Authors: Robert Kirian¹, Jason Bishop¹, Jae Jung¹, Christina Jenkins¹, Marc Mitchell², Maria Caicedo², Geddy Hamblen², Sean McMillan², Art Hamfeldt², Mark Szczypka², Elie Zakhem¹, Theresa D’Souza¹, Quentin Vicard³, Jon Rowley¹. Presented at ISCT (Poster #161, 6:30pm-8:00pm)

¹ RoosterBio Inc, 5295 Westview Drive, Suite 275, Frederick, MD 21703
² Sartorius Stedim North America Inc, 565 Johnson Avenue, Bohemia, NY, 11716
³ Sartorius Stedim France SAS, Aubagne, Provence-Alpes-Côte d’Azu, France

Authors: Stephen Lenzini, Jae Jung, Robert Kirian, James Brennan, Elie Zakhem, Jon Rowley. Presented at ISCT (Poster #316, 6:30pm-8:00pm) and ISEV.

Authors: Mary T. Doolin, Terri Willstaedt, Jon Rowley. Presented at ISCT (Poster #138, 6:30pm-8:00pm) and ASGCT.

Authors: Terri Willstaedt, Amy Walde, James Brennan, Jon Rowley, Katrina Adlerz. Presented at ISCT (Poster #159, 6:30pm-8:00pm) and ASGCT.

Above, a word cloud based on the ISCT-2021’s Annual Meeting Program [1] (IMAGE SOURCE: https://monkeylearn.com/word-cloud)

San Francisco and Washington, DC are going to be busy forums for regenmed’s pioneers and partners in May-2022, with ISCT’s (5/3 to 5/7) [2] and ASGCT’s (5/16 to 5/19) [3] annual conferences revving up for post-COVID full throttle. Closing out the month, Lyon, France deserves an especially noteworthy mention, being host to ISEV (5/25 to 5/29). [4] Yes, RoosterBio is going to be present physically at all three events—to learn about the fascinating science, and to gain insights on where it’s leading our new customers. Not only that, but we’re also thrilled to contribute to this dialogue with our own poster abstracts that feature some highlights of recent progress in the lab. It’s often said that “No one is an island,” [5] and so this aphorism precisely fits our relationships with others, far and near. Thus, we’re very happy to report at these venues how our collaboration with Sartorius [6] ramifies into several initiatives that begin to bear fruit in the form of useful, practical data.

The ISEV2022 (International Society for Extracellular Vesicles) event rightfully claims the title of “world’s largest and the premier meeting in EV research,” which “features the best in vesicle science, covering all aspects of fundamental, translational, and clinical research, disseminating cutting-edge developments in EV research.” Given the meteoric rise in global interest pertaining to therapeutic exosomes and EVs, RoosterBio eagerly looks forward to interfacing with our international colleagues on a different continent. This was true during our consecutive attendance at past years’ ISEV meetings, but it’s uniquely timely today. In the last year, a sizable majority of our product and services revenues came from institutions that had purchased our specialized EV production systems.

The “three pillars” of the ISCT (International Society for Cell and Gene Therapy) [7] uphold (i) Translational Science, (ii) Regulatory & Quality Operations, and (iii) Commercialization. These aim to promote “cell and gene therapies through a focus on translational value, bridging experts across scientific, regulatory, and commercial fields to drive consensus and action at a global level.” ASGCT is now known as “the premier event for professionals in cell and gene therapy,” having “grown to serve a wide community encompassing clinicians, bio-industry development, regulatory agencies, equipment manufacturers, patient advocates, and more.” Accordingly, we’ll present poster abstracts as posters in San Francisco and Washington that help fortify this foundation of excellence, because our new findings directly remove some technical and QC barriers that hinder (1) lower-cost growth of clinic-size batches of adherent primary cells, and (2) genetic modification of therapeutically relevant primary cells. Further, RoosterBio’s posters offer viewers a perspective that transcends the ubiquitous immune-oncology cell therapy milieu with something a little different, if not perhaps even complimentary. [8]

One abstract, “Scale-Up of a Xeno-Free Fed-Batch Stirred-Tank Bioreactor Process for hMSC Manufacturing,” depicts how we collaboratively optimized upstream MSC bioproduction parameters with Sartorius Stedim via their Ambr® product systems and MODDE DoE design and analysis software. “Developing a microcarrier stirred tank process for large-scale hMSC-EV production” further develops out of our first poster’s cutting edge work that used Ambr®250 vessels, showing scalability between 2D and 3D mesenchymal stromal cell (hMSC) culture for exosomes/extracellular vesicles (EVs) in future biotherapeutic applications. “Development of an Optimized Lentiviral Transduction Medium and Process to Manufacture Genetically Modified MSC Working Cell Banks” and “Optimization of Transient Transfection of Mesenchymal Stromal Cells with plasmid DNA or messenger RNA” advance the science of viral transduction or transfection of genetically engineered hMSCs.                                        

In “Scale-Up of a Xeno-Free Fed-Batch…” (Kirian, et al.), we describe the collaborative work with Sartorius to use its  Ambr250 as a testbed for optimizing process parameters of hMSC growth on microcarriers. Then we show how this small volume’s conditions (0.25L) can translate to full-scale bioreactor culture into a 50L Biostat STR® Generation 3 Bioreactor from Sartorius. Lastly, we demonstrate how hMSC quality and functional attributes are maintained as the bioprocess is scaled from 0.25L to 50L.

“The data generated in this study demonstrated the scalability of hMSC culture in stirred-tank bioreactors, providing significant time and cost savings as a standardized system for translational researchers and product developers in the regenerative medicine, tissue engineering, and cell therapy fields,” said Robert Kirian, Senior Process Engineer at RoosterBio.

In “Developing a microcarrier stirred tank process…” (Lenzini, et al.), we demonstrate the steps taken towards optimizing a process for hMSC-EV production in microcarrier-based stirred tank bioreactors. If viewers happen to miss this poster at ISCT in San Francisco, no worries! You can catch it again in Lyon, France at the ISEV annual meeting. This work was performed in the Sartorius Ambr250 system, a 250mL scale-down model of the Biostat STR, which is a system scalable up to 2,000L. Process parameters were optimized to enable high-density cell culture of more than 2 million cells/mL, with EVs collected in yields exceeding 10¹⁰ /mL. We have determined previously that doses of hMSC-EVs average to an approximate 4×10¹⁰ EVs/dose, [9] and recently written about techniques to hone upstream process development with EVs to accelerate clinical production. [10]  Since we soon intend to scale up this bioprocess to a 50L vessel and beyond, these methods will become readily transferred to a GMP environment for supply of quality EV material to future clinical trials.

“Enthusiasm around hMSC-EVs as therapies is accelerating rapidly and therefore, we are very pleased to present our timely process development results for hMSC-EV production in stirred tank bioreactors. Our processes for large-scale production of hMSC-EVs will be based on the work presented here, a critical step towards making hMSC-EVs widely available for research, development, and clinical testing,” said Stephen Lenzini, Ph.D., Process Development Scientist.

Our next two posters showcase our enhancements of how exemplar primary cells (here, hMSCs) can take up genetic material via lentivirus particles, plasmid DNA (pDNA), or mRNA. This is of critical importance since hMSCs are not easy to transfect or transduce, by most accounts. Such progress also helps open the door to a new paradigm in hMSC clinical translation, in which the cells are no longer the sole “drug” material; [11] instead, these MSC or MSC-EV doses serve as a micro-delivery system for genetically programmed medicines à la “MSC 2.0.” [12] Hence, RoosterBio will be at Washington DC’s ASGCT to talk our guests through these stories, as well, in addition to engaging with ISCT.

“Optimization of Transient Transfection…” (Doolin, et al.) details chemical, non-viral approaches to transfect hMSCs with pDNA or mRNA using Lipofectamine 3000 (Thermo) or PEI. We found that certain protocol parameters—such as the use of RoosterGEM® for mRNA transfection and culture of cells in RoosterNourish® for pDNA transfection—enhance % transfection as measured by counts of GFP+ cells. Notably, conditions optimized with RoosterGEM for mRNA achieve 80%.

“We have developed optimized protocols for the transient transfection of hMSCs with pDNA or mRNA. These protocols will enable investigators to quickly implement an efficient transfection process that scales from preclinical studies to clinical trials,” said Mary Doolin, Ph.D., Scientist II in Product Development.

“Development of an Optimized Lentiviral Transduction Medium and Process…” portrays our ongoing work to characterize our RoosterGEM gene transfer enhancement media to transduce lentivirus—and in turn, expand stably-modified hMSCs into a PDL that could translate to a clinically-relevant cell volume for dosing. Using RoosterGEM, we can achieve 90% efficiency in lentivirus transduction as measured by %GFP+ cells, amounting to two- to five-fold improvements over conventional methods. This can facilitate practical and significant reductions in the MOI, an advancement that could reduce cost and/or extend assay design parameters of lentiviral stock solutions for R&D or clinical translation uses.

“RoosterGEM, a novel formulation available from RoosterBio, optimizes a critical unit operation of gene modification and provides a useful tool for the generation and expansion of engineered hMSCs for use in clinical therapies,” said Terri Willstaedt, M.S., Manager of Product Development.

All in all, RoosterBio is appearing at three conferences in May 2022: ISCT, ASGCT, and the annual International Society for Extracellular Vesicles (ISEV) meeting in Lyon, France. As in years past, we fully expect to be amazed by the pace of research, innovation, and commercialization at these venues, and deeply look forward to rekindling old conversations—and to making new friends. With large crowds and strong coffee animating the vicinity of our booth and posters, we’re just itching to get on the road again!

References

1. ISCT. Program. 2021; Available from: https://isct2021.com/program/.
2. ISCT. ISCT 2022 San Francisco May 4-7. isctglobal.org 2022; Available from: https://www.isctglobal.org/isct2022/home.
3. ASGCT. ASGCT 25th Annual Meeting. 2022; Available from: https://annualmeeting.asgct.org/.
4. ISEV. ISEV2022 Annual Meeting. 2022; Available from: https://www.eventscribe.net/2022/ISEV2022/index.asp?launcher=1.
5. Donne, John. No Man is an Island. 1624; Available from: https://www.youtube.com/watch?v=_mfus7QCeWU.
6. Sartorius. Sartorius Partners With RoosterBio to Advance Cell and Gene Therapy Manufacturing. 2021; Available from: https://www.sartorius.com/en/company/newsroom/corporate-news/sartorius-partners-with-roosterbio-to-advance-cell-and-gene-therapy-manufacturing-640284.
7. ISCT. Clinical Translation – Our Three Pillars. 2021; Available from: https://annualreport.isctglobal.org/.
8. Carson, J. MSCs “Versus” or MSCs “Via” T Cells for Cell & Gene Therapy? RoosterBio Blog 2022; Available from: https://www.roosterbio.com/blog/mscs-versus-or-mscs-via-t-cells-for-cell-gene-therapy/.
9. Lenzini, S. Establishing a Working Range for Effective MSC-EV Dose. RoosterBio Blog 2021; Available from: https://www.roosterbio.com/blog/establishing-a-working-range-for-effective-msc-ev-dose/.
10. Lenzini, S. Extracellular Vesicle/Exosome Upstream Process Development: Maximizing Productivity to Accelerate Clinical Adoption. RoosterBio Blog 2022; Available from: https://www.roosterbio.com/blog/extracellular-vesicle-exosome-upstream-process-development-maximizing-productivity-to-accelerate-clinical-adoption/.
11. RoosterBio. RoosterGEM™: Addressing Key Challenges in Efficient Gene Transfer to Primary Cells. 2021; Available from: https://bit.ly/3NxS5XG.
12. Olsen, T. R., et al., Peak MSC-Are We There Yet? Front Med (Lausanne), 2018. 5: p. 178. 10.3389/fmed.2018.00178