Graduate research assistant Mason Chilmonczyk examines a dynamic-sampling device after plasma etching in a cleanroom at the Georgia Tech Institute of Electronics and Nanotechnology’s Marcus Building. (Rob Felt, Georgia Tech)

Sponsored by BioProcess International and its sister publication BioProcess Insider, the “Tech of Tomorrow Zone” at Phacilitate 2020 played host to a number of companies showcasing platforms and ideas that they believe can revolutionize cell and gene therapy (CGT) manufacturing. Some common themes arose in this diverse zone, highlighting technologies from stem-cell supply solutions to viral-vector filling.

Participating companies are aware of the complexities involved in producing regenerative medicines, and each proposed solution was intended to reduce the burden on CGT developers in bringing their products into and through clinical testing. Cost of goods (CoG) was a major talking point in current processes, so every company was conscious of how its ideas — whether for a microfluidic cell separator or a bioreactor monitoring system — could fit with industry’s drive to reduce CoG and increase cell and gene therapy accessibility to patients.

Another commonality was the connection with the overall biologics sector. Some participating companies were created or spun out from others focused on bioprocessing after their technologies seemed better placed to serve the demands of the advanced-therapy sector. Meanwhile, technologies created to aid CGT production directly often tipped their hats back to the antibody space, revealing opportunities for drug developers to leverage their platforms in a more traditional biopharmaceutical setting.

As the cell and gene therapy field evolves, some of these companies are sure to partner with larger vendors and/or license their technologies to biomanufacturers. Others might fall by the wayside or take their ideas in different directions. But some of these platforms could help set standards in cell and gene processing. They might even help to drive down production costs and bring these exciting new medicines to a broad patient population.

Understanding What Cells Are Saying
The Dynamic Sampling Platform (DSP) is a technology for cell-state analysis and real-time bioreactor monitoring (1). This system was developed as a PhD thesis in Andrei Fedorov’s laboratory in the George W. Woodruff School of Mechanical Engineering at the Georgia Institute of Technology in Atlanta, GA. Mason Chilmonczyk, the PhD student working on the project, now hopes to bring it to industry.

“As cells grow and as they mature,” Chilmonczyk told us, “they’re secreting different types of molecules. This can be thought of as ‘they’re telling us something.’ Right now, we don’t have a great way to understand what the cells are trying to tell us as they grow and mature.” Bioindustry commonly uses a number of sensors to measure attributes such as acidity or alkalinity, temperature, oxygen, and glucose, but those are all targeted criteria. For less-characterized CGTs, no suitable way is yet available to monitor in real time how cells are progressing during manufacturing.

Chilmonczyk explained that the DSP takes a sample of liquid within a bioreactor without affecting sterility or eventual product outcomes. Samples are treated rapidly — spending about a second inside a microfabricated device for direct mass-spectrometry (MS) analysis — and the advantage is that the method “is completely untargeted, so we don’t need to know what we are looking for beforehand. That enables us to use it as a monitoring tool. Manufacturers are really interested in something that can provide real-time feedback. Right now, a lot of these manufacturing workflows take between a week and three weeks.” Those are basically endpoint assays that are designed to determine whether a product is bad. “Imagine if we could close that feedback loop and what amazing things we could really do [related to] FDA chemistry, manufacturing, and control (CMC) standards, for example.”

The DSP system can be used as a discovery tool for identifying attributes that indicate cell state and therapeutic potential, he added. Although the platform was designed with the cell therapy space in mind, it also could be well suited to monitor protein production and might have applications in other industries.

Sorting Out the Apheresis Process
Aimed at shaking up the production of CAR-T therapies, GPB Scientific’s Curate cell-processing system is a microfluidic device for separating and washing cells from apheresis material. Jason Walsh (chief commercial officer at GPB) told BioProcess Insider that currently up to 90% of cells are lost in postapheresis white-cell isolation, the initial stage of the CAR-T production process. He called today’s process inefficient, slow, and costly — all problems that the Curate system is intended to resolve.

“What it does is replace current processes that are used to [perform] apheresis and separate out the white blood cells. Through our microfluidic system, we can achieve 95% or better recovery of white blood cells and 99% elimination of red cells and platelets. We’re able to get very effective separations.”

The closed system is said to eliminate up to six steps and process a full apheresis pack in under an hour. It uses microfluidic technology based on deterministic lateral displacement that separates blood cells based on their size (similar to how mechanical coin-sorting machines separate coins based on their size). The system’s washing function is based on lateral movement of cells directly into a parallel second fluid stream, which according to Walsh provides an equivalent to several rounds of washing otherwise required with traditional centrifugal processing. This is also a much gentler process, operating at around 10–20 psi. “You end up with an extremely pure and repeatable, high-quality cell product.”

GPB plans to begin placements for clinical evaluation of the Curate system in the second quarter of 2020. With adoption of a regulatory evaluation, Walsh said his company anticipates the system will be ready for use with clinical and commercial products by the end of the year. “Given the life-and-death considerations that come with efficiently producing CAR-T cells, the medical community deserves an automatable solution that can robustly and reliably deliver high-quality cells.”

An Off-the-Shelf Personalized Platform
Another company hoping to change the nature of CAR T-cell therapy is ImmTune Therapies. Its apheresis-free platform could turn this segment of the industry on its head by eliminating the ex vivo process entirely.

“We are doing in vivo CAR-Ts, carrying out the entire genetic engineering of T cells inside the patient’s body and eliminating the need for isolating, expanding, and activating T cells outside,” explained founding director Bakul Gupta. She hopes that will make CAR-T therapies both more potent and less expensive, ultimately improving patient accessibility.

ImmTune’s platform technology is based on an injectable vector targeted to T cells and administered directly to a patient. When it selectively identifies the person’s T-cells, it engineers them in vivo to express the CAR and then attack cancer cells.

One problem with CAR-T development is how complicated the logistics can be from receipt of leukapheresed material at a biomanufacturing facility to return of the final product to the clinical site and the same patient that starting material came from. ImmTune’s one-step therapy negates the need for that multistep procedure and simplifies the logistics, also minimizing patient hospital visits.

“Essentially this is an off-the-shelf personalized medicine,” Gupta says, “a combination of autologous and allogeneic. We’ve taken the best of both worlds and combined them into one.” If successful, this platform would disrupt the current manufacturing infrastructure typical for commercialized CAR-T therapies such as Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel), which rely on a network of apheresis centers and logistics.

ImmTune Therapies plans to license its platform to next-generation CAR developers. “Right now we are doing preclinical work and set to go into in vivo studies in the next few months,” she concluded, “where we will check safety, efficacy, and how well the transfection is happening. By June, we should get some preliminary data on how well it’s working and see what we may need to improve on.”

Cell Engineering for Delivery of Payloads
Spun out from the Massachusetts Institute of Technology in 2017, Kytopen is a company focused on advancing and accelerating discoveries in manufacturing within the cell and gene engineering space. “The foundational technology was built on fluid mechanics focused on engineering bacteria,” says George Eastwood (vice president of business development and partnerships). “Through customer discovery, our founders backed their way into the CGT space, where we found that this technology could be influential for entering genetic material into human primary cells, CD34 stem cells, and induced pluripotent stem cells (iPSCs).”

Kytopen’s patent-pending, nonviral Flowfect technology combines continuous fluid flow with electrical fields for payload delivery. Synchronization of pulse delivery with fluid flow eliminates harmful effects of traditional transfection through static electroporation, creating a process that yields billions of engineered cells in minutes.

“There are a number of problems within manufacturing of cell and gene therapies,” said Eastwood, “[related to] efficiencies, starting materials, scalability, and so on. We provide a gentle, scalable solution for entering these genetic payloads into human primary and stem cells, which creates a seamless transition from discovery through manufacturing.”

Kytopen’s customers are in academia and industry, but its location in the Boston/Cambridge area of Massachusetts makes it easy to provide Flowfect technology to the heart of CGT development. “We are [within] walking distance to many gene-editing companies,” Eastwood says, “and there are a lot of large cell-therapy companies on our street as well, offering easy access for collaboration. Customers can walk their cells and payloads over, and we can carry out experiments quickly with them present.”

Customizable Bioreactor Control
Born out of a project carried out about 10 years ago by its parent company Automated Control Concepts (ACC), the Lab Owl remote-access bioreactor control system is designed for cell culture and fermentation applications. ACC was on site for MedImmune at its good manufacturing practice (GMP) plant in Frederick, MD, when scientists at their process development laboratories asked them to come up with a control-system solution. Kurt Elam, senior director of sales and marketing at Lab Owl, explained that needed to work for 30 scientists performing different experiments using a number of bioreactor systems, all requiring different parameters to be monitored.

“After about a year of meetings, we came up with a system that allowed scientists to keep their data secret [from others] while sharing [among themselves],” Elam said, “with all the features and capabilities for what those teams needed.” The company implemented 40 units running 80 bench-scale bioreactors (2-L to 10-L volumes) in process development, then provided the same model for several other companies.

About three years ago, ACC decided to spin the technology out as a separate business, and Lab Owl was born. Since then, the platform has become a potential solution for the growing CGT sector. “As personalized medicine changes how R&D is done,” Elam said, “our customizable system is perfect to support this because we can build all the features these scientists need into our system. We work in a very consultative way, and we can point out features of our system that will help them. And if that feature doesn’t exist, our software engineers can build it in.” The open platform can integrate with any type of bioreactor or shaker-flask system, he added. “That’s important for the type of research these scientists are doing.”

A Marriage of Engineering and Biology
Founded as a plasmid catalog business eight years ago, UK company Oxford Genetics recently introduced the trading name of OXGENE. The goal of its business is to deliver scalable manufacturing solutions using transient and stable expression platforms for customer projects from laboratory bench to GMP scale.

“We start with custom plasmid design and engineering — we have plasmid sets optimized for AAV and lentiviral production — and then we pair that with our GMP-banked clonal suspension HEK293 cell lines and engineered derivatives,” says Sophie Lutter, scientific marketing and communications manager. “We take them through to process development, where we can support scales of up to 10 L.” With downstream purification as part of its platform, the company offers a full viral-vector package. “This leaves the customer not only with the final viral vector, but also [with] the processes and protocols to take that through to GMP manufacture.”

With a growing number of gene therapy platform technologies entering the market, OXGENE stands out because it brings together scientific expertise with the latest automation technologies intended to increase efficiency, enable scale-up, and reduce human error. “We automate what we can, where we can,” Lutter explains. “This is not just about owning robots, but rather about having the experts and scientists who work with them to marry up the automation with the biology, making sure that everything communicates and runs smoothly.”

Ready-to-Differentiate Stem Cells
Pluristyx formed in 2018 as a consultancy offering contract development services. Since then, the Seattle-based company has created a process to make PSCs as a ready-to-use product for early stage CGT companies. Phacilitate 2020 marked the first occasion for the company to showcase its fully characterized, ready-to-differentiate offering to the industry.

“When you’re ready to work with PSCs — to manufacture product or do basic research — you can just thaw a vial instead of having to go through the process of expanding stem cells for two to four weeks,” said chief executive officer Benjamin Fryer. The service has been described as a high-density working cell bank. “Normally, with a working cell bank, you’d thaw the vial and then have to grow the cells for 14–28 days to generate enough cells to do any sort of manufacturing. You end up having to do iterations with several different sources of cells, and then every passage the cells will be slightly different. The advantage of our product is that you are always starting with the same source material.”

Pluristyx works with both embryonic stem cells and iPSCs, both offering them as catalog items and serving as a contract manufacturer for exclusive supplies.
Although many other companies supply pluripotent cells, Fryer said most source them from seed vials with 1–5 × 106 cells or as a master cell bank made up of many vials.

“Those require that you do your own expansion,” he explains. “We take away the workflow required for that expansion so a company developing a product can just [work on] the valuable part of manufacturing — the end product — without the hassle, labor, and expense involved in growing cells in the front end.” Pluristyx currently makes research-use–only (RUO) cells at its in-house research laboratory. In the coming year, it expects to launch a GMP product line made in a third-party GMP facility.

Looking Beyond Manual Fill
In partnership with Biolife Solutions, Casdin Capital, and BioCrossroads, Sexton Technologies emerged out of the Cook Regentec incubator–accelerator hub late last year to launch enabling technologies aligned with the CGT industry. The company has a range of technologies, two of which were on show at Phacilitate: human-platelet lysate (hPL) media products and the CellSeal multifluid handling system for final formulation and filling.

Regarding the latter, Sexton’s cofounder and vice president of sales and product management, Steven Thompson, said it is set to solve the major problem of fill–finish in advanced therapies. “At the moment, the industry is quite happy with manual fill. When we’re treating a few hundred patients a year, that is acceptable. But as we go into higher-demand indications such as solid tumors, you will start to manufacture products to treat tens of thousands of patients per year. At that time, you are going to need something that is closed-process and flexible.”

Sexton’s T-Liven-PR media launched in September 2019. It has demonstrated improved phenotype and enhanced in vivo efficacy through CAR-T expansion research carried out in collaboration with Baylor College of Medicine (Houston, TX). “AB-negative units are used regularly for CAR-T,” Thompson explained, “but we wanted to look to our hPL as an alternative.” He added that this product is the first commercial hPL medium to take advantage of benefits in downstream cell products and that it can improve manufacturing outcomes and efficiencies for CAR-T customers.

Ancillary Reagent Products
VitaCyte makes defined enzyme products that are used in recovery of islet cells from tissue dissociation. The technology platform was born out of a project at Boehringer Mannheim in 2000 that was intended to solve the problem of isolating enough cells for human pancreatic islet transplantation.

“The project I worked on was actually the first purified enzyme mixture that allowed sufficient recovery of islets from human tissue,” explained VitaCyte president Robert McCarthy. That work brought about the first successful islet transplants, “ending the whole issue of using islet cell transplantation for therapy to manage diabetes patients.”
Launched in 2004, VitaCyte now offers a broad line of purified cell- and tissue-dissociation enzymes for academic, cell therapy, and preclinical applications. “We isolate adipose stromal vascular cells from human adipose tissue for cell therapy,” said McCarthy. “We also isolate hepatocyte enzyme products to sell to companies that isolate hepatocytes for drug development.” That is critical, she says, because the FDA requires human drug-metabolism study results in new-drug applications.

McCarthy said his company is leading a movement to improve the quality of reagents in the CGT sector. “People use crude collagenase and very ill-defined products, and we’re trying to move the field to start designing new formulations for these applications that require enzymes from cell tissue.” VitaCyte sells its ancillary reagent products to a broad range of industry players. “Only a handful of suppliers make defined reagents,” said McCarthy. “Our goal is to offer the broadest line and consider cost and productivity for people to use these reagents in whatever application they need.”

Reference
1 Chilmonczyk MA, et al. Dynamic Mass Spectrometry Probe (DMSP) for ESI/MS Monitoring of Bioreactors for Therapeutic Cell Manufacturing. Biotechnol. Bioeng. 116(1) 2019: 121-131; doi:10.1002/bit.26832.

Based in Montpellier, France, Dan Stanton is editor of BioProcess Insider. Contact him with an email to [email protected], or follow him on Twitter: @Dan5tanton.