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T Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . 2 Terry Pizzie
Has Lentiviral Purification Been Left Behind?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Sujeong Yang and Ian Scanlon
Increasing Processing Efficiency, Purity, and Recovery of Lentiviral Particles for Viral Vector Development . . . . . . . . . . . . 5 Daniella Steel
A Radical Change in Bioprocessing for Cell and Gene Therapies. . . . . . . . . . . 8 Bilal S. Ahmad
The Changing State of Bioprocessing. . . 10 Marc Hummersone
hanks for taking the time to read our special year-end report. This year has been one of the most exciting ones in the history of Astrea Bioseparations. We recently launched a game-changing, fit-for-purpose solution for lentiviral vector purification: the Nereus LentiHERO system, which uses our proprietary nanofiber AstreAdept technology. The LentiHERO lentiviral vector purification spin column enables vector developers to increase throughput of sample processing and gain higher yields and recovery, unlike legacy methods, which were developed for the purification of monoclonal antibodies (MAbs). This technology significantly shortens processing times. Although this special report focuses on our new cuttingedge purification technology, the company has made other achievements throughout the year which deserve equal recognition. Our team has almost finalized the development of a mixed-mode, host-cell protein clearance adsorbent that simplifies removal of unwanted proteins from MAb workflows. That reduces production costs and enables finished products to be released quickly and easily. This product will wrap development shortly and be available for purchase in late 2022/early 2023. Our global commercial operations are rapidly expanding, allowing us to further our reach and provide quality service to customers in Europe, the United States, and the Asia–Pacific region. Our new 27,000-ft2 headquarters in Boston, MA, provide warehousing and production capability to further support our growing US market. Finally, our manufacturing operations are increasing production to handle our rapidly scaling business. Our team on the Isle of Man has manufactured and delivered a 78% increase in manufactured product this year over 2021. I am proud of the entire team at Astrea Bioseparations for their hard work and dedication to making 2022 a year that will stand out in company history. With much more to come for 2023, I wish you all the best in the new year. Terry Pizzie is chief executive officer of Astrea Bioseparations, Ltd.; https://www. astreabioseparations. com.
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Has Lentiviral Purification Been Left Behind? Sujeong Yang and Ian Scanlon
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urrent technologies for laboratory-scale lentivirus (LV) feedstock preparations are inefficient and not fit for purpose. Why have lentiviral vector (LVV) developers not benefited from standardized purification processes emerging from other viral vector fields? Below, Sujeong Yang and Ian Scanlon, viral vector purification experts at Astrea Bioseparations, discuss the importance of laboratory-scale LV preparation, highlight common industry practices, and share their experiences. Why is laboratory-scale LV production crucial to gene and cell therapy development? Sujeong Yang: Adoptive cell therapies such as chimeric antigen receptor T-cell therapies, (CAR-T therapies) and nextgeneration versions, including CAR natural killer cells (CAR-NKs), CAR macrophage cells (CAR-Ms), and other engineered T-cell receptor treatments require genetic modification of cells. LVVs are the preferred vehicle for delivering a gene of interest (GoI) because they can infect cells that other viruses cannot (e.g., adenoassociated viruses, AAVs). Developers of adoptive cell therapies want LVVs that can deliver a GoI to a target cell to express a transgene optimally and produce an expected biological effect. Several factors will affect the efficiency of an LVV to deliver a GOI or “transduce” a target cell, including the level of efficiency to which a vector genome is packaged and whether cell-type– specific promoters are used. LVV design optimization requires analysis of many discrete LVV batches that are produced at small scale in tissue-culture flasks, shake flasks, or 100–250-mL miniature bioreactors that can be operated in parallel to generate sufficient material. After an LVV construct is developed, laboratoryscale LVV production provides material for cell line development and preclinical studies to determine thresholds for dosing efficacy, biodistribution, and pharmacokinetics and safety studies. Ultimately, those studies support investigational new drug (IND) filing that will advance an LVV therapy candidate to clinical-trial testing. How do LV characteristics impact purification strategies? Ian Scanlon: LVVs are different in several Sponsored
respects from other viral vectors used in cell and gene therapies. Whereas AAV capsids, at only 25 nm in diameter, are comparatively similar in scale to large proteins, LVVs are considerably larger at 80–100 nm. The larger size of LVVs is an advantage because it allows them to incorporate genetic constructs up to 10 kb. However, that size also poses a particular challenge with respect to purification. Although standard resin-based separation methods can be used to purify AAVs, LVVs are too large to achieve sufficient capacity with traditional chromatography methods, so their purification requires the use of different types of adsorbents. LVVs also tend to be more unstable than nonenveloped viruses such as AAVs. The LVV envelope plays an important role in vector functionality, including transduction and tropism. But that functionality is compromised by some processing conditions such as changes in temperature, pH, and ionic strength of bioprocessing fluids. Sujeong Yang: At the vector design stage, LVVs generally are produced through transfection of human embryonic kidney (HEK) 293 cells using plasmid DNA under adherent–cell-culture conditions. The presence of plasmid DNA complicates purification strategies because at a working pH, the negative surface charge of the LVV is similar to that of nucleic acid impurities. Host cell proteins (HCPs) are other contaminants that can be problematic because they can lead to undesired immunogenic effects. An LVV envelope comprises the host cell membrane, and the similarity of the LVV and those contaminants can cause difficulties for achieving a pure LVV product. What are other challenges for achieving efficient LVV purification? Sujeong Yang: The biggest challenge to LVV purification is low functional or physical recovery. LVVs are produced at titers that are lower than those of other viral vectors. Because LVVs also are fragile, processing time and number of unit operations should be minimized to reduce LVV degradation during purification steps. If too many purification steps are used at laboratory scale, then recovered product quantities could be extremely small. Such overall poor recoveries can lead to
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oversized and expensive batches of LVV productions. Ian Scanlon: Low recovery also means that the number of process steps in a purification protocol must be limited, sacrificing purity in turn. What are the limitations of existing laboratoryscale purification approaches? Sujeong Yang: Ultracentrifugation and density-gradient ultracentrifugation can purify and concentrate LVVs and commonly are used for laboratory-scale LVV preparation. But these methods have limitations, including a lack of scalability and the need for highpowdered centrifuges and special operational expertise. The processes also are time consuming, so throughput is limited. Ian Scanlon: There is no standard downstream purification process for small volumes. Molecularweight cut-off (MWCO) filters can be used with benchtop centrifuges to concentrate clarified feedstocks, but such filters have limited capability to remove impurities. The same considerations apply for tangential-flow filtration (TFF), but LVVs are concentrated along with salts, proteins, and DNA — generally for extended periods because of the length of time required for concentration. Precipitation sometimes is used to remove impurities selectively. PEGylation of a vector can enable selective product concentration, for instance. Again, the problem is that adding unit operations with LVVs typically reduces considerably final yield of a product. Alternatively, ion-exchange membrane adsorbers enable a purification process to be more aligned with chromatography steps used at typical clinical-scale LVV manufacturing. However, functional product recovery can be compromised when high-salt elution steps are used. What would be the effect of improved laboratoryscale LVV purification on therapeutic development? Ian Scanlon: When comparing LVV discovery workflows to those applied in protein and antibody drug discovery, it is clear that there is much room for improvement. Antibody discovery leverages highthroughput technologies with multiwell plates for rapid separation and analysis. Transferring that to the LVV space would be highly beneficial. Sujeong Yang: Although upstream technologies can work in parallel to produce vector targets, the capabilities of existing downstream purification solutions restrict throughput of LVV purification for use in preclinical studies, thus extending the time needed to obtain material for clinical and commercial applications. A more efficient 4
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purification process would shorten vector development time, thereby reducing costs and helping therapies progress quickly to clinical testing. How could a laboratory-scale chromatography solution facilitate lentiviral therapeutic development? Sujeong Yang: First, a process must be easy to implement with equipment generally available in a laboratory. Many LVV purification methods that can provide pure, highly functional material are not practical. A chromatography process also should be adapted to reduce LVV degradation. For example, mild elution conditions can be used to prevent the degradation observed at high salt concentrations, and a short process time should boost LVV recovery. Increased product recovery would increase production efficiency and reduce costs and reduce pressure on an upstream process to increase volumes of viral feedstocks. Ian Scanlon: A chromatography process also would need to increase throughput so that researchers could advance targets and assets that are most likely to succeed through drug development faster, ultimately getting more therapies in the hands of patients. A chromatography-based purification technology that could be implemented simply and easily, be readily scalable, and afford robust LVV purifications rapidly would be the best solution. A simplified chromatography solution that does not require extensive training to use would facilitate adoption in discovery and early development laboratories. After all, processes will need to be scaled up to generate clinical and commercial quantities, which usually requires chromatography methods. Starting with a scalable chromatographic purification from the outset will ensure that the same results are obtained in discovery as in manufacturing and reduce time required for process development. In the end, that will accelerate progress to clinical trials.
Meeting LVV Purification Needs Now
The discussion herein emphasizes a pressing need to address current laboratory-scale purification issues. A time-efficient process that simultaneously increases LVV recovery would serve as an enabling technology for future LVV-based drug development. Astrea Bioseparations is addressing those needs through our development of an unprecedented and proprietary fiber-based technology that empowers therapeutic innovators with the tools they need to purify the quantities they desire. c
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Increasing Processing Efficiency, Purity, and Recovery of Lentiviral Particles for Viral Vector Development Daniella Steel
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herapeutic developers face significant challenges in purifying cell and gene therapy (CGT) modalities. Current processes rely on solutions developed for monoclonal antibodies (MAbs) and are not fit for purpose for viral vector purification. Resin chromatography has significant limitations: Yields are low, processing times are long, and costs are high (Figure 1). To meet patients’ needs for affordable, novel therapies, developers require significant improvements over current methods. One way to meet those needs is with novel adsorbents that provide the difficult-to-purify large molecules used in CGTs with ready-access to considerably more binding surface area than lastgeneration technologies. AstreAdept proprietary nanofiber material is electrospun with two polymers, which makes the surface area highly accessible to viral vectors and enabling significantly faster flow rates compared to traditional bead- or membrane-based adsorbents (Figure 2). Astrea Bioseparations has incorporated the AstreAdept technology into the easy-to-use Nereus LentiHERO spin-column format to bring the advantages of functionalized nanofibers to viral vector purification at laboratory scale (Figure 3). Increased lentiviral vector (LVV) capture is essential to maximizing LVV production for viral vector development. Functionalized nanofibers
provide dynamic binding capacities of 1.6 × 1011 lentiviral (LV) particles per milliliter of adsorbent, enabling users to increase the amount of recovered materials from their current feedstock volumes and, in turn, reducing pressure to expand upstream production (Figure 4). In addition to the superior capture characteristics of the underlying nanofiber technology, the Nereus LentiHERO solution enables gentle processing, including low centrifugal speed, reduced processing times, and mild elution conditions for high LVV recovery. The Nereus LentiHERO technology achieves yields of 60% of loaded LV particles which is a significant improvement over the 15–30% recoveries observed with other methods (Figure 5). Minimizing host cell protein (HCP) contamination is important for limiting immunogenicity, even at laboratory scale. However, low process recovery has driven the practice of limiting the number of process steps, so purity often is sacrificed. The success rates of vector development will be affected by unwanted immunological effects on the outcomes of preclinical studies. The Nereus LentiHERO solution can reduce HCP contamination of LVV feedstocks by 95% without compromising recovery, time, or throughput (Figure 6). The structure and infectivity of purified LV particles are uncompromised when they are
Figure 1: Legacy processing solutions place pressures on upstream production and lentiviral quality.
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Figure 2: Immediate access to high binding surface area, with nanofiber technology, enables more effective processing of lentiviral vectors Diffusion
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Figure 4: High binding capacity enables efficient use of laboratory-scale lentiviral production. A Nereus LentiHERO unit was loaded at 20 mv/min. Flow-through was collected in 10-mL aliquots and analyzed using p24 ELISA to calculate percentage of virus-particle breakthrough against total virus-particle load.
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Figure 5: Nereus LentiHERO spin column is suitable for serum-free and serum-containing feedstocks. Percentage of lentiviral (LV) recovery yield, relative to input, from LV feedstock produced from an HEK293 LV production line, without serum (–) (n = 3) or with serum (+) (n = 2). 90
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processed with the Nereus LentiHERO spin-column format, thereby retaining both functionality and morphology. Figure 7 shows that the size of important proteins such as group-specific antigen (gag), encoding capsid, matrix, and nucleocapsid 6
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Figure 6: Nereus LentiHERO technology vastly decreases host-cell protein (HCP) contamination. Total protein profiles were analyzed using load, flowthrough, and eluate fraction. Samples were run on SDS-PAGE gel and visualized by a silver stain (a). HEK293 HCP levels were measured by ELISA from lentivirus input and eluate from (–) serum-free (n = 3) and (+) serumcontaining (n = 2) systems (b).
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Figure 7: Analysis is shown for lentivirus-specific p24 Gag detection (a). Transmission electron microscope (TEM) images were taken from load and eluate fraction using a negative staining method (b). Infectivity of lentivirus particles from the purified eluates was tested by transduction assay to HEK293 cell lines (c).
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Reducing both contaminant levels and sample volumes facilitates subsequent concentration. Because the Nereus LentiHERO technology comes in a spin-column format, sample throughput can be increased easily by scaling out and maximizing benchtop centrifuge capacity. By incorporating the Nereus LentiHERO solution into LVV feedstock preparation workflows, multiple samples can be processed simultaneously, and bottlenecks can be reduced. The current options to address laboratory-scale LVV purification have limited capabilities for use with CGT modalities. To address the increasing demands on therapy developers, novel solutions that are fit-for purpose are essential. The Nereus Sponsored
LentiHERO solution for LVV development and production laboratories is designed to increase sample-processing throughput and improve upon LV particle recoveries. This nanofiber-based technology reduces bottlenecks in the development pipeline, ultimately helping the CGT industry to bring therapies to patients faster than ever before. Daniella Steel is senior manager, product strategy, at Astrea Bioseparations; d.steel@astrea-bio.com. c
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A Radical Change in Bioprocessing for Cell and Gene Therapies Bilal S. Ahmad
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ith the rapidly growing number of novel therapeutics, the gaps in bioprocessing workflows are more noticeable than ever. To address the current and future challenges of throughput and yield in cell and gene therapies (CGTs), Astrea Bioseparations has developed AstreAdept fit-for-purpose purification technology, which is based on a proprietary nanofiber material. Astrea Bioseparations has used this nanofiber to create a fit-for-purpose purification spin column for lentiviral vector development. Herein, we explore how the AstreAdept technology can be used to change how bioprocessing for CGT is approached. Bioprocess adsorbents, both bead- and membranebased, are limited by their capacities, flow rates, and pressure drops. That has been the case for many years and has been widely accepted as a challenge that is “just dealt with.” Such adsorbents also are expensive to manufacture and can easily become unstable under different process conditions (e.g., temperature, and pH). Research has shown that membranes constructed from electrospun nanofibers have shown great promise when applied to the separation of biological substances, particularly for fragile vectors used in CGTs (1, 2). Single-component nanofibers are superior to single-component microfibers because pore sizes, affinity characteristics, and other performance criteria can be controlled more precisely. However, single-component nanofiber structures are often less efficient in terms of stability and time requirements. In fact, many nanofiber solutions for biological research that are currently on the market suffer from the workarounds used to tackle the stability problem. Thus, a need exists to further improve the stability of such single-component nanofiber structures for the purification efficiency of biological products.
Dual Electrospinning Technique for Fabricating Materials
The AstreAdept composite nanofiber material can increase flow rates and binding capacities for biological product purification. The nanofiber matrix is created by electrospinning a proprietary blend of 8
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Figure 1: Schematic showing the production of a composite nanofiber Polymer A
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two different polymers together, combing cellulosic and noncellulosic materials. Figure 1 shows how this composite nanofiber structure is made. Two polymeric solutions (one is a derivative of cellulose and the other a noncellulose synthetic polymer) are fed through a metallic needle under an applied voltage. Both polymer nanofibers are deposited onto the same substrate material in sequential layers thus forming a composite membrane.
Open Structure Enables Immediate Access to Large Surface Area for Binding
The nanodimensions of nanofiber naturally gives it a high surface-area-to-volume ratio. This characteristic makes it very attractive in applications in which large surface areas are highly desirable, such as in affinity membranes. Figure 2 shows a scanning electron microscopy (SEM) photo of the composite nanofiber membrane, illustrating the nanofiber’s high surface-area-to-volume ratio and improved permeability. Those characteristics provide a large accessible binding surface area, with potential for high flow rates.
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Figure 3: Comparing specific strength and strain to failure of the cellulose, noncellulose, and composite nanofibers
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AstreAdept Nanofiber Shows High Specific Strength While Retaining Flexibility
Single-component nanofibers have high fragility and poor mechanical strength. The AstreAdept nanofiber combines the advantages of cellulosic and noncellulosic nanofiber materials to create a nanofiber matrix that exhibits a high specific strength while retaining flexibility (Figure 3), thus eliminating the limitations of traditional singlecomponent nanofiber membranes.
AstreAdept Nanofiber Improves Purification Efficiency
To demonstrate its bioseparation performance, the AstreAdept nanofiber was functionalized with a weak anion exchange and tested using a chromatography system. Extremely high flow rates were obtained without a decrease in dynamic binding capacity. Those results exemplify the purification efficiency that the AstreAdept nanofiber brings to bioseparation processes (Figure 4). Sponsored
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The unique advantages offered by the AstreAdept nanofiber will affect other areas of biomanufacturing. For example, higher recovery reduces pressure to expand upstream production, so productivity can increase in current facilities. With improved capacity and fast process runs, bioprocessing equipment size can be minimized, resulting in positive effects on process economics. Furthermore, devices housing this material have smaller physical footprints, which reduce facility use, water use, and waste removal. Thus, such industrial processes can progress to greener manufacturing. c Bilal S. Ahmad is a subject matter expert at Astrea Bioseparations; b.ahmad@astrea-bio.com
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The Changing State of Bioprocessing Marc Hummersone
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ew industries have made such astronomical gains as the biopharmaceutical industry has in the past 20 years. In the not-so-distant past, manufacturing was well known as the biggest bottleneck for the progression of a therapeutic. Inefficient processes were being wrestled with and generally accepted as unsolvable while low yields and nonreproducible processes were the norm. With the implementation of quality by design (QbD) and the development of improved expression systems, the industry has gradually shifted toward higher yields with more reproducible outputs. Such gradual improvements have created processes that are now capable of delivering more grams of protein per liter of culture without having to increase the number or size of bioprocessors. This improvement in manufacturing efficiency is in part a result of the introduction of single-use technologies (SUTs) and the widespread adoption of continuous bioprocessing techniques. However, the use of SUTs in improved productivities comes with its own set of challenges. For example, although the use of SUTs can reduce batch-to batch contamination and provide flexibility to users, SUTs are made from plastic or plastic derivatives and are disposed of after a single use. That can lead to significant environmental impacts for waste stream and raw-material usage. SUTs also are often difficult to scale up or down because many vendors them in only a standard size. Nonetheless, such advances in bioprocessing technologies are noteworthy because of how they have shifted the bottlenecks in biopharmaceutical manufacturing, thereby forcing the focus to downstream processes. That has enabled some agile life science companies to focus on downstream applications of novel therapeutics by reevaluating the classical approach to sorbent delivery and development. Novel modalities, particularly in the realm of cell and gene therapies (CGTs), are underserved by current well-established approaches, which are largely based on the manufacturing of 10
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Schematic of devices suitable for process development at pilot and manufacturing scales.
monoclonal antibodies. To solve the unmet need in CGT, many companies have shifted their business paradigms to focus on process compression. That is an innovative and disruptive method of product development whereby agile product teams are dedicated to taking lengthy workflows and creating fit-for-purpose and novel solutions that allow for the compression of those workflows. The output of products address current and unmet needs in the industry. Process compression has been introduced to the general public by the “moonshot” approach used in the recent mRNA vaccine for COVID-19, which fueled
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the unparalleled development of the vaccine from bench to bedside in record time. That approach saw human trials within weeks of the virus sequence being published. However, progress always finds itself up against difficulties, and healthcare regulators have struggled to keep up with the technological advancements in next-generation therapies. The bottleneck in therapies is evershifting from upstream to downstream and ultimately to regulators. Key developments and paradigm shifts in regulatory approaches to the new wave of therapeutics will be the only way in which the leading regulatory agencies such as the US Food and Drug Administration (FDA), the Medicines and Healthcare products Regulatory Agency (MHRA), and the European Medicines Agency (EMA) define the scope for their entry into clinical trials and beyond (e.g., approval and market entry). The regulatory roadmap (particularly within CGTs) is evolving rapidly, and the set of rules defined by global healthcare regulators will direct the quality and characteristics of the products that many life science companies are developing rapidly to support next-generation therapeutics. As an industry, life science suppliers historically have been closed from the regulatory agencies that pharmaceutical and biotechnology companies work with frequently. To begin eliminating bottlenecks, life sciences companies need to work closely with regulators by establishing constant communications and “datafeedback loops.” Only by doing that will regulators be up to speed with developments and capabilities of the industry to deliver next-generation therapeutics to the clinic with the correct level of quality and safety for patients. In lieu of working directly with the regulators, life science vendors also have established their own action plans and roadmaps to meet the ever-shifting needs of the biopharmaceutical manufacturing industry. Astrea Bioseparations, for example, is trying to address the gap in efficiency and processing in viral vector manufacturing. By working with nanofiber technologies (and proprietary AstreAdept technology), Astrea Bioseparations has developed the ability to “supercharge” already established ligands that have been refined to a point at which they are as efficient as possible. Astrea Bioseparations has focused on sorbents rather than on ligands, thus disrupting the status quo of resins that have dominated the Sponsored
industry for the past 70 years and making minor gains in efficiency year over year. Nanofibers have surface-area-to-volume ratios that are larger than those of resins, and nanofibers feature open porous networks that are considerably larger than the small pores of traditional resins. Nanofibers have negated the need for target molecules to passively diffuse into pores, but they allow large target molecules (such as those in cell and gene therapies) to permeate freely within a macrostructure. Because there is no passive diffusion associated with the binding event between target molecules and ligands on the surface of fibers, bind-and-elute residence times are a fraction of those required for traditional resins. With such low residence times, flow rates can be increased, which in turn significantly increases purification efficiencies. The highly porous nature of nanofibers enables large flow rates accompanied by low operating pressures, which are a fraction of those associated with resin use. That means that that nanofiber technology can be used with established laboratory hardware. Using AstreAdept technology, Astrea Bioseparations has developed the first nanofiberbased product for the purification of lentivirus, the Nereus LentiHERO spin column, which negates the need for regular pump technology. It has been designed to enable to research scientists to purify their lentivirus samples with nothing more than a bench-top centrifuge. The Nereus LentiHERO spin column is the first product in a family of nextgeneration purification devices designed to leverage the properties of AstreAdept nanofiber chromatography. c Marc Hummersone is senior director of research and development at Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, United Kingdom; m.hummersone@astrea-bio.com.
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