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Fresh or Frozen? Navigating the Cryopreservation Dilemma for CGT.

Cell and gene therapy development grapples with a paradox: fresh starting material is often equated with higher-quality drug products, but cryopreservation is crucial for scale-up. In fact, every approved autologous cell-based therapy relies on freezing cells. Reconciling these truths can be key for the success of any commercial therapy based on human cells, reveals Priya Baraniak, Head of Process Development and Manufacturing at OrganaBio, in this European Pharmaceutical Review article.

Although fuelled by unprecedented impact on patients, scaling up cell and gene therapies (CGTs) for commercial accessibility has proven to be an enormous challenge. This nascent industry still has much to learn about what makes the best therapies, and what constitutes an optimal manufacturing process.

To date, cryopreservation has been a key part of the logistics chain for CGT manufacturing, even though early-stage developers are often wary of freezing cells for transport. Early development efforts are largely focused on producing a therapy with the best chance for positive clinical outcomes, and developers are hesitant to introduce manipulations that could impact cell potency. Cost, infrastructure, skill limitations and added time can also be decisive factors in the decision on whether or not to cryopreserve.

As companies pivot to larger, multi-centre trials and commercial-scale manufacturing and, for allogeneic therapies, an exponentially growing need for more donors and drug product doses, it becomes apparent that cryopreservation is the only sustainable path to scaling. Therefore, the question may not be whether but rather when and how to implement freezing into process and product development.

The case against cryopreservation

Ex vivo manipulation of cells can introduce unintended changes in their properties and function. Some cell types, like natural killer (NK) cells, are particularly sensitive to cryopreservation. Studies show a significant decline in NK cell viability and function 24 hours after thawing.1 These challenges can also complicate regulatory approval for cell therapies.

In addition, cryopreservation services may be difficult to access. Many hospitals, clinical sites and blood centres that draw patient/donor tissues or blood lack the infrastructure or knowledge for cell isolation and/or cryopreservation. Processing adds hours to manufacturing and necessitates on-site, validated cryopreservation processes and storage of frozen materials, incurring additional labour and equipment costs. This is especially true if these processes must be GMP‑compliant and performed in a cleanroom setting. For companies looking to minimise cash burn, freezing and cold-chain logistics may seem cost prohibitive. Thus, developers often adopt the ‘fresh is best’ mantra.

Beyond fresh vs. frozen: optimising cell therapy manufacturing

The cell therapy industry is evolving, with a growing understanding of how different unit operations – from cell collection via apheresis to manufacturing and shipping – impact the final product’s quality and potency. Researchers are actively identifying critical process parameters (CPPs) and product critical quality attributes (CQAs) and developing solutions to address challenges associated with them.

Cell therapies will likely require cryopreservation to facilitate large-scale production and distribution. Developers are therefore investigating the potential effects of freezing and thawing on their therapies. This involves analysing specific technologies and processes through data-driven methods and minimising any negative impacts by developing mitigation strategies early. For example: NK cell therapy developers have realised that a steep drop‑off in viability may not be a reason to work around cryopreservation. Working from the assumption they will see a 50 percent decline in functional NK cells post-thaw, developers are exploring ways to double the number isolated from blood or differentiated from stem cells to offset this loss.

Further, recent research suggests other factors may have a bigger influence on cell function than cryopreservation itself. Katy Rezvani and her team at The University of Texas MD Anderson Cancer Center recently published results from an allogeneic chimeric antigen receptor (CAR)‑NK cell therapy trial in patients with B-cell blood cancers. For a cord blood-derived NK cell therapy, the number of nucleated red blood cells and time to cryopreservation emerged as predictors of patient outcomes. Specifically, units frozen within 24 hours of collection yielded highly functional cells with superior antitumour activity and significantly better clinical outcomes.1

The realisation that processing plays a major role in cell therapy outcomes is not novel. Other studies have suggested, for example, that shorter manufacturing cycles for CAR-Ts may produce better therapeutic outcomes,2 even if the final doses are smaller due to less expansion time.3 Another recent publication highlighted how the manufacturing platform and parameters such as oxygenation significantly influence CAR T-cell expansion and differentiation.4

The implication is that developers need to interrogate assumptions about the qualitative superiority of fresh cells, including the hypothesis of superior functionality, and fully explore the tradeoffs before committing to fresh over frozen cellular starting materials. Coordination with partners to optimise manufacturing and logistics – including choice of donors, tissue procurement/apheresis protocols and timing and method of cryopreservation – could yield desirable therapeutic safety and efficacy results. These may also be advantageous compared to use of cells that, although not cryopreserved, have experienced extended transit times prior to manufacturing. Given that transition to a freezing process may be inevitable for scalability, developers who explore the impacts of and account for effects of cryopreservation early in development are likely to avoid costly adjustments later.

Cold truths

The logistical advantages of cryopreservation tend to drive its use in CGTs during scale up and commercial operations for both autologous and allogeneic cell manufacturing, given the complexity of scheduling patients or donors, respectively.

Allogeneic therapy developers often seek to identify a single or small pool of donors for development and early-phase clinical trials, taxing the logistics chain when working with fresh cells. Working backwards, this means scheduling a donor at such a time that your manufacturing team is ready to process the cells upon arrival, which may mean thawing and preparing thousands of dollars‑worth of reagents and materials. Autologous therapies may entail even greater complexity, scheduling sick patients for tissue collection and ensuring that they are fit to withstand the procedure. Altogether, this necessitates a flawless symphony of tissue collection partners, shipping partners and internal staff ready for seamless hand-offs.

However, there are many uncontrollable variables and nodes of potential failure in this model. What if the donor could not arrange childcare or change a work shift? What if the individual presents with a fever, fails a complete blood count (CBC) test or experiences an adverse event during apheresis? In the case of birth tissues, babies come when they’re ready; there is an ever‑present question mark around timing. Additionally, what if transport is delayed by the courier service, TSA/customs, or a natural event? Even when the quality of fresh cells is found to be superior at the start, viability dwindles with time.

Seasoned partners may be able to plan for and adapt to some delays, scheduling a backup donor (not an option for autologous therapies or birth tissues) or working with specialty couriers who understand the urgency of timing and how cells must be handled. Also, experienced providers will maintain real-time communications with the manufacturing team to update on any surprise delays, helping limit downstream costs. Still, the delicate balance of the process should make developers question how much risk they can really afford.

Cryopreservation can mitigate many of these risks, permitting flexibility to account for unforeseen delays. Companies will not have to worry about the impact on manufacturing team schedules and resources, or squandering cleanroom time they have already paid a contract manufacturer for. The buffer period afforded by cryopreservation can also be beneficial in terms of sterility testing done on the day of collection: it can take up to three weeks to get results, and manufacturing will be well underway by the time a viral or bacterial contaminant is discovered in fresh cells. These advantages multiply as a therapy scales, minimising manufacturing failure rates and saving companies significant costs.

Today’s commercial autologous CAR T-cell therapies all use frozen cells. Compromised patients may not produce enough cells even once for a therapeutic dose, due to their late-stage blood cancers and taxing earlier-stage treatments. A leukopak squandered due to a logistics failure can be the deciding factor between life and death for these individuals.

Timing is everything

Developers considering the switch from fresh to frozen cellular starting materials must weigh up the long-term impacts on their programmes. Early changes might preclude the need for comparability studies and may only require an amendment rather than an entirely new regulatory filing.

In our industry, every change comes with cost. Avoiding significant changes to raw materials and processes is typically ideal, but when it cannot be avoided, it is critical to fully understand the implication of changes to determine when they must be made.

References

  1. Saults N, Otegbeye F. Optimizing The Cryopreservation And Post-Thaw Recovery Of Natural Killer Cells Is Critical For The Success Of Off-The-Shelf Platforms. Front. Immunol. 2023;14:1304689.
  2. Watanabe N, Mo F, McKenna MK. Impact of Manufacturing Procedures on CAR T Cell Functionality. Front. Immunol. 2022;13:876339.
  3. Ghassemi S, Nunez-Cruz S, O’Connor RS, et al. Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer Immunol Res. 2018;6(9):1100-1109.
  4. Song HW, Prochazhova M, Shao L, et al. CAR-T Cell Expansion Platforms Yield Distinct T Cell Differentiation States. Cytotherapy. 2024.

Andrew Larson

Managing Director, CPC Services

Andrew joins OrganaBio as a project manager with varied experience in project management, client relations, and process improvement.

Prior to OrganaBio, Andrew was a client relations manager for the cGMP nucleic acids business unit at Aldevron, coordinating and managing contracts at each stage of the contract lifecycle in support of cell and gene therapy program development. Andrew supported small- and large-scale biotechnology and pharmaceutical clients anywhere from pre-IND work through commercial supply chain establishment. Before Aldevron, Andrew was a project manager for the commercialization and business development department for Sanford Health, a worldwide hospital institution. At Sanford Health, Andrew helped manage medical device patent and prototype development efforts for employee innovations primarily in the cardiovascular, neurovascular, and software spaces. Andrew was also an engineer for Atirix Medical Systems and supported the buildout of automated analysis worksheets to streamline radiology department quality control procedures.

Andrew received his Bachelor of Science in Physics from Minnesota State University Moorhead and his Master of Science in Biomedical Engineering from the University of Minnesota. At the University of Minnesota, Andrew was part of the Center for Magnetic Resonance Research, assisting efforts to automate MRI dataset registration and workflow improvement.

Michael Dee

Associate Director, QC and Analytical Development

Michael Dee has spent the last 17 years researching the immune system. Initially studying the recombinant cytokine IL-2 and its role in T cell subset differentiation and function at the University of Miami. He also helped elucidate the lower level of TCR diversity of T regs required to prevent autoimmunity in mice. Michael also supported construction, cloning, production, purification, and testing both in vitro and in vivo a novel IL-2/IL2Rα complex currently under clinical development with BMS. Michael also was a member of the department of immunology’s program project delineating the effect of a novel Eg7GP96 heat shock protein vaccine on tumor immunity.

While at Immunity Bio (formerly Altor Biosciences), he helped to characterize over 20 novel drugs for immune modulation and treatment of cancer.  After Immunity Bio, Michael was a founding team member of HCW Biologics, where he continued his role in design and initial production and characterization of several novel biologics. He has experience with proof of principle experiments with the generation CAR-NK and CAR T cells. His research at HCW was highlighted by his discovery of a process using novel biologics to activate and expand CIML NK cells. The process and rights were sold to Wugen and is currently in Phase I clinical trials. He also is listed as an Inventor on patent number: US20210268022A1 on method of activating regulatory T cells.

Meram Alamoudi

Senior Cell Processing Specialist

Meram received her master’s degree in biomedical sciences from Barry University and bachelor’s in Biology from Palm Beach Atlantic University.

Before her position at OrganaBio, Meram conducted research at Larkin University where she worked on assessing the impact of Hurricane Maria on respiratory diseases in Puerto Rico, which provided her with insight into research investigation and analysis along with generation of grant documentation.

Valeria Beckhoff-Ferrero

Senior Bioprocess Scientist

Valeria Beckhoff Ferrero has over 5 years of experience in the fields of stem cell research and tissue engineering. Valeria received her Bachelor of Science in Biomedical Engineering, specializing in Biomaterials and Tissue Engineering, from Drexel University in Philadelphia. Valeria has expertise in problem solving and finding manufacturing solutions for isolating various types stem cells and other cell derived products from different tissues.

Before joining OrganaBio, Valeria was a lead manufacturing engineer at the Amnion Foundation. She aided in instituting a GMP infrastructure, including documentation, to manufacture clinical grade placental derived stem cells. In her role, she worked in perfecting isolation, culture, selection and cell maintenance processes for perinatal derived stem cells.

Valeria’s experience includes working as an Automation Engineer at the New York Stem Cell Foundation, where she aided in the creation and coding procedures for liquid handlers to manufacture induced pluripotent stem cells. At NYSF, Valeria researched new methods of sorting, reprogramming and differentiating iPSCs.

During her studies, Valeria worked at Thomas Jefferson University Hospital’s Radiation Oncology department, where she engineered various devices to aid in hyperthermia treatments. Additionally, Valeria co-authored multiple publications on magnetic resonance guided focused ultrasound and radiation antennas for hyperthermia treatments.

Marisa Reinoso

Director, Regional Scientific Sales

Marisa has experience leading marketing and sales life sciences programs for over a decade. Originally a lab researcher, she made the jump to marketing & sales in life sciences and never looked back.

At OrganaBio, she connects cell therapy developers on the West coast and in Asia with the healthy donor starting materials they need to develop their therapies. Prior to OrganaBio, she was the cell therapy marketing lead at Invetech, heading the launch of the company’s first cell therapy product. Marisa has led marketing programs at clinical supply companies Sherpa Clinical Packaging and PCI Pharma Services. In her spare time, Marisa enjoys traveling, eating, and pretending she’s a tennis player. She has a Bachelor of Arts in Biology from Reed College and an MBA from Portland State University.

Thelma Cela

Senior Director, Tissue Procurement

Thelma Cela is a top performing professional with over 25 years’ experience in management, leadership, business development and marketing fields with business acumen and skills in driving revenue and profit growth in multiple corporate cultures. Prior to joining OrganaBio, Thelma served as Senior Director for Health and Human Services for the Seminole Tribe of Florida. Her role had oversight for health clinics, health plan administration, the behavioral health department, and elder services. In this governmental administrative capacity, Thelma had primarily responsibility for the HHS’ divisions’ budget, capital projects, utilization management, efficiency, and efficacy.

Thelma’s prior work experiences include Vice President of Clinical Operations for OrthoNOW. In this role, she provided guidance on all clinical matters, set direction on clinical policies and procedures and monitoring healthcare policy changes. As the national Vice President of Clinical Operations, Thelma also designed, developed, and implemented guidelines and protocols and ensured compliance regarding overall patient experience.

Before joining OrthoNOW, Thelma had been recruited by Leon Medical Centers, a private healthcare company operating comprehensive medical centers to launch a new business line addressing the health and wellness of an aging population. As Director, Thelma researched, created, and launched the company’s Health Living Centers which provided first of its kind facilities in the South Florida market to offer services to the community of health aging.

Thelma has a proven track record in multiple corporate healthcare cultures having worked for Mercy Hospital where she was Senior Program Director of their Diabetes Treatment Center and Director of their Surgical Weight Loss Program. She enhanced these service lines awareness in the community, improved both lines’ clinical outcomes, and built volume growth while maintaining ongoing physician support. She served in a similar capacity for American Healthways.

Thelma earned her MBA from Miami Regional University where she graduated Cum Laude and her undergraduate degree in Psychology is from the University of Miami.

She serves on the advisory panel for Florida International University’s Women in Business Leadership Program helping future women become future business leaders through thought leadership, barrier destruction, and the power of influence.

Dominic Mancini

Vice President, Operations

Dominic Mancini brings 12 years of experience working the interfaces between Analytical Development, Process Development, Quality, and Manufacturing Science to OrganaBio. A lifelong learner, Dominic enjoys solving the many scientific and operational challenges presented in the field of cell and gene therapy.

Prior to OrganaBio, Dominic spent 8 years at Bluebird Bio as the company grew from 45 to 1200+ employees and from 1 clinical asset to a robust commercial pipeline. At Bluebird, Dominic initially supported the development and technology transfer of lentiviral vector manufacturing processes. As demand grew for lentiviral process and product characterization, Dominic led the development, qualification, transfer, and validation two commercial release methods. Dominic transitioned back to the Process Development organization to lead the vector manufacturing core team, increasing operational efficiency through a 5S implementation, process schedule intensification, and reverse technology transfer initiative. More recently, Dominic supported the build-out of bluebird’s Manufacturing Science & Technology team followed by the Data Systems & Analytics team, handling late-stage commercial asset support.

Dominic received his Bachelor of Chemical Engineering with Distinction from the University of Delaware. Dominic’s undergraduate research culminated in his thesis on heterologous expression of G-protein coupled receptors in Saccharomyces cerevisiae. After graduation, Dominic was the premier hire of the Zhou Laboratory at Brigham and Women’s hospital in Boston, MA. In three years, Dominic established an animal model of COPD and co-authored several papers with his collaborators in the Pulmonary division.

Christopher B. Goodman

Vice President, Quality & Regulatory Affairs

Christopher B. Goodman is a biopharmaceutical consultant and executive making a global impact in the cellular therapy technology arena. The scope of Christopher’s expertise encompasses Cellular Therapeutic Operations, Quality and Regulatory Affairs, Global Corporate Operations, Scientific Strategic Planning, Scientific R&D Collaborations, and Marketing & Commercialization.

Christopher recently joined OrganaBio as their Vice President of Regulatory Affairs. In this role, Christopher will be helping the company, its clients and partners navigate the complexities of the domestic and international regulatory requirements governing advanced cellular therapy products and manufacturing.

Previously, Christopher held positions with the Association for the Advancement of Blood and Biotherapies (AABB), Virgin Health Bank, Ventana Medical Systems, and Celgene.

While with AABB, he held the positions of Senior Director of New Products and Lead Quality Assessor, auditing both domestic and international organizations to known standards in an effort to promote and ensure patient quality care and manufactured product consistency and standardization within Cellular Therapy, Blood Banking, Transfusion Services, Perioperative and Donor Center industries and operations. He contributed greatly to the work of AABB’s accreditation program providing his deep breadth of knowledge and technical acumen on many committees during his tenure. His pioneering work in the realm of virtual assessments during the COVID pandemic allowed AABB to flex into the planning and execution of this novel approach to the maintenance of accreditation activities during a global travel crisis. His agile thinking and approach to planning provided as minimal disruption as possible to AABB’s customer facilities.

While working with Virgin Health Bank in the State of Qatar and the United Kingdom, Christopher advanced through a series of executive roles. He joined Virgin Health Bank as the Director of Operations, during which time he managed the successful design, and build out of a new state-of-the-art cGMP facility, the first in the Middle East. As Director and Chief Executive Officer, he directed the launch of the first Arab-centric stem cell bank, and strategically guided the organization to enhanced shareholder value and expansion across the Middle East and UK. In these roles, he also oversaw global corporate operations, research collaborations, product portfolio expansion, and regulatory framework.

Christopher managed the Detection and Chemistry Assay Development Group for Ventana Medical Systems, a global leader and innovator of tissue-based diagnostic solutions. In this role, he directed overall program goals, optimized resources, and guided technical and product direction in global regulated environments.

Prior to Ventana Medical Systems, he held the position of Director of Operations for the high-growth Cellular Therapeutics Division of Celgene. As a senior-level scientist and member of the executive team, he directed divisional operations, medical affairs and executed business and scientific strategic planning.

Danielle Smyla

Senior Director, Quality Assurance

Danielle Smyla, M.S., brings 14 years of Quality Assurance and GMP experience in the Biotechnology and Medical Device industries. Ms. Smyla is an established Quality Leader with expertise in the implementation, management and continuous improvement of Quality Management Systems for GMP operations.

Prior to joining OrganaBio, Danielle was a key member of the Quality Management team at Canon BioMedical, where she led the cross-functional development and implementation of their Quality Management System. She also managed a team of Quality Specialists and Sr. Specialists, coaching them in the implementation, management and identification of improvements to quality processes.

Ms. Smyla’s Quality-focused career is complimented by valuable hands-on experience in GMP product manufacturing, as well as R&D laboratory experimentation and formulation work in support of product development.

Danielle has earned a Master’s in Biotechnology from the Johns Hopkins University and a Bachelor of Science in Chemistry from the George Washington University.

Priya Baraniak, Ph.D.

Chief Business Officer

Dr. Baraniak is a proven strategic thinker, problem solver and leader who brings 20 years of expertise in stem cells and tissue engineering, coupled with a keen business acumen, to OrganaBio. Dr. Baraniak has published multiple peer-reviewed papers and book chapters on the use of stem cells and biomaterials in cardiac repair and regeneration and is routinely invited to speak at conferences.

Before joining OrganaBio, Priya was a founding member of RoosterBio and was a vital member of the company’s Leadership Team. At RoosterBio, Priya leveraged her technical expertise to build and rapidly scale the company’s sales and marketing engines in a fast-paced start-up environment, delivering impressive growth in revenue year-over-year. Additionally, in her role as Business Development lead at RoosterBio, Priya structured, negotiated and executed multiple strategic partnerships for aggressive growth of the organization.

Priya’s industry experience includes a role as Senior Director of R&D for Garnet BioTherapeutics, a clinical-stage stem cell-based regenerative medicine company, where Priya led multiple projects on tissue repair and regeneration using mesenchymal stem cell (MSC)-based therapeutics and devices. While at Garnet Bio, Priya also worked on the company’s FDA filings, contributed to drafting and prosecuting the company’s patent portfolio, managed CRO, CMO and industry partner relationships and actively participated in establishing Garnet’s strategic R&D plan, thereby gaining critical insights into business operations across a small organization.

Priya’s scientific training began as an undergraduate student at Duke University, where she earned a Bachelor of Science in Engineering (BSE) from Duke University in 2001 after double majoring in Electrical Engineering and Biomedical Engineering. While at Duke, Priya conducted research in the lab of Dr. Doris A. Taylor on the use of skeletal myoblasts and stem cells for cardiac repair and regeneration. Priya went on to receive her Ph.D. in Bioengineering from the University of Pittsburgh in 2008. She completed her dissertation research in the laboratory of Dr. William R. Wagner working on developing a controlled release biodegradable elastomer for applications in cardiovascular regenerative medicine. In 2008, Priya joined Dr. Todd McDevitt’s lab in the Department of Biomedical Engineering at Georgia Tech and Emory University as a post-doctoral fellow. Her post-doctoral research as an American Heart Association Fellow focused on harnessing the secretome and isolating the extracellular matrix from MSCs and other cell types, including pluripotent stem cells, for cardiac tissue repair and regeneration. Priya co-authored many grants while a post-doc and went on to contribute critical sections to a NIST grant that resulted in the first ever National Cell Manufacturing Consortium in the United States.

Sarah Alter, Ph.D.

Senior Director, Scientific Affairs

Sarah Alter, Ph.D., has 15 years of immunology research experience which includes autoimmunity, cancer, and infectious disease.

Before her position at OrganaBio, Sarah was responsible for leading a team of scientists at Altor Bioscience where she facilitated the advancement of Altor’s technologies. As a Research and Development Manager, Dr. Alter coordinated immunotherapy-focused preclinical and clinical studies and contributed to the progress of Altor’s drug discovery and therapeutic applications.

Sarah received her Doctor of Philosophy from the University of Miami, Miller School of Medicine. She is also a registered Patent Agent, licensed to practice before the United States Patent and Trademark Office. Her work was published in many peer-reviewed journals and presented at national and international business and scientific meetings.

Carlos Carballosa, Ph.D

Director, Scientific Sales

Carlos Carballosa is a biomedical engineer with over 8 years of stem cell research experience with broad expertise in the culture, differentiation, and cryopreservation of adult stem cell populations. Carlos earned his bachelors, masters and doctorate degrees in Biomedical Engineering from the University of Miami, where his research focused on the effects of nicotine and electronic cigarette vapor exposure on the regeneration potential of adult stem cells. In addition to his dissertations, Dr. Carballosa has authored numerous publications related to stem cell biology.

Oscar Robles

Director, Quality Systems

Oscar Robles has over thirty years of experience in pharmaceutical and medical device industries. His main areas of expertise are in Quality Systems, Quality Assurance, Manufacturing Systems Validation, Computerized Systems Validation, implementation of GxP Computerized Systems and ERP Systems such as TrackWise, Electronic Document Management, JDEwards, SAP, and Oracle. Prior to joining OrganaBio, Oscar was a member of the Quality Management team at Apotex – Aveva Drug Delivery Systems for ten years. Oscar has earned a Master’s in Business Administration from Nova Southeastern University and a Bachelor of Science in Electrical Engineering from Florida International University.