R&D innovation in the Bioproduction Group at Thermo Fisher Scientific, notes that recent launches of single-use centrifuges are now starting to show promise at the 500-L+ scale and are commercially available.

For chromatography operations, meanwhile, adoption levels are mixed, says Lalit Saxena, senior director of MSAT at Samsung Biologics. He attributes this fact to the technical challenges to scaling, issues with the device size availability per batch size, and operational costs.

While Weichang Zhou, CTO and president of global biologics development and operations at WuXi Biologics, notes that disposable single-use anion-exchange and hydrophobic interaction chromatography membranes are used for a small number of projects that allow for flow-through mode, and a newly launched single-use Protein A membrane is currently under evaluation. He points to the low binding capacity and high cost of these SUTs compared to reusable resins as being major hindrances. “Additional efforts are required to reduce the cost of these technologies to be competitive,” Zhou comments.

Not everyone agrees, however. Goldstein points to excellent, scalable prepacked columns up to 60-cm in diameter from at least two suppliers.

Saxena suggests, however, that the limitations with scale up for more than 5-L membrane adsorbers and 80-cm prepacked columns can mean that process scientists need to develop an alternative approach when moving to large-scale manufacturing.

In addition to chromatography, Aparajithan notes that SUTs for large-scale tangential-flow filtration (TFF) may have reduced implementation due to specific technical challenges associated with scalability and pressure limitations. Here again, Goldstein takes a different view, noting that today single-use solutions for ultrafiltration/diafiltration (UF/DF) up to the 2 x 2000-L scale are quite capable.

Looking at different modalities, stainless-steel is more broadly adopted for large-scale, established antibody processes in late development phases or commercialized, according to Nieder. “Dedicated facilities with dedicated hardware make economic sense for these products,” he explains. Disposable solutions are, however, well-developed for smaller-scale mAb platform processes, but less advanced for microbial modalities, according to Nagy.

SUTs, meanwhile, are frequently used for more recently developed modalities, such as viral vectors and cell therapies, Noverraz observes. Antibody-drug conjugates are also often processed using SUTs, says Saxena, where closed-containment devices such as powder handling and single-use transfer manifolds help minimize the risk of operator exposure and cross-contamination.

In fact, Goldstein believes that given the specific advantages afforded by SUTs, they have been shown to be beneficial and cost-effective in multi-product facilities and in instances where process flexibility, in terms of volumes and product requirements, is required.

Finally, Saxena highlights the fact that SUTs are facilitating advances in strategies and applications for continuous and connected manufacturing concepts.

Advances in SUTs for harvest/clarification

Harvest clarification via depth filtration has been the most common form of clarification for decades across all modalities. Depth filters are designed as high-capacity dead-end filters for removing cells, cellular debris, and process impurities and generally easy to use. Advances in high-performance depth filtration as well as in-line monitoring systems have offered customers the ability to increase operational efficiency, improve product quality and productivity, and enhance scalability, according to Aparajithan.

“Efficient removal of impurities such as host cell proteins and DNA, availability of small-scale development systems with corresponding large-scale systems for clinical and commercial manufacturing, and increased productivity with decreased processing times are a few examples of typical performance gaps addressed by the advances in high-performance depth filtration systems,” Aparajithan says.

Advances have been made in both device formats and filtration media, according to Saxena. For instance, increased filter throughputs and capacities are being achieved using different membrane materials, while encapsulated contained devices are reaching the market. There also has been progress in reducing the content of extractables and β-glucan impurities of single-use depth filters, he observes.

In addition, Thomas Page, senior VP of small-scale operations and process technology at FUJIFILM Diosynth Biotechnologies, comments that improvements in single-use centrifuge technology have made it possible to reduce the area needed for subsequent depth-filtration area, which in turn is enabling higher-volume upstream mammalian cell-culture processes.

Goldstein adds that the use of single-use centrifuges in combination with depth filtration increases efficiency and reduces filter and liquid waste relative to traditional depth filtration alone. “The resulting raw material and equipment reduction can reduce the need for extra manufacturing site space and warehouse space, which contributes to reduced environmental impacts,” he states.

Saxena disagrees, however; he believes there is still much room for further improvement before single-use centrifuges will be used as the primary choice for clarification. “There is a desire in the industry to use pre-sterilized, single-use centrifuges for some large, production-scale operations, but the mere geometric scale-up of existing smaller designs has not been successful beyond a processing rate of one to two liters-per-minute. As a result, the technical limitations inherent in these existing smaller designs make a direct geometric scale-up approach one of the major restraints for deployment of single-use centrifuge systems beyond the 2000-liter scale,” he explains.

A new technology gaining traction for viral-vector manufacturing is tangential flow depth filtration (TFDF), according to Nieder. It uses a TFF-style cassette with a filter thickness akin to a depth filter and is operated in a TFF mode. “The growing interest in TFDF in the viral-vector world is due to its potential for high capacity and high recovery by sweeping of potential foulants off of the filter membrane combined with the use of non-adsorptive materials of construction,” he explains.

Meanwhile, an important trend in the industry noted by Paul Cashen, process technology consultant for separation technologies with Sartorius, is an an increasing demand for clarification media that can be provided completely closed and gamma-sterile and more synthetic or chemically defined media that do not contain any additives. He believes both gaps are particularly important for cell and gene therapies, and thus anticipates next advances in single-use harvest/clarification solutions in the short to medium term will focus on addressing these needs.

SUTs for UF/DF prior to chromatography

UF/DF typically is performed using TFF, with single-pass TFF being adopted for continuous processing. This approach involves lower flow rates, requires less expensive pumps and tubing, reduces exposure of drug substances to shear, and eliminates the recirculation loop, decreasing risk of aggregation and lowering bioburden, according to Page.

Next-generation TFF systems, observes Cashen, promote lower hold-up volumes, lower processing volumes, more options for closed processing, and greater flexibility. He believes these advances can be largely attributed to the increasing prevalence of advances in the manufacturing space, such as messenger RNA (mRNA) and gene therapies, which generally have lower manufacturing volumes compared to traditional proteins and a greater desire/need for closed processing. He also notes that TFDF is attracting interest for UF/DF applications as well.

Nieder adds that TFF prior to chromatography is more often used for viral vectors due to the lack of available vector-specific affinity resins for removing impurities and/or the need to reduce the load volume onto the chromatography column. At larger scales, he notes that the larger filter area means that traditional modular cassettes installed in stainless-steel holders with single-use product-contact components often make the most sense. For smaller production scales with low filter installation areas, meanwhile, single-use encapsulated TFF cassettes are an option and offer the benefit of being pre-sterilized, which saves cost and time. In addition, Nieder notes that most encapsulated cassettes are designed to scale to traditional cassette format, providing flexibility during process development.

Specific advances in SUTs for TFF prior to chromatography of note to Aparajithan include implementation of semi- and fully automated TFF systems with integrated single-use sensors for streamline operations through real-time continuous data monitoring and control of key parameters such as transmembrane pressure (TMP), flux, and conductivity. She also points to the development of high-flux membranes, which Aparajithan says contribute to addressing performance gaps in productivity, product recovery, and scalability.

SUT solutions for chromatography

Chromatography is a complex operation, and different modalities have unique requirements. Even within the same biomolecule class, variations in structural and physicochemical properties can lead to the need for different buffer and resin/membrane/monolith chemistries.

Advances in single-use chromatography systems are helping to simplify some aspects of chromatography, at least for R&D and clinical manufacturing applications, with larger solutions under development.

Advances in pre-packed columns have led to the introduction of high-capacity single-use solutions which offer increased binding capacity and enhanced resolution, according to Aparajithan. Pre-packed columns also allow for a reduced footprint in manufacturing and minimize the set-up time, she notes.

Membrane adsorbers and other non-resin-based solutions are attracting more attention than disposable column chromatography technologies, though. “While resin-based chromatography has been seen as the standard for chromatography, the desire for process intensification for mAbs to increase productivity, combined with the increasing popularity and prevalence of gene, mRNA, and cell therapies at manufacturing scale has really opened the door for different chromatography technologies to find their place,” Cashen contends. For mAbs, membrane- and fiber-based Protein-A chromatography devices, when combined with rapid cycling chromatography, promote faster process times and higher productivity compared to resin-based chromatography, Cashen says.

That is possible, Yifeng Li, head of downstream process development at WuXi Biologics, adds because while the binding capacities of Protein A membranes are slightly lower than those of Protein A resins, the residence times are extremely short, and multiple cycles can be conducted in a relatively short time. As a result, the full lifetime of the membrane can be used in a single batch, reducing the purification cost for clinical manufacturing, according to Li.

Goldstein also notes that high-flow-rate resins are being developed that greatly reduce the residence time on the column, allowing for quicker turn-around of equipment.

Advances that enable cleaning and cleaning validation are key to making it possible to use membrane adsorbers for multiple cycles, observes Saxena. “There is a technical challenge presented by the nonlinear flow paths found in membrane adsorbers, however. More complex hydrodynamic models are needed to better understand the residence time distribution, before it will be possible to realize practical large-scale membrane adsorbers,” he explains. Saxena adds that as a result, these SUTs are mostly used in small- and pilot-scale plants and very rarely observed in applications greater than 5000 liters.

For viral vectors and mRNA vaccines, advances in affinity technology and the development of new membranes and monoliths tailored specifically for these modalities are beginning to raise the standard of purification by improving product recovery and quality, notes Cashen.

Nieder adds that while affinity resins have been on the market for adeno-associated viral vector purification, there remains a need for affinity resins for adenoviral and lentiviral vectors, for which often the primary purification step involves the use of non-optimal, bead-based ion exchange resins. He does note, though, that single-use monoliths are now available in a variety of chemistries and porosities that are potentially better-suited for primary and secondary purification of viral vectors, as well as the separation of full and empty capsids, while also having the added benefit of short processing times since the interaction with the ligand is not dependent on diffusion.

Finally, Cashen observes that an increase in the desire for intensified processing combined with greater regulatory understanding of continuous processing over the past couple of years has led to greater focus on chromatography systems that can enable more advanced forms of purification, such as multi-column chromatography. “With increasingly more options coming to the table, manufacturers can make decisions on what chromatography formats best fit their process, rather than having to brute force a specific product into their process that may yield sub-optimal performance,” he concludes.

Gaps in SUTs for TFF prior to fill/finish

Single-use UF/DF membranes typically have lower costs than reusable systems, and thus help reduce raw material costs, according to Hang Zhou, head of bioprocess research and development of WuXi Biologics. “It is preferable,” he adds, “to use single-use UF/DF membranes at early development stages and change to reusable ones suitable for large-scale processing when multiple good manufacturing practice (GMP) batches are required, as the switch should be straightforward without any need for additional development.” Goldstein finds, however, that there often is limited additional development needed to optimize performance at large scale.

Encapsulated single-use TFF cassettes are a significant advance that allows for sterile UF/DF operations where that option did not exist as of a few years ago, emphasizes Nieder. “The only alternative in a sterile, single-use format has been hollow fiber filters, which have a much lower mass transfer and thus longer processing times,” he says. One potential downside is reduced modularity compared to cassettes with stainless-steel holders. This issue can be addressed by stringing the encapsulated cassettes together; however, Neider points out that an increase in void volumes typically results, which can be challenging for modalities such as viral vectors that are produced in smaller process volumes at this step.

Overall, though, Aparajithan believes that the development of fully closed single-use flow paths with integrated in-line sensors and the strides made forward in disposable flat-sheet cassette and hollow-fiber technologies have been important advancements in SUTs for TFF before fill/finish. “The implementation of these gamma-irradiated flow paths and pre-sterilized flat-sheet cassettes and hollow fibers with several different options for aseptic connectors offer reduced risk of contamination, increased flexibility/ability to customize, and increased precision and process consistency by providing real-time continuous data monitoring and control of key parameters such as transmembrane pressure, flux, conductivity, and flow-path hold-up volumes,” she concludes.

Disposable sensor technologies

Advances in sensor technologies incorporated into single-use downstream processing technologies are occurring, but effective solutions remain limited and much more work is needed. Recent advances have focused on improving the accuracy, reliability, and robustness of sensors and are helping to increase the value of sensors installed in SUTs, according to Nagy. “The trend toward integrating [single use] sensors with automation and control systems is a significant process improvement that will ultimately support real-time release during continuous manufacturing,” he comments.

Zhou points to a multi-column chromatography system for continuous processing that uses a disposable flow kit equipped with single-use sensors for pressure, flow rate, conductivity, pH, and UV signatures. “This system enables extended continuous processing because it mitigates bioburden contamination risk and eliminates the need for cleaning validation,” he notes.

The increased focus on improving process monitoring/control and data integrity is not limited to continuous processes, according to Aparajithan. She points to the growing use of single-use sensors for pH, conductivity, pressure, and turbidity in various downstream unit operations. “The ability to provide real-time detection of deviations from key process parameters and respond accordingly in a timely manner is essential in increasing overall process understanding and enabling process optimization,” she states.

There are many process parameters for which single-use sensors are not yet available, however. “Real-time measurement of key quality attributes such as protein concentration or aggregation and overall data integration and correlation of process parameters to these quality attributes seem to be areas that are lacking in the industry,” says Aparajithan.

There are many more single-use sensor technologies deployed for upstream manufacturing that have not yet been proven for downstream applications, Saxena adds. “As proteins are gradually purified during downstream processing, greater analytical sensitivity and precision [are] needed to make decisions regarding process- and product-related impurities present at nano-scale concentrations. This level of capability has yet to be realized for most single-use sensor technologies. Another challenge is the lack of scalability of many single-use sensors, with few solutions available for use in large-scale equipment,” he says.

“Even fewer single-use sensors are available for detecting process parameters and product quality attributes for emerging therapies,” Saxena continues. That is particularly true for viral vectors, notes Neider, where there is a real need for real-time analytics (titer, aggregation, and %-full capsids) to drive decision-making, especially in a process development environment. He does observe that UV-based systems have been developed that can rapidly provide data, but they are not single-use sensors integrated into the processing equipment.

On a positive note, Saxena emphasizes that science is evolving every day, so it can be expected that ongoing research will lead to the introduction of additions single-use sensor technologies to the market that are designed for use in downstream unit operations. “The next step will be for manufacturers to assess the risk/benefit ratio associated with their implementation at large scale as process analytical technology tools,” he concludes.

Sustainability of SUTs

While SUTs eliminate the need for cleaning and often facilitate the implementation of continuous processing, which requires a much smaller footprint, the use of large quantities of plastics raises concerns with respect to sustainability impacts of disposable systems. This issue is being closely examined as biopharmaceutical companies seek to find means for implementing more sustainable practices. Consequently, economic and environmental considerations, as well as the specific needs and goals of a given production process, drive the selection of single-use or stainless-steel equipment, according to Goldstein.

What is needed when assessing the sustainability of SUTs is the use of a holistic approach that considers all aspects of the affected process and generates a quantitative result, such as equivalents of carbon dioxide (CO₂) per dose or batch, states Joel Eichmann, co-founder and managing director of Green Elephant Biotech.

On the positive side, Eichmann notes that in addition to not consuming water and energy for cleaning like stainless-steel equipment, SUTs in some cases allow for processing under less stringent cleanroom conditions, resulting in reduced energy consumption and less waste from gowning. Incineration of used disposable equipment does, however, lead to CO₂ emissions. They can also, Goldstein says, reduce upfront capital investments and operational costs, and thereby drive better utilization of a plant’s overall resources.

The ability of SUTs to enable process intensification is another important factor, according to Noverraz. “When intensification methods are able to decrease the size of consumables and the equipment footprint, they also decrease the size requirements of the facility housing them, which decreases the consumption of material to build it and energy to operate it,” he explains. That is important, Noverraz adds, because according to a study (1) on mAb manufacturing, electricity consumption has the greatest environmental impact.

Using standardized and comprehensive life-cycle assessment (LCA) methods such as those outlined in the ISO 14000 guidance documents, says Noverraz, is an ideal approach as they assure consideration of all aspects from “cradle to grave.” Conducting such assessment is quite complex, however, and requires extensive data that might be readily available, he notes. “Alternative strategies include focusing on the impact of the consumption of key resources (water, energy) or determining the process mass intensity (ratio of waste and product masses), which are simpler to evaluate,” he adds.

Waste disposal is of course an important issue associated with the use of SUTs, emphasizes Erin Dunn, senior manager, biosafety and EHS at FUJIFILM Diosynth Biotechnologies. “Reducing waste by employing recyclable materials and more robust flow paths that can be reused several times is an important approach to improving their sustainability. The latter would make SUTs less disposable than actually single-use,” she notes. If these two approaches are combined, calculation of sustainability benefits would then need to consider the percentage of the flow path that is recyclable and how many times or how long the material can be used, as well as the other factors outlined above.

In that vein, Aparajithan observes that manufacturers are developing new recyclable, biodegradable, or alternative materials to help reduce plastic waste and prioritizing sustainability by offering SUT recycling programs and environmentally friendly packaging. As a specific example, Eichmann highlights the use of medical-grade bioplastics such as polylactic acid that have reduced CO2 footprints. Noverraz also points to initiatives by SUT suppliers to redesign products to reduce their material use.

“SUTs have enabled real financial savings in the cost-of-goods manufactured. The increased focus on sustainability is now driving investment of some of those savings into the design, manufacturing, packaging, distribution, and disposal of SUTs to achieve further reductions of carbon footprints,” Goldstein observes. He believes that as the adoption of SUTs increases, manufacturers will continue to pursue ways to improve the economics and sustainability of bioproduction and the materials used in those processes.

Ongoing SUT innovation

While SUTs offer many advantages for downstream processing of biologics, there are some limitations that, along with the need for increased sustainability, are driving significant innovation efforts. Up-front cost considerations are often a challenge for the implementation of SUTs, according to Aparajithan, as are material expiry and sterility issues. “Complex or sensitive components specifically used near the fill/finish operations of downstream processing can provide a challenge to sterilize and can have a short shelf life,” she explains.

For some unit operations, Saxena adds, SUTs are not available and/or those that are may be limited by performance, scalability, and cost. For instance, he points to the need for affinity and multi-modal chromatography beads that are sufficiently cost-effective to be classified as single-use. Scalable pre-packed chromatography systems that can be used for commercial manufacturing, disposable sensors for real-time measurement of protein concentration, and issues with subvisible particle formation in single-use containers are others. Affinity ligands applied to membrane adsorbers or monoliths are needed for viral vector purification, Nieder adds.

Extractables and leachables remain a primary concern as well, according to Saxena. “Regulatory requirements are becoming increasingly stringent with respect to patient safety, and thus more in-depth risk assessment to implement single-use technologies for late-stage downstream unit operations is required,” he contends.

As an example of increasing regulatory expectations, Saxena highlights the extension of the scope of the Annex 1 regulatory guidance regarding drug substances in the European Union. He also notes that required compliance with United States Pharmacopeia (USP) <788> (for particulate matter) and USP <85> (for endotoxin certification) for single-use materials is creating challenges, particularly for suppliers of single-use manifolds and consumables used for fluid transfer during downstream processing. In addition, the complexity increases with customized single-use assemblies, particularly for assemblies comprising components from multiple vendors, Saxena observes.

Regulations can, however, help achieve greater standardization, says Nagy. Collaboration between biopharma manufacturers and SUT vendors will be important for driving further advances in SUT solutions, Nieder adds. “Such relationships allow sharing of information about needs and pain points, which can help guide efforts and accelerate product development timelines,” he comments.

Vendors do understand the challenges and are continuously improving to meet the needs of process scientists, Saxena observes. Recent advances in customization and modular designs of SUTs are enabling efficiency, scalability, and flexibility in downstream processing by allowing companies the ability to incorporate SUTs into current facility layouts and process requirements, adds Aparajithan. “These advances in turn help to accelerate the development and manufacturing of life-saving therapies for patients,” she concludes.

Reference

1. Budzinski, K. et al. Streamlined Life Cycle Assessment of Single Use Technologies in Biopharmaceutical Manufacture. New Biotech, 2022 68, pp. 28-36. DOI: 10.1016/j.nbt.2022.01.002

About the author

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.

Article details

BioPharm International®
Vol. 37, No. 4
April 2024
Pages: 8-13

Citation

When referring to this article, please cite it as Challener, C.A. Benefiting from Single-Use Tech Downstream. BioPharm International® 2024 37 (4).