Solving The Leukopak Supply Chain Issue With Cryopreservation

By Jennifer Chain, Ph.D., CABP, Consultant, CSM Consulting LLC

Two major supply chain challenges for allogeneic cell therapy are the quality and reliability of cellular starting materials used in manufacturing. Previously, I explored factors influencing starting material quality and their impact on therapy manufacturing and efficacy.¹

This article focuses on the reliability of starting materials, particularly leukopaks, and proposes a fundamental solution to this issue.

What Is The Impact of Unreliable Leukopak Delivery?

Over several years, conversations with allogeneic cell therapy developers have underscored the severe impact of unreliable leukopak deliveries on their manufacturing success and costs. Developers report that too many leukopaks ordered for crucial manufacturing runs arrive at the wrong temperatures, arrive late, or fail to arrive altogether. These issues, including late deliveries, result in tens of thousands of dollars in wasted reagents, supplies, and staff time, and add costs to reserve new manufacturing slots.

Additionally, developers producing allogeneic therapies for active clinical trials note that leukopak delays affect the timing of patient treatment.

Why Is The Leukopak Supply Chain Unreliable?

Donors

The unreliability of the allogeneic leukopak supply chain stems from various causes, with donors being the most common. Most leukopak donors are recruited from blood or biospecimen donor databases. While volunteer donors of whole blood, plasma, or platelets can schedule their appointments at their convenience, including evenings and weekends, the same flexibility does not apply to leukopak collections.

When donors are scheduled for leukopak collections on specific dates to meet shipping deadlines, they often treat these appointments like their volunteer donations. If something arises in their personal lives, they may not show up or may request to reschedule. By the time this happens, it is usually too late to qualify and schedule a new donor, resulting in a failure to collect the leukopak just before it is needed for manufacturing.

Leukopak collections also require donors to stay well-hydrated for several days beforehand. If they do not prepare adequately, it can be a challenge to gain or maintain venous access, potentially slowing or halting the collection. Overhydration just before the appointment can lead to requests to stop the collection early for a bathroom break.

Additionally, donors can end their participation at any time as part of the informed consent process. This can happen in the middle of the collection, especially if donors begin to experience significant side effects from the anticoagulant or if the donation is taking longer than expected. Consequently, donor participation in leukopak collections can be quite unpredictable.

Staff training in logistics

Shipping leukopaks is more complex than it appears, especially when ensuring regulatory compliance for clinical therapy manufacturing. While there are regulations for handling, packing, and paperwork, logistics professionals responsible for shipping cell therapy starting materials often lack standardized training beyond Department of Transportation-compliant courses for shipping dangerous goods.

Depending on the developer’s requirements, freshly collected leukopaks may need to be shipped at ambient temperatures (18-26 degrees C) or refrigerated temperatures (2-8 degrees C). The challenge of shipping fresh leukopaks from different donors at different temperatures for different developers on the same day often goes unrecognized until errors occur.

One developer reported that nearly half of their leukopaks arrived at the wrong temperature, despite close collaboration with the shipping staff. Other developers have experienced mix-ups in the destinations of leukopaks from donors qualified for their collections.

Courier issues

Once the collection organization hands over the leukopak to the courier, its delivery largely depends on the courier’s skills and efficiency. Shipping delays are often caused by adverse weather conditions along the shipping lane. The right courier can mitigate many issues by having multiple shipping lanes planned, using a tracking system to locate lost packages, and engaging in real-time communication with the consignee.

Despite advancements in courier services for cell therapy products, delays in flights or pickups are sometimes unavoidable. Developers consider fresh leukopaks expired within 48 hours of collection due to increased cell death over time.² Therefore, any delays in delivery can result in the leukopak expiring before it can be used.

Fundamental Solution: Cryopreservation Of Leukopaks

Implementing cryopreservation in the manufacturing process is the most widespread solution to these and other logistical challenges. Using cryopreserved leukopaks eliminates the impact of donor delays on product delivery. Collection centers can schedule donors well in advance of the shipping date, allowing time to qualify and schedule a new donor if necessary. Cryopreserved leukopaks are shipped in liquid nitrogen dewars, simplifying the packing process. They can be shipped several days before they are needed, mitigating issues related to shipping delays. The dewars can maintain cryogenic temperatures for seven to 10 days, providing ample time to account for unexpected delays or to be used as short-term storage.

Developers would benefit from incorporating cryopreserved leukopaks early in their manufacturing process, rather than switching later once logistical issues are realized and unnecessary costs mount. Transitioning from fresh to cryopreserved leukopaks as starting material requires some redevelopment efforts, so the earlier this occurs, the less impact it will have on the timing and cost of therapy development. Fortunately, cryopreservation has significantly advanced in recent years, overcoming historically cited disadvantages.

Cryopreservation is progressing

Cellular cryopreservation has its origins in the late 19th century, with significant advancements beginning in the 1950s.^3-5 These advancements include determining the optimal cooling rate of 1 degree C per minute, using cryoprotective agents like dimethyl sulfoxide (DMSO) to prevent ice crystal formation inside cells during freezing, and rapidly thawing to prevent ice from damaging the cells.

The most recent advancement in cryopreservation is the use of control rate freezers, which enhance temperature regulation and track temperature decreases and heat release during ice crystal formation.³ Sophisticated cooling programs (freeze curves) for these freezers are validated to maximize cell viability. Control rate freezing has become the gold standard for preserving cell-based therapeutic products.

Cryopreservation is being used more frequently

The use of cryopreservation for cellular starting materials in cell-based therapies has steadily increased over the years, with a significant surge during and after the COVID-19 pandemic.^6,7 The pandemic posed challenges for transplant physicians in accessing resources and safely performing bone marrow transplants, making the cryopreservation of collected bone marrow and mobilized leukopak products crucial for the success of both autologous and allogeneic transplants during that time.

Prior to the pandemic, Novartis identified major supply chain issues with using fresh starting materials. As a result, it developed its FDA-approved CAR T therapy, Kymriah, to be manufactured with cryopreserved patient starting material (non-mobilized leukopaks). This approach provided Novartis greater flexibility in collections and simplified shipping logistics by cryopreserving patient cells within hours of collection.²

Cryopreservation stops the clock on cell death

Cells in freshly collected leukopaks shipped at ambient or refrigerated temperatures die increasingly over 48 hours. The cellular composition of the leukopak affects cell death rates; lymphocytes and monocytes die faster in leukopaks with higher red blood cell and granulocyte contamination.^8-10 By the time a fresh leukopak reaches the manufacturing facility, cellular viability is at least somewhat compromised. Cryopreserving the leukopak within hours of collection essentially halts cell death in the key therapeutic populations that will be isolated during manufacturing.

Stopping the clock on cell death is dependent on starting with a leukopak low in red blood cell, granulocyte, and platelet contamination¹¹ and using a well-optimized cryopreservation protocol, which includes a high-quality cryoprotective agent and a control rate freezer with a validated freeze curve.^4,5 Although cryopreservation is a complex technique, skilled scientists and technicians with proper training in cryopreserving blood products with high cellular concentrations can perform it competently. Additionally, optimizing the thawing procedure is crucial to maximize cell viability.

Cryopreservation does not negatively impact CAR T manufacturing or efficacy

It is commonly believed that cryopreserved cells are less viable or functional than fresh cells. Indeed, studies have shown that T cells from thawed leukopaks have lower viability and recovery compared to fresh leukopaks, and they exhibit a slower start to expansion.^2,12,15 However, these same studies and others demonstrated no correlations between post-thaw viability or T cell recovery with cellular phenotype, transduction efficiency, cell yield after expansion, or cell function in vitro or in vivo. Cryopreservation also did not alter gene expression or the proportions of key cell types. This suggests that while cryopreservation may cause initial delays in cellular activity after thawing, the cells eventually recover and match the expansion and function of their never-frozen counterparts.

When Novartis transitioned its clinical manufacturing from academic processes, it compared fresh and cryopreserved starting materials.² It conducted multiple studies to establish the best cryopreservation protocol to maximize the manufacturing success of Kymriah. Cryopreserved patient material and healthy donor test material met the acceptance criteria established with fresh leukopaks, and thawed cells exhibited comparable overall growth kinetics to fresh cells. Manufactured cell therapy products from both cryopreserved and fresh leukopaks had similar cellular composition, proportions of transduced cells, CD19 CAR expression levels, and functional responses to target cells. It also demonstrated that cryopreserved leukopaks remained stable for at least 30 months, showing comparable results to leukopaks stored for only six weeks. Novartis reported that cryopreservation helped create manufacturing consistency despite variable patient material.

Novartis also demonstrated comparable clinical outcomes between early trials with Kymriah manufactured using fresh leukopaks and later trials using cryopreserved starting material.² Other studies have shown similar results,^13,14 indicating that clinical outcomes are not impacted by using cryopreserved starting material or drug product: CAR T cells remained active against their tumor targets and patients achieved disease remissions.

Conclusion

Issues that affect the timely delivery of fresh leukopaks can significantly impact developers of allogeneic cell therapies. Delays result in wasted reagents, supplies, and staff costs, affecting the financial bottom line of clinical trials. For some developers, these delays can also hinder patient access to treatment. Cryopreservation of leukopaks addresses many logistical problems causing these delays. It offers flexibility in scheduling donor collections and shipping materials, ensuring availability for critical manufacturing runs.

Historical concerns about cryopreserved cells, such as reduced viability and recovery, do not seem to affect the cellular expansion, function, or quality of the final manufactured product. Cryopreservation can enhance the consistency of donor-derived starting materials across different manufacturing runs. Therapies made from cryopreserved cells perform well in patients.

Developers who integrate cryopreservation early in the development of their manufacturing process are likely to experience significant cost savings compared to those who switch after developing their process with fresh material or those who never switch and continue to face the challenges of unreliable fresh leukopak delivery.

References:

1. What’s In The Leukopak Matters for Cell Therapy Manufacturing
2. Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture
3. Cryopreservation: An emerging paradigm change
4. Cryopreservation: An Overview of Principles and Cell-Specific Considerations
5. Chemical approaches to cryopreservation
6. Transplantation of Allogeneic Cryopreserved Hematopoietic Cell Grafts during the COVID-19 Pandemic: a National Marrow Donor Program Perspective
7. Cryopreservation of unrelated donor hematopoietic stem cells: the right answer for transplantations during the COVID-19 pandemic?
8. Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function
9. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function
10. Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells
11. Cryopreserving human peripheral blood progenitor cells
12. Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics
13. Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients
14. CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production
15. Impact of cryopreservation on CAR T production and clinical response

About The Author:

Jennifer Chain, Ph.D., CABP, is a cellular therapy expert with 26 years of experience in T-cell immunology, product development, blood banking, and consulting. She holds a Ph.D. in immunology and a Certified Advanced Biotherapies Professional credential from the Association for the Advancement of Blood and Biotherapies (AABB). She currently works as a consultant in the cellular starting material space, helping blood centers and cell therapy companies develop CSM collection and procurement programs and donor screening strategies. From 2016 to early 2024, she led efforts to collect leukopaks and bone marrow from more than 1,000 healthy donors and developed novel blood- and cell-based culture materials for early-stage cell therapy companies. She volunteers and consults for AABB, where she engages in educational program development, strategic planning, and advocacy efforts in the field of cellular therapy. Reach her on LinkedIn or CSM Consulting’s website, www.cellsmatter.com.