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.

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.

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.

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.

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 (CO

) 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 CO

 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.

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 

 <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.

1. Budzinski, K. et al. Streamlined Life Cycle Assessment of Single Use Technologies in Biopharmaceutical Manufacture. 

 68, pp. 28-36. DOI: 10.1016/j.nbt.2022.01.002 

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

When referring to this article, please cite it as Challener, C.A. Benefiting from Single-Use Tech Downstream. 

Articles

Cover Story

Increases in efficiencies, flexibility, scalability, and sustainability are impacting adoption.

Benefiting from Single-Use Tech Downstream 

In the rapidly evolving landscape of healthcare, nucleic acid therapeutics have emerged as a promising frontier. These innovative treatments, which include modalities such as antisense oligonucleotides, messenger RNAs (mRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, and gene therapies, have the potential to address a wide range of diseases, from rare and infectious diseases to cancers (1), and offer hope for many patients worldwide.

Each modality presents unique mechanisms of action and therapeutic potential, broadening the scope of diseases that can be targeted. However, the journey from concept to clinic is fraught with challenges, particularly in the areas of manufacturing, regulatory compliance, and technological innovation.

Ensuring GMP compliance and regulatory adherence are paramount in the manufacture of any medicine. When considering a nucleic acid therapeutic, the intricate nature of manufacturing processes and the high standards required to produce safe and effective therapies only emphasize this importance. GMP guidelines provide a framework for maintaining consistency, quality, and safety throughout the manufacturing process, mitigating risks of contamination, variability, and product failure. Regulatory requirements further ensure that nucleic acid therapeutics meet these stringent standards before they can be brought to market.

Navigating the intricate landscape of nucleic acid manufacturing regulations, guidelines, and best practices requires specialist knowledge and expertise. Partnering with a knowledgeable contract development and manufacturing organization (CDMO) specializing in nucleic acid therapeutics becomes imperative. An experienced CDMO partner brings a thorough understanding of GMP compliance and regulatory affairs with which to offer guidance and support throughout the manufacturing process. A CDMO’s expertise ensures that all regulatory requirements are met, which minimizes the risk of delays, rejections, or compliance issues that could hinder the progress of nucleic acid therapeutic development.

The core of GMP lies in ensuring accountability, documentation, and traceability throughout the manufacturing process. This involves meticulously documenting who performs each task and tracking all materials and manipulation steps involved. For example, when producing mRNA therapeutics, adherence to GMP principles remains critical, mirroring the requirements for any drug product.

However, certain aspects of GMP, particularly regarding DNA starting material, may require interpretation due to the lack of clear guidance in both the United States and Europe. The industry currently relies on a risk-based approach outlined in a question and answer series provided by the European Medicines Agency (EMA) (2). While this resource provides a starting point, further clarification is needed.

The principles of GMP revolve around the philosophy of work execution, while regulatory guidance provides the framework for compliance. Within this framework, quality systems follow the attributable, legible, contemporaneous, original, and accurate principles, emphasizing the importance of knowing who does what, when, and with what, and ensuring comprehensive documentation.

Regulatory requirements dictate the systems necessary to meet these principles, including the design of risk-based approaches and the identification and control of risks throughout the manufacturing process. This extends to demonstrating process control for the manufacturing process itself, identifying critical process parameters and quality attributes, and defining parameters to maintain during drug manufacturing.

In essence, GMP principles serve as the philosophy guiding work execution. At the same time, regulatory requirements define the systems and approaches necessary for compliance—essential to ensuring the quality, safety, and efficacy of nucleic acid therapeutics.

Challenges and strategies to maintain compliance

The nucleic acid therapeutic manufacturing landscape presents specific challenges in maintaining GMP compliance and meeting broader regulatory expectations. One of the primary challenges lies in the manual processes traditionally used for mRNA and encapsulated RNA production, leading to human error and potential contamination. To address this, leveraging automated robotic equipment, minimizes human intervention, reduces errors, and mitigates contamination risks.

A significant industry-wide challenge in the mRNA space pertains to analytics, particularly regarding double-stranded RNA. Understanding the immunogenicity and tolerance levels of double-stranded RNA for different indications remains a hurdle. The current variability in analytical tests, ranging from characterized antibodies to rudimentary dot plot assays, further complicates the situation. To overcome these challenges, industry efforts are focused on enhancing analytics for double-stranded RNA.

Best practices for ensuring GMP compliance revolve around automation to minimize human interaction with the product. By removing or reducing human intervention, the risk of errors and contamination is significantly reduced. Additionally, traditional mRNA production involves scaling up in volume, which poses complexities due to non-linear scaling of parameters such as shear and thermodynamics.

An ideal approach streamlines this process by maintaining a set volume and process. An effective strategy for this is scaling out using robotic parallel reactions to maintain consistency in production regardless of batch size. This simplified approach streamlines production and simplifies the chemistry, manufacturing, and controls process by keeping process parameters consistent across different material volumes.

Investment in novel and innovative technologies is pivotal in enhancing the efficiency, scalability, and quality of nucleic acid therapeutics manufacturing. By leveraging these technologies, such as continuous, optimized, and high-yield manufacturing platforms, significant improvements are observed across various aspects of the manufacturing process.

Firstly, these innovative technologies enable more concentrated reactions with higher yields, leading to a decrease in costs of goods by efficiently converting raw ingredients into final products. Additionally, the reduction in manipulation time and faster reaction rates further contribute to cost savings, as time is a critical factor in manufacturing operations.

The integration of continuous manufacturing platforms allows for decentralized manufacturing, as the set process and equipment can be deployed at any global location to achieve consistent results. This flexibility not only enhances efficiency but also opens doors to global collaboration and market access.

Although cost reduction is a significant benefit of these technologies, their impact extends beyond financial metrics. Consistency of the product is also enhanced, as the process remains standardized and repeatable. This not only ensures product quality but also minimizes the need for expensive rework or adjustments.

In addition to these tangible benefits, there is a theoretical potential to reduce raw material costs for the actual in-vitro transcription process. The efficiency of the manufacturing process means that less starting material is required, leading to potential savings in raw material expenses. As time goes by and continuous manufacturing programs are more widely adopted, there is potential to collect a greater amount of real-world data to add support to this hypothesis, presenting a promising avenue for further exploration and optimization.

Furthermore, it is crucial to highlight other pertinent technologies in the field of nucleic acid manufacturing. Several groups and companies are actively working on similar aspects of RNA manufacturing, utilizing innovative approaches and automation. For instance, similar continuous manufacturing platforms are being developed, with pioneering microfluidics-based approaches for RNA production representing one of these exciting technologies. Additionally, academic groups worldwide are also exploring automated equipment to streamline nucleic acid manufacturing processes, showcasing the collaborative efforts driving innovation in the industry.

Navigating the complex regulatory landscape to ensure compliance with diverse global, regional, and industry-specific regulations is essential. Companies with dedicated expertise in these areas play a pivotal role in guiding regulatory compliance across markets such as the US, Europe, Japan, and others. A CDMO that extends this expertise as a service to clients can be critical to project success, assisting companies in meeting regulatory requirements and facilitating regulatory filings.

This service is particularly crucial for drug developers who may lack extensive quality teams or experience in navigating regulatory complexities. By offering regulatory assistance, clients are free and empowered to focus on bringing their therapeutics to patients efficiently. Whether it’s providing documentation support for regulatory filings or representing clients at regulatory meetings, streamlining the regulatory process and accelerating the path to market are vital facets to remaining competitive in a modern market.

Part of an industry-wide effort to ease the pathway of medicines to approval is seen in international harmonization. The role of international harmonization efforts in shaping GMP standards and regulatory frameworks holds immense significance in nucleic acid therapeutic development and commercialization. Diseases and conditions, including cancer and infectious diseases such as coronaviruses, transcend borders, thus necessitating a streamlined regulatory approach. Harmonizing regulations allows for a more straightforward path to bringing therapeutics to market, ensuring consistency in defining indications and manufacturing techniques across different regions.

In recent discussions at mRNA meetings convened by both FDA and EMA, there has been a concerted effort to integrate and align regulatory approaches globally (3). The goal is to establish a unified set of rules that can be applied on a global scale, fostering a conducive environment for the development and commercialization of nucleic acid therapeutics.

Effectively integrating novel technologies

As pioneers in nucleic acid therapeutic manufacturing, it is important to lead the charge in effectively integrating novel technologies—all while upholding stringent regulatory standards. Specialized companies dedicated to advancing nucleic acid technologies are pivotal in bringing innovative solutions to the market, with exciting advances such as continuous manufacturing laying the groundwork for faster mRNA generation with high quality.

Although these advancements are impressive, it is essential to recognize that the ultimate goal is to make biologics available and affordable to all patients in need. Therefore, alongside the pursuit of cutting-edge technologies, there is a critical need for services aimed at ensuring the accessibility and affordability of these therapies. By combining innovation with a commitment to equitable access, the industry can revolutionize healthcare and improve patient outcomes on a global scale.

A foundational approach to achieving this integration lies in adopting a quality-by-design (QbD) methodology. By embracing QbD principles, manufacturers can ensure that novel technologies are seamlessly incorporated into the manufacturing process without compromising regulatory standards. This includes rigorous compliance with regulations such as 21 Code of Federal Regulations Part 11, which governs the use of software in manufacturing processes, ensuring robust control and governance over electronic records and data acquisition. Features such as access control, version control, and audit trails are vital components in maintaining the reliability and integrity of electronic records, ensuring they meet or exceed the standards set for traditional paper records.

Moreover, the implementation of a design of experiments (DoE) approach further strengthens the integration of novel technologies. Through DoE, critical factors in the manufacturing process are identified and optimized, ensuring that the benefits of new technologies are maximized while adhering to regulatory requirements. This systematic approach not only facilitates the seamless integration of novel technologies but also ensures that regulatory considerations are embedded into the development process from the outset.

With the continual approval of new nucleic acid therapeutics globally, regulatory standards are becoming increasingly stringent, necessitating industry-wide adherence. Regulatory agencies emphasize the control of critical process parameters and demand additional robustness data for analytical assays. Notably, a trend toward standardization of quality control assays is evident, particularly in advancements in the double-stranded RNA assay. To address assay variability, companies explore innovative solutions such as manufacturing internal standards and fostering consistency in assay results across different sites.

Proactive preparation for evolving regulatory requirements is crucial for industry stakeholders. Demonstrating unwavering commitment to consistency and adherence to standards through standardized, scalable processes enhances regulatory confidence and streamlines compliance efforts. Moreover, engagement with stakeholders, including regulatory bodies, during the early stages of product development is essential. Open communication and knowledge-sharing about emerging techniques facilitate collaborative efforts to address potential concerns and pave the way for smoother regulatory approval processes.

Looking ahead, the future of nucleic acid therapeutic manufacturing holds great promise, fueled by advancements in GMP compliance, technological innovation, and regulatory success. As the industry continues to navigate the complexities of this evolving landscape, it is clear that collaboration between industry stakeholders, along with expert guidance from specialized CDMOs, will be crucial in driving forward innovation and ensuring the success of nucleic acid therapeutics in improving patient outcomes.

Furthermore, the role of technological innovation in enhancing efficiency, scalability, and quality in nucleic acid manufacturing is essential. Continuous advancements in technology, including optimized and high-yield manufacturing platforms, are driving innovation and setting new standards in the industry.

By remaining committed to GMP compliance principles in achieving regulatory success, and embracing technological innovation, the future of nucleic acid therapeutic manufacturing is bright and poised to revolutionize healthcare.

1. ASGCT. Gene, Cell, + RNA Therapy Landscape Report: Q4 2023 Quarterly Data Report. ASGCT, 2023.
2. EMA. EMA/246400/2021, Questions and Answers on the Principles of GMP for the Manufacturing of Starting Materials of Biological Origin Used to Transfer Genetic Material for the Manufacturing of ATMPs (Feb. 24, 2021).
3. EMA. Regulatory and Scientific Virtual Conference on RNA-based Medicines (Feb. 3, 2023).

Patrick Thiavilleis chief technology officer of Exothera.


Volume 37, No.4
April 2024
Pages: 14–16, 24

When referring to this article, please cite it as Thiaville, P. Driving Innovation in Nucleic Acid Therapeutics. 

Development

Innovation in this space depends on strategizing for GMP compliance and market access.

Driving Innovation in Nucleic Acid Therapeutics

The changeover to single-use bioreactors has become more common in biomanufacturing; however, stainless-steel bioreactors still have their place. Stainless-steel bioreactors are important for certain monoclonal antibody (mAb) products that depend on bulk manufacturing, for instance.

Although single-use technologies (SUTs) are becoming increasingly popular in biomanufacturing, stainless steel is expected to remain in use for some time, especially for universal blockbusters such as treatments for cancer, diabetes, and others in which large volume is required, says Luis Velez, executive vice-president, US Operations, AGC Biologics. Furthermore, while the focus is often on mAb production, the plasma and vaccine industries are likely to continue adopting hybrid facilities where stainless steel carries out many core purification operations, but where SUT can increase flexibility and efficiency in the bioreactor and fluid management operations, Velez explains.

Although single-use adoption is not absolute, most companies are not overhauling their facilities from stainless-steel to single-use, notes Lorenz Hasler, vice-president Global Manufacturing, Science, and Technology, KBI Biopharma. “Instead, there seems to be a sweet spot, depending on the demand, flexibility, and scales that we’ve seen really fit well leveraging the advantages that single-use technologies can bring, particularly on the front-end of product lifecycles,” he says.

In considering the cost effectiveness of building a new single-use bioreactor facility versus converting a stainless-steel facility into a single-use facility, it is generally more cost-effective to build a new (or start with) a single-use facility, rather than convert a stainless-steel facility, says Hasler. “With the cost of construction, commissioning, and automation, it often doesn’t make financial sense to convert an entire facility. It’s more likely for someone to recognize the benefits single-use can bring in addition to existing stainless-steel capability in their network,” he points out.

“The benefits of SUTs compared to stainless-steel have been compared in economic models, specifically in terms of total cost of ownership and consumables, as well as facility buildout,” explains John P. Puglia, PhD, senior director of Research and Development in Thermo Fisher Scientific’s bioprocessing business focusing on single-use technologies. “Hardware for SUTs is significantly less expensive than stainless-steel, and the lower upfront capital expenses of single-use facilities provide economic flexibility that stainless-steel facilities cannot offer when looking at total bioproduction capacity and molecule pipeline approvals.”

Build-to-suit bioproduction facilities can take several years to design, construct, and meet quality control guidelines. Single-use facilities boast the benefits of shorter facility development and deployment timeframes, Puglia observes.

Another factor to take into consideration for biomanufacturers when approaching final capacity demand is continuous product and process improvements, Puglia continues. “Because stainless-steel facilities must be built-to-suit at maximum capacity, this can lead to complete standardization of processes regardless of running from hundreds to thousands of kg/yr demand. And while standardization can be beneficial to consistency of product, this could create inefficiencies when considering advancements in bioprocess operations down the line,” he explains.

Thus, if a biomanufacturer is considering new technologies aimed at reducing costs or increasing productivity or yield, they may encounter difficulties in implementing these technologies in an established stainless-steel facility. As molecule demand increases and new single-use facilities are constructed, however, it will be become easier to implement new technologies, Puglia adds.

Meanwhile, Velez notes that, from his perspective, the cost effectiveness of single-use facilities versus stainless-steel facilities will be directly related to the product(s) to be manufactured and the annual capacities. “Manufacturers will also continue to leverage existing facilities and inherent expertise at these sites to manufacture new therapies in which single-use bioreactors could fit very well,” he states.

“Rather than convert a stainless-steel facility, single-use bioreactors can be flexible to scale and to fit each process,” adds Shunsuke Shiina, PhD, principal downstream development scientist, AGC Biologics R&D Center, who notes that single-use bioreactors can also reduce costs for utilities and operations related to cleaning and sterilization.

With the trend toward single-use bioreactors growing more common in the biomanufacturing sector today than a decade ago, is there still a strong argument for retaining large-scale, stainless-steel bioreactor facilities?

Puglia notes that, “with sizeable patient populations and demand for large product volumes, commercial-scale manufacturing via stainless-steel bioproduction has dominated the market, even after the first single-use bioreactors emerged in the late 1990s. Stainless-steel is a well-established method for large-scale biopharmaceutical production.”

Puglia goes on to say that, because of their size, stainless-steel bioreactors can support large-scale commercial manufacturers across a variety of higher volumes, including those between 10,000 L and 20,000 L. “Though stainless-steel has higher upfront cost, if you have a blockbuster drug, it could provide more long-term savings because of its durability,” he remarks.

Velez adds that, “Indeed, large-scale stainless-steel bioreactor facilities represent a solid, efficient option to manufacture large-scale volume products. That is one of the unique benefits of large-scale stainless-steel manufacturing to commercial needs.”

Hasler agrees with the sentiment of retaining stainless-steel bioreactors, saying that, “Depending on the demand and needs of your customer base, I would say there is an argument to retain large-scale stainless-steel bioreactors, if that’s what you have, especially at larger scales.” For example, Hasler explains, “with high demand of primarily large-scale commercial products, it may make more sense to retain stainless-steel bioreactors, from a capital expenditure perspective, given the number of batches required for [for instance] biosimilars.”

Hasler emphasizes, however, that each organization needs to determine where the inflection point is regarding their specific product portfolio to determine the most appropriate technology.

In the years to come, stainless-steel bioreactors still have a role to play. For example, Velez also points out that biosimilar products will be more popular in a high-demand market; therefore, large-scale stainless-steel bioreactors could be more suitable than multi-single-use bioreactors.

Shiina, however, says that it is likely that stainless-steel bioreactors will continue to be used for products that are already on the market (including biosimilars), as these products require production on a large scale. “Stainless-steel is also effective for development in which future demand and scale can be predicted (particularly those that need larger scales and yields),” Shiina adds.

“The processes being employed in the future will depend as much on economic benefit versus process risk and control,” says Puglia. “Obviously, practical deployment of a single-use system becomes more difficult beyond 10,000-L batch sizes, but with the regulatory demands for contamination and process control, the risk mitigation by using smaller single-use systems may outpace the economic advantages of larger stainless-steel systems.”

Considering the different modalities of biotherapeutics in development today, determining the correct path forward will be crucial.

Deciding between single-use and stainless-steel is unique to each biomanufacturer’s situation, Puglia emphasizes. “Factors to consider when deciding between the two types of technologies include molecule type, titer range, cell density, demand stage (pre-clinical, early-phase, late-phase clinical, commercial, etc.), patient population, quantity of product, single-product versus multi-product facility, and new versus existing facility. Other areas to consider are run rate and the number of molecules to change at the site, including tech transfer and downtime for the site involved,” he states.

Puglia notes that SUTs are ideal for multi-product facilities and instances where process flexibility, in terms of volumes and product requirements, is vital. “This includes R&D, which is not always planning for the commercialization stage. With benefits in flexibility, scalability, and overall performance, coupled with reduced water consumption and facility energy usage, SUTs are the clear option for biomanufacturers who are looking to increase yield, improve efficiencies, and make strides toward achieving ESG [environmental, social, and governance] goals,” he explains. Puglia also points out the fact that single-use bioreactors have been shown to reduce cost of consumables by 37% compared to stainless-steel (1).

Velez emphasizes the flexibility of single-use bioreactors, noting that SUT and related materials allows for one to change the configuration of a manufacturing line, exchange or add unit operations, and cover various scales of operation—adding flexibility for developers at early stages. “Flexibility helps if you are seeking to build clinical manufacturing programs and want the flexibility to hold off on scaling drug products in early development stages until demand is well understood, saving internal resources, time, and money. The benefits also extend to later stages—developers can scale out based on need and demand,” he states.

Velez adds that stainless-steel bioreactors are also effective and, as noted previously, are a powerful technology as the scenario moves into larger scales and later phases, particularly as manufacturers look for commercial-level production.

Shiina points to the production of vaccines, which, he notes, need to be flexible to respond to pandemics and changing circumstances, and the production of cell and gene therapy products. These are areas that will potentially drive more single-use market growth.

“Beyond bioreactors, even the well-established monoclonal antibody platform processes can benefit from modernized single-use technologies,” Shiina adds. For example, an R&D team at AGC Biologics has been working to scale up and implement a new disposable membrane-based protein A capture device for antibody purification, Shiina says. “By leveraging these new single-use protein A purification devices … one can reduce the cost of the protein A purification step, remove storage and bioburden challenges, condense the overall manufacturing footprint, and even quicken your timeline to the clinic,” he explains.

Hasler adds that viral vectors are most effective in single-use situations. Messenger RNA development and, generally, clinical scale-level development of new therapeutic modalities may also be better suited in a single-use bioreactor; whereas, a mammalian blockbuster or commercial product may be better suited in a stainless-steel bioreactor, Hassler states.

1. Thermo Fisher Scientific. Single-Use vs. Stainless Steel: The Biopharmaceutical Manufacturing Debate. 

Feliza Mirasol is the science editor for 


Volume 37, No.4
April 2024
Pages: 17–20

When referring to this article, please cite it as Mirasol, F. Evaluating Uses for Both Single-Use and Stainless-Steel Bioreactors. 

Upstream Processing

Despite a growing trend toward single-use bioreactors, stainless-steel retains its relevance for certain products.

Evaluating Uses for Both Single-Use and Stainless-Steel Bioreactors

Affinity ligands play an exciting role in the ever-growing sphere of downstream processing. In speaking with 

®, Phillip Elliott, PhD, associate director of Process Development, BioCina; Jan Bekker, PhD, director, Business Development, Commercial and Technical Operations, BioCina; and Parviz Shamlou, PhD, senior vice-president of Science & Technology, Abzena illuminate the newest innovations with affinity ligands and provide a deeper understanding of the role affinity ligands play in the future of downstream processing.

 What are affinity ligands and how do they contribute to the downstream purification process?

 Affinity ligands are molecules that can bind with high affinity to specific targets. Affinity ligands can be used for various purposes, such as detection, purification, or modulation of biological activities. Affinity chromatography is a separation method based on the specific binding interaction between an immobilized affinity ligand and its binding partner. The most common example in the realm of bioprocessing is Protein A, which has an extremely high affinity for some immunoglobulin molecules. Other examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. The degree of purification achieved by affinity purification can be quite high depending on the specificity of the interaction. Consequently, it is generally the first step in a purification strategy.

Release of the protein from the ligand is generally achieved by altering parameters such as pH, and sodium chloride concentration or by the addition of a competitor for ligand binding.

In nature, there is a vast array of biomolecule interactions where binding of one molecule to another through receptor–ligand interactions is critical for biological function. This concept is applied to the drug development sector and revolutionized manufacture of large complex medicines where use of ligands that have a specific affinity for the drug are used for purification—with antibodies being the most notable success story.

Large-scale manufacturing of a therapeutic monoclonal antibody (mAb) starts with the expression of the antibody in mammalian cells, typically CHO [Chinese hamster ovary] cells, followed by a series of steps to purify the antibody from all the impurities in the cell culture media. Following the removal of cells and cell debris from the media, the first chromatography purification step is based on the specificity (affinity) properties of the antibody. Bioprocess scientists and engineers realized the importance of affinity chromatography in purification of mAbs in the early 1980s and identified a small, 42 kD protein (i.e., Protein A) that has exceptionally high affinity for mAbs in the Fc region.This is an important property that allows for most antibodies to be purified irrespective of their specificity.We know a lot about Protein A ligand since it was first discovered in 1958. Today, Protein A ligand is manufactured and purified on a large scale using recombinant DNA technology. Purfied Protein A ligand is covalently coupled (conjugated) to a solid, porous support to form a matrix that can uniquely and specifically bind to mAbs under the correct biophysical conditions. The matrix is typically in the form of small 50–150 micron beads that are highly porous to provide a large surface area, maximizing antibody binding potential for higher yields from fixed-volume columns.

 Why are affinity ligands preferred for biologics purification over other downstream purification techniques?

 Affinity ligands can produce a very high purification factor in a single unit operation and capture product from a dilute process stream. This makes affinity purification very attractive in biopharmaceutical manufacturing operations as it can help to reduce time and cost. Most other ligands used in chromatography operations rely on more general properties of analytes (proteins and other biological molecules), such as charge and hydrophobicity. This can result in many other molecules besides the target molecule binding to and eluting from the stationary phase and hence being carried forward in the purification scheme. The same drawbacks apply to purification processes that do not rely on ligands, such as the size separation performed by size-exclusion chromatography or filtration.

In addition, affinity separations are generally simple to operate, not requiring gradient or other complex operations, and are rapid in execution.

 Affinity chromatography is based on highly specific attractions between two biomolecules. Importantly, the interactions between the two biomolecules are reversible making them an ideal choice for purification. This is achieved by attaching one of the biomolecules, known as the affinity ligand to a solid matrix which acts as the stationary phase while the other biomolecule, referred to as the target molecule, is in the mobile phase. While there are many ways affinity ligands may be exploited for purification, Protein A affinity ligand for purification of antibodies from is one of the best examples and has proven a powerful mode of chromatography.

Protein A ligand affinity chromatography is simple to operate, robust, rapid, and highly selective with very good resolution. Importantly, and unlike other modes of preparative chromatography, binding of antibody to Protein A affinity ligand is not sensitive to either cell free culture media pH or its osmolality so there is no need for conditioning the feed. The high specificity and ease of operation of Protein A ligand affinity chromatography has made it the technology of choice and the “darling” of the mAb purification platform
process in industry.

 What are the most cutting-edge innovations in the manufacturing process for affinity ligands, and what drove those innovations?

 One of the challenges of traditional protein A affinity chromatography is the use of acidic, low pH, elution. The low pH can lead to instability of certain antibodies potentially causing aggregation. A recent innovation is the development of a new generation of Affinity ligands that are like traditional Protein A in all respects except that elution can be performed at around pH 5.

Another challenge with traditional Protein A affinity ligands is the leaching of protein A from the solid support. This reduces the resin lifetime and introduces a process related impurity into the product mainstream. Leached protein A is considered a CQA (critical quality attribute) to measure for with a release specification that must be met before the product can be released.Much progress has been made in recent years to address this issue and the new generations of protein A are more robust and stable.

 A very interesting recent development from Cytiva is the ProteinSelect system which consists of a tag protein sequence added to the N-terminus of a target protein and an affinity ligand for that tag. Once the protein of interest binds to the affinity ligand on the resin via the tag, refolding and self-cleavage of the tag-ligand complex occurs, such that the protein of interest is eluted without any trace of the tag. This will allow more widespread use of this affinity ligand with proteins that do not have a current natural affinity ligand as well as more rapid purification of new drug targets. The need to have more rapid purification of a variety of proteins for drug development drove this innovation, as well as the availability of these self-cleaving sequences.

 In what direction is affinity ligand development headed moving forward?

 Another popular affinity ligand for purification of antibodies is Protein G, which has a molecular mass of 21.9 kDa and has a different binding affinity to antibodies compared to Protein A.

Affinity ligand is a powerful technology and, beyond Protein A and G affinity ligands, in recent years, affinity chromatography matrices have been developed with high selectivity for purification of, for example, recombinant viral vectors for gene therapy applications.In one example, camelid single domain antibodies that recognize adeno-associated virus (AAV) viral vector capsids are proving very effective. Another affinity matrix also provides high affinity for recombinant human growth hormone (hHGH) by using an immobilized 14 kD single variable domain of heavy chain (VHH) antibody that is recombinantly produced in yeast. The single-domain heavy chain antibody fragments from Camelidae have also been utilized successfully as affinity ligands for purification coagulation factors to help patients with hemophilia type A and B.

Several factors must be considered to obtain a successful separation in affinity chromatography. For instance, it is necessary to immobilize the ligand onto or within a chromatographic support. The immobilization process should ideally create a stable affinity ligand without significantly altering the binding properties of the ligand. If the affinity column is to be reused for multiple cycles, care must also be taken to select application and elution conditions that allow both effective dissociation of retained targets and good column regeneration without permanently damaging the ligand. As an example, much engineering has recently gone into modifying the Protein A ligand to ensure it can withstand the stringent cleaning and sanitization procedures required for use in a pharmaceutical setting without becoming inactive and no longer being able to bind its target. Similarly, a ligand should not bind its corresponding analyte too strongly, such that the target analyte cannot be eluted without using conditions that damage the analyte. These issues represent areas of ongoing research in the development of affinity ligands. Continued growth is expected in the future for affinity chromatography as further advances are made in the ligands, supports, immobilization schemes, and potential applications.

Recent work has explored the use of molecularly imprinted polymer surfaces (MIPS) for the purification of proteins. MIPS are polymer stationary phases that contain pockets or cavities specific to the target analyte. Due to the lower cost of these surfaces compared with biological affinity ligands, developments in this area are expected to continue and be highly beneficial.

 What role do you see affinity ligands playing in the future of downstream purification, especially with emerging biologic therapeutic modalities?

 Due to their specificity and high purification factor, affinity ligands will continue to play a major role in the downstream purification of biological molecules. Cell affinity chromatography is another important application involving target purification by affinity-based separations. Examples of affinity ligands that have been used for this purpose are lectins, which can interact with glycoproteins on cell membranes, and antibodies, which can bind to specific surface proteins. With the expected increase in cell-based therapies, the development of ligands for these purposes should see them continue to find a large role in the future.

 Recently, oligo-deoxythymidine acid (oligo-dT) ligands have been developed and immobilized to chromatography stationary phases to provide a highly specific ligand for the polyadenylated tail of mRNA [messenger RNA] molecules. The emergence of this class of therapeutic molecules (mRNAs) drove the development of the corresponding affinity ligand that could simplify their purification. As with cell therapies, this new ligand will play an important role in mRNA’s future development.

 Gene therapy is an emerging therapeutic modality that needs significant process development including the application of novel affinity ligands during purification. A key step in the production of gene therapy products is the use of high-quality viral vectors with [AAV] vector and lentiviral vector (LV) being the most popular platforms. Robust manufacture of high-quality vectors has proven to be a major bottleneck in the growth of gene therapy. Titers are relatively low during production and robust purification of the viral particles from impurities has proven to be a challenge. For example, viral particles are physically much larger than proteins and antibodies. Using traditional resins with small pore size leads to low binding capacity because of very low diffusion rates into the pores of the resin. LV particles are also exceptionally sensitive to pH, salt concentration, shear, and temperature making their purification more challenging. These challenges currently mean there is no ‘platform’ downstream purification for viral particles.

A new generation of vector serotype specific and non-specific affinity ligand chromatography resins are proving successful in capturing LV and AAV particles from cell-free media and promise the potential of a platform approach to purification of AAV and LV particles. AAV and LV affinity ligands are, however, exceptionally expensive making commercial scale an issue for many drug manufacturers.

When referring to this article, please cite it as Husni, D. A Closer Look at Affinity Ligands. 

Downstream Processing

A look at the newest innovations offers a deeper understanding of affinity ligands and their role in the future of downstream processing.

A Closer Look at Affinity Ligands

There are more than 60 peptide therapeutics approved around the world and hundreds currently in clinical studies (1). A peptide is a polymer consisting of less than 100 amino acids (AA) linked by an amide or peptide bond. These therapeutics are desirable as they exhibit low toxicity and high biological activity. The two major peptide manufacturing pathways consist of recombinant and synthetic. Recombinant manufacturing uses an engineered living microorganism, such as 

), to synthesize the peptide. Synthetic peptide manufacturing originated with solution-phase peptide synthesis to groundbreaking solid-phase peptide synthesis (SPPS) and hybrid methods (2). Further advances in the SPPS technology improved the deprotection and cleavage steps (see 

), resulting in the ability to safely manufacture synthetic peptides for personalized medicine to commercial manufacturing of greater than 100 kilograms (3).

The manufacturing of commercial peptide therapeutics requires current good manufacturing practices (CGMPs) including validation of the manufacturing and cleaning process (4,5). Cleaning validation consists of three phases: the design phase to understand critical cleaning parameters as well as defining cleaning quality attributes; the qualification phase to ensure the cleaning process consistently meets pre-established acceptance criteria; and the continuous monitoring phase to ensure the cleaning process remains in a state of control (6,7). If multiple peptides are manufactured in shared equipment, then a grouping or bracketing approach can be used to identify the worst-case peptide for validation (8–10). The most common factors used for grouping drug actives are solubility, toxicity, and difficulty of cleaning. Other considerations could include structure, molecular weight, availability of analytical methods, bioavailability, permeability, and degradation profiles (11).

 provides a grouping or bracketing matrix of nine peptide therapeutics that range in molecular weight from 987.5 to 4568 g/mol along with their solubility in water and cleaning procedures. Eight of the nine peptides were easily cleaned with 1% v/v formulated alkaline detergent at 50 

C for 15 minutes (cleaning procedure A), and one peptide required a second cleaning step using 1% v/v formulated acid detergent at 50 

C for 15 minutes (cleaning procedure B). In this example, cleaning procedure B was selected for all nine peptides, and a second formulated chlorinated alkaline wash was added to ensure degradation of the peptides. With the grouping strategy, a single cleaning procedure for all the products was implemented. The addition of the acid wash also eliminates the requirement for wastewater neutralization, reduces bioburden, and provides continual maintenance of the stainless-steel equipment (12).

This study was performed almost 10 years ago, and therapeutic peptides are continuing to increase in length and complexity, which warrants revisiting the worst-case ranking with each additional peptide introduced. Within the past few years, a large biopharmaceutical company expressed an interest in a process and cleaner evaluation to perform solubility and cleaning evaluation of a new peptide as well as the process reagents used in the synthesis. In this article, the authors explore the solubility, difficulty of cleaning, and degradation of a new therapeutic peptide with a C225HNO structure), which includes the use of a modified sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method for the analysis of lower molecular weight molecules.

Organic solvents are commonly used in the manufacture of peptide therapeutics but can present operational, safety, and environmental concerns with their use in cleaning commercial manufacturing equipment. The building blocks of peptides, 20 different amino acids, can vary in size, polarity, pH, and other factors included in 

. The larger molecular weight amino acids are hydrophobic in nature, reducing the solubility in water. Other factors that affect solubility include the number of amino acids present, negative (acidic) or positive (basic) charge, pH, and temperature of the solution. The cleaning method, such as sonication, can also affect solubility. Laboratory testing evaluating the solubility of the peptide in water and/or cleaning agent may provide helpful information on cleaning as well as rinse and swab analysis.

While other solvents in the manufacturing process show good solubility, aqueous detergents may clean at lower temperatures and concentrations. Optimizing this cleaning process reduces the risk of cross-contamination and saves the manufacturer cleaning time and overall costs. Specific parameters for solubility of the new peptide and reagents within the peptide manufacturing process can be explored through laboratory studies (see 

Organic solvents are traditionally used in peptide manufacturing as well as cleaning of manufacturing equipment. Due to the potential safety issues with using solvents, the solubility of the peptides and process reagents were investigated using various solvents and aqueous-based pharmaceutical detergents.

Reagents evaluated in the experiments are: Ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), Hexaflurophosphate Azabenzotriazole Tetramethyl Uronium (HATU), Dithiothreitol (DTT), and Tri-isopropyl silane (TIPS) along with a peptide API and peptide fragment.

Solvents used in the experiments were as follows:

Solvent 1 is 70% v/v dimethyl sulfoxide (DMSO)/30% acetonitrile (ACN) at ambient temperature.

Solvent 2 is 70% v/v DMSO/30 % ACN at 50 °C.

Solvent 3 is 30% ACN at ambient temperature.

Solvent 5 is 100% ACN at ambient temperature.

Solvent 6 is formulated alkaline detergent at ambient temperature.

Solvent 7 is formulated alkaline detergent at 50 °C.

The following procedure was used in the experiment:

This article explores a grouping strategy for therapeutic peptides incorporating theoretical and experimental methodology and results to define a practical and scientifically justified cleaning procedure.

BioPharm International, April 2024

Benefiting from Single-Use Tech Downstream

Driving Innovation in Nucleic Acid Therapeutics

Evaluating Uses for Both Single-Use and Stainless-Steel Bioreactors

A Closer Look at Affinity Ligands

Cleaning Process Design for Peptide Therapeutics

Taking Full Advantage of Service Providers

May I Present the Neoantigen

Focusing on Effectiveness for CAPAs