Category: Microfluidics

Microfluidic technologies proficient in handling exceedingly small volumes of liquids and gases with precision have emerged as a pivotal innovation offering new possibilities across the life sciences. Their capacity for consistent and measured fluid dynamics, coupled with a significant reduction in the consumption of materials and energy, positions these devices to enhance the efficiency and precision of various processes at a diminished cost and to offer new solutions to propel advancements in drug discovery, diagnostics, and manufacturing. By integrating cutting-edge micro- and nanofabrication techniques with sophisticated sensing capabilities, microfluidics technology is forging new pathways in high-throughput screening, precise analytical assessment, organ-on-a-chip simulations, and drug delivery systems, underscoring a significant potential to revolutionize aspects of personalized medicine and lab automation, as well as optimizing bioprocessing and healthcare.

Overview of Microfluidics in Biopharma

Microfluidic devices are designed to manipulate micro-, nano-, and/or picoliter volumes of liquids and gases within microscale channels and chambers with precise control.¹ Microscale pumps, valves, mixers, and other systems mix, transport, separate, and heat these liquids and gases, while microsensors provide control. Microfluidic devices leverage laminar rather than turbulent flow, leading to more consistent fluid movement. They also make use of the surface-to-volume ratios and surface interactions that are unique to the microscale.²

The benefits of microfluidic technologies include more consistent and predictable liquid flows, reduced material and energy consumption, and greater control — all of which result in improved efficiency and accuracy at reduced costs.³ They also enable the simultaneous performance of multiple operations, facilitating both process automation and rapid implementation and analysis of physical experiments while ensuring high data quality.^1,4

In the biopharmaceutical industry, potential applications for microfluidics include diagnostics; analytics; process development, including high-throughput screening; biomanufacturing; and drug delivery, as well as therapeutics. This technology is enabling personalized medicine and point-of-care diagnostics, automation of a wide variety of laboratory workflows for greater speed and efficiency, unique approaches to cell culture, and continuous manufacturing of lipid nanoparticles and microparticles for oral solid dosage formulations.¹

Of particular interest in many biopharmaceutical applications is droplet microfluidics, which involves the generation of droplets of a specified diameter.⁵ This technology is used in high-throughput screening, bioprocessing, and analytical applications.

Within microfluidic devices, fluid flow can be pressure driven or based on electroosmotic flow.⁵ Construction materials include both rigid and flexible polymers. Several methods have been investigated for the manufacture of microfluidic devices. One of the most attractive options is 3D printing, as it allows for construction of complex devices without requiring highly skilled workers operating at difficult scales.

Enhancing Analytical Capabilities with Microfluidics Technologies

One of the common challenges in early drug development and diagnostic method development is access to only minute quantities of material. The inherent nature of microfluidics overcomes this issue; analyses not only require minute sample quantities but often can be completed extremely rapidly and in parallel within a very small footprint, saving both time and money.⁵ In addition, unlike miniature liquid-handling systems, microfluidics, owing to the unique behaviors of materials at the micro scale, can provide significantly more information about the physical properties and behaviors of molecules. Furthermore, the greater design freedom available in the microfluidics space enables implementation of a wider array of scale-down models and the use of many different analytical techniques, as they can be integrated with many spectroscopy instruments that do not work with microplates.

Any analytical methods incorporated into microfluidic devices must be extremely sensitive and accurate. One of the most common techniques, therefore, is fluorescence.⁵ Microfluidic devices have also been used for DNA and protein analysis, and, when linked to mass spectrometry instruments, have enabled accurate evaluation of picomole quantities of peptides.⁶

Microfluidic sensors leveraging cells, enzymes, antibodies, aptamers, and other biomolecules can also be used as sensors for the detection of small molecules, proteins, antigens, RNA/DNA, electrolytes, gases, and other substances of interest for many different types of applications, including clinical diagnostics.⁴ In fact, some microfluidic devices integrate cell isolation, cell lysis, DNA extraction, polymerase chain reaction (PCR), and detection processes.

Digital droplet PCR (ddPCR) is one of the best-known and most widely adopted methods leveraging microfluidic technologies in the biopharma industry today,⁴ particularly for analysis of viral vectors. With this technique, it is possible to evaluate samples much more rapidly, with the potential to increase throughput from hundreds to millions of analyses in the same timeframe.

Examples of microfluidic analytical devices include the 2100 BioAnalyzer (Agilent Technologies) for automated electrophoresis, the LabChip GXII (Perkin Elmer) for automated sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the Biacore™ X100 (Cytiva) for surface plasmon resonance analysis.⁵

Meanwhile, researchers at Texas A&M University and the U.S. Army Combat Capabilities Development Command Army Research Laboratory (ARL) have developed a droplet microfluidic device (DNA ENhanced TRAnsfer Platform) that automatically transports DNA from one cell to another across a wide variety of cell types, including highly engineered cells and uncommon natural strains, enabling rapid analysis of genetic modifications, which is typically a time- and labor-intensive activity.⁷

Benefits of Microfluidics for Process Development

One of the main foci of biopharmaceutical companies today is increasing the efficiency and productivity of process development to reduce time to the clinic and the market and to reduce the costs of new drugs. Unsurprisingly, microfluidics has attracted significant attention for its potential to facilitate achievement of these goals. Integrated devices can be used for rapid process characterization and optimization.⁸

Examples of microfluidic devices under investigation for use in bioprocess development include devices for DNA assembly and transformation, high-throughput screening of cell libraries, and bioreactors for process optimization.⁹ Many perform operations on single cells using the unique fluid-flow properties within microfluidic devices comprising picoliter-scale compartments, gel beads, and/or single-cell imaging capabilities. Underlying all these microfluidic technologies are solutions for increasing throughput, parallelization, and automation. A key application highly sought in the industry is acceleration of cell line development, a typical bottleneck in chemistry, manufacturing, and controls (CMC) for drug development.

Some technologies leverage combinatorial DNA synthesis in droplets.⁹ This may be coupled with electrowetting and digital droplet microfluidics techniques for transferring DNA into cells. Transfection of hard-to-transfect cells has also been shown to be facilitated within microfluidic devices. Even cell sorting can now be performed in microfluidic devices using technologies such as fluorescence-activated cell sorting (FACS), magnetic sorting, cell size–based sorting by deterministic lateral displacement or inertial microfluidics, acoustophoretic sorting, and droplet sorting. Label-free detection methods that can be used within microfluidic devices are also being developed, including some based on Raman spectroscopy. Cell growth–based selection assessing the quantity of cells or metabolic profiles is also being explored.

Efforts are also being directed to the development of microfluidic bioreactors.^4,5,8–10 Theoretically, microfluidic systems can be used to mimic batch, fed-batch, and perfusion processes.⁹ Furthermore, the design of these devices and the choice of materials can provide not only temporal and spatial control but also specific surface conditions and nano-structured topographies to meet specific needs. Devices have been generally focused on adherent cell culture and designed to mimic both 2D and 3D systems.¹⁰ Standardization will be essential if such microfluidic devices are to see widespread adoption for bioprocess development.

Microfluidic devices serving as miniaturized bioreactors that mimic batch manufacturing are often based in microwells and use microfluidic droplets for cell culture.⁹ Some are designed to enable cell culture of single cells in a high-throughput manner to rapidly identify the highest-performing clones.⁴ These devices are at an early development stage, but various types of cells have been shown to grow within such devices. For instance, picoliter bioreactors assembled for single-cell fermentation set up within microscopes have been used to screen cell culture processes with real-time visualization.⁵

Microfluidics can also benefit process development of downstream operations.⁵ Researchers have demonstrated the potential for microfluidic solutions for chromatography, including the purification of monoclonal antibodies and parallel chromatography runs for nine different protein concentrations.

One important key to the success for microfluidic devices designed to facilitate bioprocess development is the incorporation of effective microsensors. Such devices could not only increase throughput and reduce cost but also provide more reliable and accurate data than what is obtainable using miniature bioreactor systems.^10,11 Typical examples include optical, spectroscopic, and image-analysis techniques. The best solutions involve non-invasive methods that do not disturb the cell culture.

Advantages of Integrating Microfluidics in Biomanufacturing Operations

The use of microfluidic devices for actual biopharmaceutical manufacturing and not just high-throughput screening in process development remains largely conceptual at this point, although many academic and industrial research groups are actively investigation the deployment of various microfluidic technologies.

The intent is to miniaturize existing production systems and protocols in microfluidic cartridges to leverage the precise control, parallelization, and other benefits that microfluidics offers.⁸ Two leading potential applications are parallel production of cell lines and continuous drug substance encapsulation to generate drug products in novel delivery systems.

Researchers at the Massachusetts Institute of Technology have developed an industrial-scale spiral inertial microfluidic device for continuous clarification of proteins following perfusion-based mammalian cell culture that can achieve clog-free cell retention and high product recovery at a volume process rate of 1 L/min.¹² The microfluidic cell retention device could therefore potentially be scaled to process harvest fluid from a 1,000-L bioreactor.

A microfluidic device capable of high-throughput size-based cell separation using inertial sorting was then developed to enable microfluidic perfusion cell cutlure.¹³ It was shown to successfully achieve high-throughput and high-concentration removal of dead cells from culture. The microfluidic perfusion and clarification systems were integrated with a novel nanofluidic filter array providing continuous online purity monitoring of the proteins in the cell culture supernatant. The researchers believe that the microfluidic clarification device and coupled process analytical technology (PAT) solution addresses key issues with conventional membrane filiation, including filter clogging, low product recovery, manual sample preparation, and offline analysis. They also suggest that the nanofluidic filter array could replace the existing offline analytical technologies for protein purity monitoring.

Improving Drug-Delivery Systems with Microfluidic Device Technology

One of the most promising potential applications for microfluidics in drug manufacturing is the production of encapsulated drug substances as nanoparticles that can facilitate drug delivery.¹⁴ Mixing of two or more immiscible fluids in laminar flow in the presence of an appropriate surfactant results in the formation of nanoscale emulsion droplets with uniform properties. As a result, microfluidic technologies are attractive for the generation of nanoparticles in which a drug substance is encapsulated. They can also be used for encapsulation of cells into hydrogels to generate artificial organs. In a similar vein, microfibers generated using microfluidics can be constructed into implantable patches for drug delivery. It is also worth noting that this technology may be useful for enhancing the efficiency of transfection processes.

Enabling Organ-on-a-Chip Technologies with Microfluidics

Another significant application of microfluidics involves organ-on-a-chip technologies, which are designed to mimic the functioning of human organs and are used in diagnostic and drug discovery and development applications, as well to increase understanding of organ physiology.⁴ They often comprise three-dimensional channels lined with living cells and appropriate mechanical and biochemical microenvironments to replicate the interactions and interfaces that take place in a specific type of tissue. Media flowing through the channels mimics the bloodstream and is injected with a compound of interest to see how it impact the behavior of the cells.

Potential Diagnostic and Therapeutic Applications for Microfluidics

Microfluidics can be used not only in bioprocess research, development, and manufacturing but within diagnostic devices and drug products themselves.

One diagnostic technology of note enables “minimally disruptive fluid and cell manipulations within living cultures.”¹⁵ The device was shown to seed and culture multiple types of cells in specific special patterns and enable removal of undesired adherent cells using a focused trypsin flow. Fluids can also be sampled, and “biopsies” of cells anywhere with the culture chamber can be performed for external analysis. External electronics automate device operation, enabling computer-controlled tissue engineering. The researchers anticipate scaling the technology to three-dimensional microfluidic scaffolds to facilitate the development of tissue-engineering technologies with clinical applications.

Additionally, microfluidic cell-separation devices are being employed for diagnostic applications in which certain cells must be isolated from blood.⁴ In particular, microfluidic technologies are useful for detecting bacteria in blood, which is critical for the diagnosis of blood sepsis. Similar approaches can be used to separate white and red blood cells, an important step in many medical diagnostic applications.

The Business of Microfluidics

Several companies are pursuing the commercialization of microfluidic technologies for various biopharmaceutical applications. Selected examples include:

• Finnadvance: multichannel 3D microfluidic organ-on-a-chip systems that simulate the activities, mechanics, and physiological response of entire organs and organ systems;¹⁶
• MicroMedicine: Sorterra, an automated microfluidic, cell-isolation technology that rapidly processes millions of cells;¹⁶
• CellFe Biotech: a scalable, high-throughput microfluidic technology for the efficient delivery of gene-editing molecules into cells;¹⁷
• MFX (previously MicrofluidX): Microfluidic cell culture technology for parallel implementation (the Cyto Engine™ Stack) to enable more efficient biomanufacturing of cell and gene therapies;¹⁸
• Microfluidics: Lab-, pilot-, and production-scale Microfluidizer^® Processors for cGMP-compliant fluid homogenization;¹⁹
• OneCyte Biotechnologies: a high-throughput integrated microfluidic platform for single-cell screening, culture, and selection enabling rapid single-cell analysis with during drug discovery and development;²⁰ and
• Stämm: a novel bioprocessor incorporating cell line on-a-chip and bubble-free bioreactor and leveraging microfluidics and 3D-printing technologies for continuous industrial production of biologics and cell therapies.²¹

Conclusion

Significant advances in micro- and nanoscale fabrication and sensor technologies are enabling the design and construction of highly complex microfluidics devices with a broad array of applications in the biopharmaceutical industry. New solutions are being explored in both academic and industrial settings, with much progress being driven by innovative small and emerging pharma and biotech companies targeting applications in precision medicine, point-of-care-diagnostics, drug discovery and development, lab automation, bioprocessing, drug delivery, and more.

Underlying the growing interest in microfluidics technologies is their potential to increase the efficiency and productivity of all aspects of drug discovery, development, and manufacturing, as well as some aspects within healthcare. With ever-growing pressures to reduce the time and cost of drug development and manufacturing and the move toward targeted and personalized medicine, microfluidic technologies will likely play an increasing role in future advances within the biopharmaceutical and overall healthcare industries.

References

1. “The Microfluidics Revolution: Transforming Research and Industry.” 3D AG Blog. Accessed 25 Mar. 2024.
2. Bahnemann, Janina and Alexander Grünberger. “Microfluidics in Biotechnology: Overview and Status Quo.” in Microfluidics in Biotechnology. Springer International Publishing. 2022.
3. Chan, HonFai, Siying Ma, Ka, andW. Leong. “Can microfluidics address biomanufacturing challenges in drug/gene/cell therapies?” Regenerative Biomaterials. 3:87–8 (2016).
4. Enders, Anton, Alexander Grünberger, and Janina Bahnemann. “Towards small scale: overview and applications of microfluidics in biotechnology.” Molecular Biotechnology. 14 Dec. 2022.
5. Silva, Tiago Castanheira, Michel Eppink, and Marcel Ottens. “Automation and miniaturization: enabling tools for fast, high-throughput process development in integrated continuous biomanufacturing,” Chem. Technol. Biotechnol. 2021. DOI 10.1002/jctb.6792.
6. Barry, Richard andDimitri Ivanov. “Microfluidics in biotechnology.” Nanobiotechnology. 2:2 (2004). doi: 10.1186/1477-3155-2-2.
7. Rose, Rachel. “Microfluidics and synthetic biology expanding capabilities of biotechnology.” Texas A&M Engineering News. 2 Sep. 2021.
8. Gutierrez, Lorenzo. “Integrating Microfluidics into Biomanufacturing.” Starfish Medical Blog. 2 Feb. 2022.
9. Bjork, Sara M., and Haakan N. Joensson. “Microfluidics for cell factory and bioprocess development.” Current Opinion in Biotechnology. 55: 95-102 (2019).
10. Marques, Marco PC and Nicolas Szita. “Bioprocess microfluidics: applying microfluidic devices for bioprocessing.” Current Opinion in Chemical Engineering. 18: 61–68 (2017)
11. Tsai, Ang. “Bioprocess Microfluidics: The Use of Microfluidic Devices in Bioprocessing.” Bioengineer. & Biomedical Sci 12:8 (2022).
12. “Industrial-Scale Spiral Inertial Microfluidics Boost Productivity.” Gen Eng News. 10 Aug. 2022.
13. Kwon, Taehong. “Advanced Micro/nanofluidic System for Continuous Bioprocessing.” Micro/Nanofluidic BioMEMS Group.” Accessed 25 Mar. 2024.
14. Chan, HonFai, Siying Ma, W. Leong. “Can microfluidics address biomanufacturing challenges in drug/gene/cell therapies?” Regenerative Biomaterials. 3: 87–98 (2016).
15. Tong, Quang et al. “An Automated Addressable Microfluidics Device for Minimally Disruptive Manipulation of Cells and Fluids within Living Cultures.” ACS Biomaterials Science & Engineering. 27 2020.
16. “5 Top Microfluidics Startups Impacting The BioTech Industry.” StartUs Insights. Accessed 24 Mar. 2024.
17. “Building the future through microfluidics.” CellFe Biotech. Accessed 25 Mar. 2024.
18. Kirk, David. “UK Startup to Manufacture Cell and Gene Therapies with Microfluidics.” Labiotech. 24 Apr. 2020.
19. “Biopharmaceutical Manufacturing Equipment (cGMP).” Microfluidics. Accessed 25 Mar. 2024.
20. “Forging the Future of Single-Cell Discovery.” OnceCyte Biotechnologies. Accessed 25 Mar. 2024.
21. Stämm. Accessed 25 Mar. 2024.
22. “Microfluidics’ Emerging Trends: Revolutionizing Research and Industry.” EnCell Global Blog. 14 Jun. 2023.

Originally published on PharmasAlmanac.com on April 17, 2024.

Current preclinical testing using assays based on cancer cells and animal models often results in high failure rates for drug candidates in the clinic. Microphysiological systems like Altis’s RepliGut™ human intestinal tissue model more accurately reflect human physiological environments, enabling more efficient and cost-effective drug discovery and development.

Using Human Tissue for Drug Screening

The inadequacies of current preclinical drug development methods have left the industry with an unacceptable clinical trials failure rate and nearly $200 billion spent every year on research and development. A key factor driving clinical failures is the use of tumor cell lines such as Caco-2 cells and animal usage in preclinical testing. Thus, too often ineffective drugs reach clinical trials, while potentially efficacious medicines do not. An alternative and more promising approach to drug discovery, preclinical testing, and toxicity screening is the use of human tissue models. 

Combining Engineering, Chemistry, and Physics

Altis Biosystems was spun out from the University of North Carolina at Chapel Hill; our company’s goal is to apply engineering, chemistry, and physics to new strategies that lead to successful, faster drug development. We utilized methods from the semiconductor and electronics industries to build small-scale devices in an efficient, scalable, and cost-effective manner, seeking to marry this approach with stem cell technology. 

We focused on the large and small intestines because they play crucial roles in the absorption and metabolism of drugs. Studies have proven that the bacterial composition in the colon can modulate immune responses and may play a role in many diseases, which also contributed to our interest.

Starting Simple

Capitalizing on research on growing organoids from human intestines, Altis has developed a next-generation intestinal platform for in vitro testing during drug development. The platform produces a layer of human intestinal stem and differentiated cells — either of the large or small intestine — that can be used for compound screening, disease modeling, and microbiome research.

RepliGut™ tissue constructs are polarized monolayers that express tight junction proteins and can be tailored to include stem/progenitor cells, differentiated cells, or both, representing all major cell lineages in physiologic ratios. Each tissue sample on the RepliGut™ kit features a patent-pending biomimetic scaffold that separates RepliGut™ cells from the cassette’s porous membrane and allows cells to survive for a prolonged period of time. Luminal and basal reservoirs allow compounds and additional cell types to interact with the epithelial cells for side-specific assays.

Providing Diversity

With our donor bank comprising tissue from multiple donors of different demographic backgrounds, Altis is able to make realistic models that recapitulate many different physiologies and thus provide a faithful representation of the diversity of the human population. We have developed a suite of platforms and assays tailored for specific applications to investigate drug absorption, transport, toxicity, and inflammatory cytokines. 

Our commercially available RepliGut™ system is compatible with the vast majority of assays commonly used in the biopharmaceutical industry. Gene expression, protein expression, cytokine production, permeability, transport, toxicity, inflammatory response, and other attributes can be evaluated using ELISA, PCR, transepithelial electrical resistance (TEER), immunofluorescence, mass spectrometry, microscopy, and other techniques.

Differentiated from Other Organs-on-a-Chip

Unlike most other human intestinal tissue models, Altis has included both stem cells and differentiated cells. The presence of stem cells is important for evaluating tissue repair and drugs to treat cancer. The RepliGut™ platform can also include cells that secrete hormones, cytokines, and other biochemicals involved in the proper function — and dysfunction — of the large and small intestine. Most other models do not have this capability. We also include mature enterocytes, goblet cells, and enteroendocrine cells. These combined features result in a more sophisticated system than other tissue models, allowing for more faithful predictions of the impact of drug candidates. 

Fits into Existing Workflows

In addition to providing a human tissue model for the large and small intestines that faithfully recapitulates the behavior of these important organs from a wide array of demographic groups and for many different applications, Altis has focused on developing a platform technology that is easy for researchers to use. 

Unlike many microfluidic devices that require the purchase of large, specialized instruments and the connection of tubes and pumps, the RepliGut™ system has been designed in a footprint that slots into existing workflows that the biopharmaceutical industry already uses. 

The RepliGut™ kit includes a Transwell multiwell plate comprising 6, 12, 24, or 96 wells, with each well containing a Transwell insert with an individual tissue sample. Each kit also includes the materials needed to culture the cells, including stem cells and media. To populate the device, the stem cells are placed on the scaffold, and a few media changes are completed via pipetting. The tissue structure self-assembles into the lineages populating the intestinal epithelium.

Moving to the Next Level

Microbiome-based therapeutics is a rapidly growing field that would benefit greatly from access to better preclinical testing methods. It is clear that bacteria in the colon influence every aspect of human body functions, from mentation and satiety to metabolism and response to chemotherapeutics.

Education of the immune system takes place to some extent in the gut. Incorporating a functioning immune system into the RepliGut™ epithelium will in the future facilitate exploration of the mechanisms involved in immune education and the impact of different microbes.

The intestine also plays a significant role in the nervous system. One of the largest sites for the production of serotonin — a neurotransmitter — is in the gut. Combining aspects of the nervous system into the RepliGut™ system is an exciting avenue currently under investigation at Altis.

Leaky gut is a significant problem from the neonate to the adult. A robust and reliable human tissue model that enables the investigation of this phenomenon and drugs that can prevent or treat gut damage holds tremendous promise. 

Developing RepliGut™ models specifically designed to enable investigation of treatments for colon cancer presents yet another opportunity for Altis. Among all types of cancer, colon cancer causes the third most deaths among both men and women.¹

In the near term, Altis has much interesting work ahead of us as we add other types of tissues and features to the RepliGut™ human tissue model for the large and small intestines. Our hope is to eventually supplant old tumor cell (Caco-2) models with models like our RepliGut™ platform that more accurately reflect what occurs in the human body, enabling more efficient and accurate drug discovery and development. 

Reference

1. “Key Statistics for Colon Cancer.” American Cancer Society. 29 Jun. 2020. Web.

Originally published on PharmasAlmanac.com on September 29, 2020.

In the company’s 10^th year of business, 908 Devices’ Chief Executive Officer Kevin Knopp, Ph.D., reflects on the company’s history of innovation and discusses how they are leveraging novel microfluidics techniques and miniaturized mass spectrometry to serve a growing user base that spans from biopharma to homeland security, in conversation with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D.

David Alvaro (DA): Can you give me a concise overview of 908 Devices and the history of the company from its inception to today?

Kevin Knopp (KK): As of February 2022, it’s been 10 years since we started the company with a mission to change how and where chemical and biochemical analysis are being done by undertaking a platform change to a gold standard technique: mass spectrometry. We see it as a disruptive platform based on a technology we call “higher-pressure mass spec.” We’re taking the gold standard, laboratory-grade mass spectrometry technology out of the centralized laboratory environment where it normally lives and bringing it to what we call the “point of need.”

In terms of the evolving technology, it’s analogous to what we see in the computer industry, where there has been a progression from large, centralized supercomputers to desktops, laptops, and even mobile devices. We see parallels for chemical and biochemical detection, and we view our products as analogous to simple and convenient handheld and desktop devices, in contrast to large, supercomputer-like devices. As a result, our products are much more accessible and serve a much broader range of customers. We joke about this, but it’s true: our customers span “the FBI to the FDA” — from forensics applications to biopharmaceutical applications.

DA: Did you start with a particular use case in mind and expand from there, or were you considering all the different possibilities for the technology from the beginning?

KK: We always thought about the many possibilities. We could see mass spectrometry and chromatography-based technologies that large instrumentation companies like Agilent and Waters were making to serve a range of customers, both in the life sciences and in applied markets, and we could draw parallels. We saw that we could launch solutions to serve clients directly who would normally send their samples to these traditional instruments in centralized labs.

We started from a technology perspective, working on high-pressure mass spectrometry itself. We launched a handheld, lunchbox-size mass spectrometer, MX908, which was an entirely new category of mass spec devices at that time. Then, we started working on the applications — it fits naturally into areas like counterfeit drug detection and forensic toxic hazard detection.

Still, most of the traditional mass spectrometers on the market have some sort of chromatography on the front end, which is vital for analyzing complex samples. So, as we desired to broaden our application set and enable innovation in the life sciences, particularly biopharma and bioprocessing, we started developing a chromatography alternative that is based in genomics — a microfluidic device similar to what is used in genetics labs, where it’s a capillary electrophoresis (CE) device. What we produced is a CE microfluidic device that separates on the basis of size and charge to perform the initial separation that would then be fed into the mass spec.

In short, our development process began with the backend mass spec, which allowed us to achieve measurements with key forensic applications, and then we developed the technology further with microfluidics, which has allowed us to process liquid phase samples, opening the door for applications in biopharma and bioprocessing.

DA: Can you expand more on the fundamental foundational technology: what is unique about high-pressure mass spec, and why it is now possible to create these devices that are smaller than what anyone had ever built before?

KK: We partnered 10 years ago with our science founder, Professor J. Michael Ramsey at the University of North Carolina at Chapel Hill, who had previously been part of a research group at the Oak Ridge National Laboratory working on making a smaller mass spec using miniaturization technology and microfluidics. The common approach at the time was to simply try to shrink the existing technology, but a major issue was that mass spec requires a powerful vacuum pump that needs to reach outer space–levels of vacuum pressure, so reducing the size of the pump was a losing battle. Instead, Mike theorized a solution that does not require such vacuum and started working on realizing an ion trap design that could enable mass spec to work at higher pressure.

High-pressure mass spec still requires a vacuum, but it operates much closer to atmospheric pressure, which allows us to use a pump that can fit in the palm of your hand. Mike was able to accomplish this by using a microscale ion trap. As you miniaturize the dimensions of the trap and increase the drive frequency, theory shows you can achieve comparable resolution even at higher pressures — and in a much smaller form factor.

DA: Within the pharma space, where can mass spec be performed with 908’s technology that it couldn’t before for practical reasons?

KK: Each of our products has enabled customers, across pharma and other industries, to use mass spec as a newly accessible solution for longstanding or ongoing problems. Today, a lot of the application of our MX908 handheld device is in the detection of counterfeit pharmaceuticals and related substances. There is currently a massive overdose problem among young adults who purchase pills of what they believe are conventional drugs like Adderall and Xanax, but in which the active ingredient has been replaced with fentanyl. The Journal of the American Medical Association conducted a study that showed that the number of adolescent deaths from drug overdose doubled between 2010 and 2021, even though teen drug use is at a historic low, and they noted that fentanyl was involved in 77% of those deaths. We have a wide range of customers for our handheld device, most often law enforcement agencies, customs organizations, or other health and safety organizations trying to stop the tide of counterfeit drugs and fentanyl from entering our communities.

With our desktop mass spec REBEL, we serve the top 20 large pharma and biopharma companies who use the power of mass spec to measure key process parameters, critical quality attributes, and ultimately product quality attributes. We’re providing this technology directly to customers who could never have purchased a mass spectrometer and would normally send samples to a laboratory and face slow, cumbersome turnarounds.

REBEL is designed to sit next to a bioreactor in a process development lab. Today, REBEL is able to measure 32 analytes: amino acids, metabolites, vitamins, and so on. Biologists are able to get that data as often as needed throughout their run of a bioreactor, which could be a 12- to 14-day process. This frequency of data collection is unheard of. Researchers would normally take samples infrequently, freeze said samples, and send some or all out for analysis after the entire run is complete. However, those data are in the rearview mirror; they are not actionable. The rapid and frequent feedback from both our desktop and handheld products allow biologists to adjust and tailor their processes. At 908 Devices, we are excited to create access to high-quality chemical and biochemical data in an actionable way that enables users to adjust their workflow.

DA: How does bringing this formerly offline technology to at line help to advance the industry’s goals around the Bioprocessing 4.0 vision?

KK: Taking the power of mass spec out of the central lab and bringing it ‘at-line’ next to the bioreactor is a major step. Now, researchers can take a sample from the bioreactor and place it in our device for measurement, which is a manual process. There is a lot of interest to push forward toward “Bioprocessing 4.0,” and an ‘on-line’ connection of analyzers and mass spec would be a powerful technique to enable this. We see our future will be to take our technologies and connect them in an on-line, integrated fashion to reduce sampling error and reduce labor.

Miniaturizing and increasing accessibility to this technology supports the Bioprocessing 4.0 revolution by closing the loop in research and development. It quickly generates a data set that can be fed into predictive models and be used as feedback to help adjust process attributes in a live, real-time fashion. By closing the loop, your entire process development time can shrink. Rather than many combinatorial, parallel design of experiments (DOEs), you can do a single timed course and adjust feeds and process in real time to get the best outcome of critical quality attributes, such as titer.

If you were to send out samples for analysis at a core lab, a typical turnaround time is two to three weeks, but there is an additional logistics burden that adds a few weeks of delay. In contrast, by having the REBEL cell culture analyzer right next to the bioreactor, you can have a meaningful result in 5–10 minutes, and then you can run it again as often as you like.

DA: Do the benefits for researchers go beyond simply making processes quicker and more efficient?

KK: Beyond the benefits of speed, the technology also enables manufacturers to use the data in ways that were not logistically practical before, and we’re working with key opinion leaders to drive its use in new areas. We work with groups like Johns Hopkins University, which wants to make in silico predictive models of their bioreactor but needs inputs into those models. The comprehensive REBEL analyte panel enables them to tailor predictive models and ultimately to more accurately predict outcomes.

We’re also collaborating with groups at Emory University that are working to optimize CAR-T cell therapies. They’re looking at these novel microbead libraries for manufacturing, and REBEL is allowing them to measure the amino acids and process attributes to look at the activation of T cells with their microbeads attached. Beyond boosting efficiency, REBEL is enabling a new type of quick monitoring to inform their feeding schedules and to control processes in a way that hasn’t been done before. And the list goes on: we’re working with groups at MIT, CPI, Boehringer Ingelheim, and even Amgen and GSK in areas of critical quality attribute monitoring in a much more rapid, simple way.

DA: Can you expand on the microfluidics technology and what it can enable?

KK: We have a technology that’s a microfluidic chip about the size of a business card and designed with an approximately 22-centimeter-long channel that weaves back and forth across the chip. It’s designed to desalt, concentrate, and separate on the basis of size and charge over a multi-minute period that can range depending on analyte size but is generally 2–8 minutes. Then, it electrosprays a mist into a mass spectrometer for analysis.

Since the vast majority of mass spec machines on the market have some variation of chromatography that helps separate the sample and spoon feed it to the mass spectrometer, allowing the mass spectrometer to decipher the results for complex samples more easily, we designed our microfluidic technology to accomplish this critical step.

Two of our products use this microfluidics technology. Our ZipChip device, which is an open-access platform that plugs directly into a mass spectrometer, allows us to work with researchers in central labs on current assays, like critical quality attributes. We then took our microfluidics technology and connected it to our high-pressure mass spec in a very purpose-built way to create our REBEL product.

Our technology also opens the possibility of working with innovators on new areas, new applications, and new use cases. We have folks in the academic space that embark on new projects using our technology who are looking beyond critical quality attributes in biopharma and are researching biomarkers for disease states in clinical samples. Other folks are using REBEL more in the context of synthetic biology, even looking at new solutions in the realm of pesticides.

We also have some very interesting next-generation products and technologies coming up in proteomics, where we are using these microfluidic chips to separate many, many proteins at low concentrations much more quickly compared with chromatography techniques.

DA: Are there any limitations or particular cases where it would make more sense to use conventional mass spec rather than REBEL?

KK: For sure. It’s analogous to a large supercomputer versus a laptop or a tablet. The large devices have more “horsepower” and performance settings, but there’s so much that can be done with a laptop or a tablet. We see REBEL as a purpose-built device, like an appliance or a medical device. We look at things like true positive rates and false positive rates, and we also pay attention to things like receiver operating characteristic (ROC) curves that quantify the system because we’ve designed it to do a particular job.

Our REBEL and handheld devices are purpose-built and meant to do a particular job: measuring 32 analytes next to a bioreactor over and over again to inform that bioreactor or testing for trace levels of counterfeit drugs. Our ZipChip device is ideal when customers need an open architecture: they want to discover and develop new application use cases. The tradeoff is that REBEL and ZipChip are much more affordable and suitable to users who are experts in their own fields but don’t want to do method development or to be experts in chromatography or in many cases even mass spectrometry.

DA: How important to you is the user interface, and for what kind of user are you ultimately designing the system?

KK: I appreciate that question because I think it touches on something that really needs to be well understood. We have discussed our hardware capabilities that shrink down the mass spectrometer and simplify the sample prep and separation for microfluidics. But if we had the same interface as a conventional mass spec with all the nobs and the squiggly lines at the output, then we wouldn’t really have enabled a broader reach. In order for our technology to actually enable customers, it is critical that we couple all the machine learning, algorithms, and automation that we provide with a good graphical user interface.

If you take the device out of the central lab and give it a smaller screen but still need an expert operator with a Ph.D. to run it, you’re only halfway there — and if you’re only halfway there, you really haven’t done it.

We are quite literally making devices that can be used by a fireman, a law enforcement professional, a mailroom operator, or someone in a health and safety organization, while also designing our tools to enable biologists to get quantified information for their bioreactor. Our devices must help each of these customers do their jobs faster and with more informed decision-making capability. When our team talks with these customers, we’re not talking about the technology; we’re talking about the analytes — the results and what these results enable them to do.

DA: Can you discuss the role that machine learning plays?

KK: At 908 Devices, we have three pillars of technology. We discussed the high-pressure mass spec technology and the fluid-handling side — including microfluidic sampling and separation — and the third is the algorithmic, machine learning, and related user experience side, which is critical to enabling the control over processes that we discussed earlier. For example, let’s say that a user is measuring a counterfeit pharmaceutical using our handheld device. When our handheld is showing the user a result, it not only indicates if it detected fentanyl, it further specifies if it found fentanyl versus carfentanil, remifentanil, or other substances that could be in the sample. And when it gives a precise identification for the drug, it literally puts a name up on the screen for the user to see. It’s not showing any squiggly lines or any other type of “science-y” data. It’s showing the user a clear identity in a reliable, robust way. To make this possible, we need all the machine learning underneath that drives the device’s ability to interpret results.

Similarly, with our REBEL device next to a bioreactor, we’re giving biologists quantified information for all of those 32 analytes. There are no calibration curves, and there is no need for manually looking at migration times and the mass spec data and then trying to do calibration curves. All that’s been taken out, so users just get a report that tells them: here are your 32 analytes, and here’s your quantitative level. We leverage all of our machine learning automation to deal with the environments to give those robust results.

DA: Do you have a clear path forward in terms of what the next generation would look like or additional functionality and connectivity that you want to develop?

KK: We have a very strong belief that smaller, faster, and simpler analytics can take us very far across all of our end markets. If we zoom in to where we are serving the life sciences sector, including biopharma and bioprocessing, we’re very excited to use those analytics and that expertise to move from at-line to on-line to offer more analytes in an on-line configuration: being able to stream a broad set that’s very informative to how users will run and optimize their processes. Today, we see clearly that analytics are a cornerstone of Bioprocessing 4.0, and we are working hard to make our analytics simpler, more connected, and more robust for measuring key process attributes all the way to critical quality attributes.

DA: Shifting gears a little, how have things evolved for 908 Devices and your team since you took the company public a couple years ago?

KK: We were very fortunate in December 2020 to become a public company, and I often say that it’s been like an injection of caffeine, because it brings dollars; it allows us to scale. We’ve grown our team to around 200 people, which has really allowed us to double down on our R&D efforts and build out both our channels and applications and our sales and commercial teams to better serve our customers. We’re working hard, and I think that we’re successfully leading innovation and maintaining our entrepreneurial spirit even as we scale.

We were recently notified that we’ve ranked number 48 [out of 100] on Fast Company’s fourth annual Best Workplaces for Innovators list; so that’s very exciting news for our company. We resonate with that, and we work hard every day. Even earlier today, we were talking about the impact that these technologies and our customers have in these marketplaces. The prospect of stopping lethal counterfeit drugs and the prospect of helping customers get therapies to market faster are exciting to us.

DA: Looking to the future, what comes next for 908 Devices?

KK: One area in which we anticipate seeing developments is the field of advanced therapies from the process development labs that use REBEL. As the pipeline of advanced therapies that these researchers are trying to develop grows and becomes more complex, these folks need have quick insights to do DoE much more rapidly to bring their therapeutics onto the market. The number of investigative new drug (IND) filings has really been increasing many-fold over the span of just a few years. During Advanced Therapy Week earlier this year, there was a conference talk about how the number of new cell and gene therapies that reach the market is expected to be 10–20 per year, and that they’re expecting to have approvals through 2025.

So, there is a lot of momentum in those areas, and we think that having REBEL’s insights is game-changing. It’s also a little bit of a Wild West right now between the directions that autologous cell therapies versus allogeneic therapies will go and how both could benefit from online and integrated sensors during manufacture. We fundamentally believe that analytics in these areas need to be simplified, because the development of these therapies requires the generation of broad sets of analytics that will be streaming off of these processes. We think our customers will do great things to enable some of these new classes of therapies.

Originally published on PharmasAlmanac.com on September 27, 2022.

Novel microfluidics cell engineering technology from CellFE provides superior delivery of the largest range of complex payloads to cells for cell therapy development from research scale to commercial manufacturing. The rapid nature of the process, which is readily scalable, reduces cell therapy vein-to-vein manufacturing time and leads to improved cell health and final product quality.

Increasing Access to Cell Therapies

Despite the tremendous transformative potential of cell therapies, only a fraction of the patients who could benefit have access to these lifesaving therapies. This is a consequence of several challenges, including the limited number of global locations that provide cell therapies, the long vein-to-vein time associated with manufacturing, and the high costs of manufacturing.

Driven by a motivation to expand access to these curative therapies, CellFE addresses the latter two issues through manufacturing innovations using its microfluidic cellular engineering technology. The CellFE platform offers the following advantages over existing technologies: gentle processing of cells, rapid cell recovery that enables complex gene-editing with high transfected cell yields, and a simple process that leads to higher quality of genetically engineered cells that are critical to robust and durable therapies.

From Academic Studies to Commercial Products

CellFE began as an academic endeavor to explore cell mechanical responses to large deformations occurring within short timescales. Our studies led to a foundational hypothesis that cells could withstand large deformations for very short periods of time with no impact on the biology. While investigating this hypothesis, we discovered a practical application of delivering large and complex payloads into cells for engineering cell therapies.

Delivering a payload into a cell requires two things: a pore in the cell membrane (i.e., a transient and reversible permeabilization) and a driving force for transporting the payload into the cell interior. The most common approaches for delivering molecules into cells for cell therapy are the use of viral vectors and electroporation. Viral vectors are the predominant platform for integrating a functional transgene into the cell, though their use results in high manufacturing costs, cumbersome logistics, and risks associated with random genetic integration. Electroporation creates pores in the cell membrane by applying repeated electrical fields, after which molecules passively diffuse or are electrophoresed into cells before the temporal pores reseal. Exposure of cells to repeated electrical pulses can cause irreversible damage to cells that, along with other harmful effects, hinder cell proliferation. With the CellFE technology, my research team found that, when cells are compressed abruptly at short timescales (<1 milliseconds), they act much like sponges in that they quickly reduce in volume and return to their original shape and thereby actively transport exogenous material into their interior. Anything contained in the cell medium is carried by this flow into the cells, providing a powerful mechanism to deliver larger, more complex gene-editing payloads.

Several applications of this technology have been investigated. With medical partners at Emory University, we found chimeric antigen receptor (CAR)-T cells could be generated, and at greater yield, a result that indicated distinct and improved outcomes from other delivery methods. In addition to the delivery of gene-editing payloads, we also studied the delivery of nanoparticles and other modifications to cells. With medical partners from Stanford University, we found that high concentrations of iron oxide nanoparticle payloads could be delivered to T cells to study the homing of CAR-T cells to the tumor in vivo. This preliminary work helped us elucidate the key features needed for an effective device that provides the best results for cell engineering. Recognizing the commercial potential of this technology to impact patients’ lives, CellFE was founded.

A Brief Overview of Microfluidics

Microfluidics refers to the control of fluids at small scales within microchannels. It is an ideal technology for achieving fine control over forces applied to cells within liquids. With the CellFE microfluidic technology, results obtained with a one-channel scenario can be quickly scaled to many channels without further need for optimization. This unlocks a range of possibilities for a rapid and seamless transition from laboratory experiments to clinical, large-volume applications. The key to success is appropriate, fit-for-purpose engineering of microfluidic devices. We believe this unparalleled seamless scalability is a game-changer as it will provide significant time and cost efficiencies in development and manufacturing of cell therapies.

CellFE’s Solution for Cell Therapy

CellFE’s technology offers a solution that optimizes delivery and cell health, representing an unmet need in the cell therapy field. Within the microfluidic chamber, cells are compressed via a sudden constriction, which induces a temporary decrease in volume, followed by their re-expansion. This results in a transfer of surrounding payload into the cells. This brief compression can also be designed to minimize perturbation to the nucleus, which could result in unwanted cell damage. In addition, throughput is increased by allowing the cells to move in a plane to pass through the constriction in parallel without being forced into a single-file flow leading to cellular roadblocks.

The unique design of CellFE’s technology has been used to deliver large DNA templates that are 15 kilobases in size into cells — approximately twice the size of a payload that a virus can carry. In one example, our partner at the University of Iowa used CellFE’s technology to simultaneously deliver a >10-kb DNA construct and CRISPR/Cas9 reagents to perform homology-directed repair (HDR) in patient-derived induced pluripotent stem cells (iPSCs) to correct a mutation of an inherited disease that causes blindness, recently published as a first-in-microfluidics application.

Key Performance Differentiators

CellFE’s technology offers several key advantages over other delivery methods, such as viral vectors, electroporation, lipofection, or other microfluidic-based approaches. One key differentiator is the simplicity of the process. Cells do not need to be placed in a special medium or buffer; they can be taken directly from cell culture and placed into the transfection device along with the payload. A second key differentiator is the high proliferative viability of the cells so that they can readily grow and proliferate following transfection or even undergo multiple editing events sequentially, opening new possibilities for performing complex edits.

A full appreciation of the benefits of the CellFE platform may be contingent on which metrics are prioritized to assess manufacturing outcomes. Conventionally, developers remain focused on legacy metrics that can be easily measured immediately after processing, including transfection efficiency and necrotic viability. However, such metrics may not be the best indicators of the yield or quality (persistence, potency) of the cells, which ultimately drive product manufacturing efficiencies. For instance, a high transfection efficiency that results in the majority of the cells being lost or apoptotic, and thus failing to expand, negates any benefits of high transfection efficiencies. In contrast, the advantages of CellFE’s microfluidic technology are clear when viewed through the lens of metrics pertinent to therapeutic success. A CAR-T researcher recently compared the CellFE platform to electroporation. This researcher was able to use the CellFE platform to generate over six-fold more CAR-T cells five days after editing, with further increases in yield observed during the 12-day monitoring period. Stable integration of a CAR transgene without using virus and with robust cell growth post-editing can open new paths to rapid manufacturing workflows.

Importantly, CellFE’s microfluidics technology has the potential to optimize the potency and durability of cell therapies by unlocking higher cell yields, enabling shorter manufacturing processes than currently required, and lowering the vein-to-vein time. Many CAR-T cell therapy manufacturing times are measured in many days or weeks, risking further decline in the patient’s health during that waiting period. Moreover, the quality of the manufactured cells plummets with long ex vivo expansion. Researchers at the University of Pennsylvania and elsewhere have found that reduced culture time leads to a higher percentage of stem memory (versus effector) T cells, among the best predictors of cell therapy success to blood cancers. CellFE’s technology shortens the current manufacturing timeline by preventing the damage caused by conventional methods that hinders edited cells’ ability to expand and grow. The impact is a greater therapy potency and durability, which further underscores the unmet need to improve cell manufacturing.

Addressing Scalability

Another challenge posed by cell therapies is the need to engineer cells with a wide range of production scales throughout process development and clinical and commercial manufacturing. Some cell therapies, such as CAR-T cell therapies, are personalized medicines in which one batch must be produced for each patient. Newer CAR-T treatments might involve clinical doses of just 10 million edited cells, while more typical products comprise a few hundred million or up to a billion edited cells. Production batches for allogeneic cell therapies require even greater numbers of cells — several billion edited cells.

CellFE’s technology is designed for simple scalability — scaling is elegantly achieved by only increasing the number of channels. Most importantly, it is engineered to ensure that all cells move through the channels in an identical manner to ensure the highest-quality results. As CellFE’s technology becomes more automated and integrated with other operations, reduced costs will also follow. The simplicity of operation of the microfluidics solution will facilitate a more distributed manufacturing approach, in which manufacturing facilities need not be centralized but can be located closer to the point of care, another key step in expanding access to cell therapies.

Future Vision—Focusing on the State of the Cell

CellFE’s technology is transformative because its manufacturing workflow results in rapid editing of cells — delivering exceptional quality and high proliferative yield. We are continuing to develop innovative and powerful workflows, including complex editing of cells in a manner that retains cell quality while exhibiting lower genotoxicity and fewer risks of chromosomal abnormalities. The CellFE technology allows new ways of performing multiple edits, with each edit performed at a different timepoint and avoiding negative impacts to cell quality. Since gene editing requires the introduction of double-strand DNA breaks, co-existing breaks introduce the possibility of unintended genetic rearrangements or translocations with mutagenic risks and additional toxicities to the cell. CellFE’s technology reduces the risk of translocations by providing optionality to when those edits occur, making it an attractive solution for manufacturers developing allogenic cell therapies or cell therapies targeting solid tumors, both of which require multiple edits.

The long-term vision for CellFE is to become the backbone of next-generation cell therapies by enabling higher-quality genetic-engineered cells that result in more robust and durable therapies.

Originally published on PharmasAlmanac.com on December 13, 2023