Introduction
In the first two blogs in this series, we looked at cryopreservation as a means of stabilizing leukopaks (LPs) for transport and storage in cell therapy. We reviewed the advantages to patients and doctors in terms of increasing the flexibility of scheduling for both leukapheresis collection and patient treatment. And also, the advantages to manufacturing pharma companies in terms of improved manufacturing scheduling and a robust supply chain. All of these benefits derive from the stability and robustness of cryopreserved leukopaks.
In our second blog, we reviewed some of the advances in cryopreservation protocols and publications that increasingly shows the minimal viability loss that can be achieved with the proper handling of leukopaks prior to freezing and during the thawing process. We highlighted the landmark paper from Novartis in which they summarize their use of cryopreserved leukopaks for a CAR-T therapeutic (Kymriah), supporting two global, multi-center, phase-3 clinical trials and subsequent commercial manufacturing. You can find links to the first two blogs in this series at the end of this article.
What drives post-thaw viability for cryopreserved leukopaks
As we saw in blog 2, from the published literature it is possible to get very similar levels of pre-freeze and post-thaw viability from the same leukopak before and after cryopreservation. This is important as the cryopreservation process essentially locks the living cells at a point in time. From the perspective of the cells, time stops from the moment of freezing until they are thawed out again. Thus the health of the cell population at the critical point when time stops is going to be a key factor in determining the health and viability of the cell population post-thaw. Several factors play into this pre-freeze viability, including the time and storage conditions of the LP prior to freezing. Cells start to lose viability from the moment they are removed from the bloodstream. No matter the storage media there is no substitute for the complex, finely tuned environment inside the human body. The key to retarding this process is time and temperature, the shorter the elapsed time from blood draw to freezing the less opportunity there is for degradation of the cell population. The second is storage conditions, especially temperature. Cells maintained in a continuous, cooled environment will fare better than those that are not. For this reason, rapid processing is critical for cryopreserved LPs.
Optimizing the collection and processing of leukopaks is critical
In recognition of these facts, Discovery’ state-of-the-art collection and processing are housed within a single building. Patients or donors undergo leukapheresis and the resultant LPs need only be transported a short distance down corridors within the same building for downstream processing. Enabling rapid processing of the living cells and maintaining them in a single, contained, and controlled environment throughout. In the simplest case where LPs are simply being cryopreserved without any additional processing, the LP will be processed and frozen within less than two hours from the time the cells were removed from the donor.
Simple cryopreservation is just the tip of the iceberg, the co-location of patient (or donor) and sample processing means that Discovery can perform even complex, multistep processing of leukapheresis products on behalf of customers, such as the isolation of individual cell types, followed by single-cell transcriptome analysis, and still achieve high pre-freeze viability. Figure 2. Shows the workflow from leukapheresis to cryopreservation including a sample of the downstream services offered by Discovery. All within a sufficiently short time window, and without leaving a controlled manufacturing environment to maintain high pre-freeze, and hence high post-thaw recovery and viability from cryopreserved leukapheresis products.
Successful clinical results using cryopreserved leukopaks
Clinical outcomes using Kymriah manufactured from cryopreserved starting material in both the ELIANA and JULIET global, multicenter clinical trials was comparable to data generated in earlier studies using fresh leukapheresis material, further supporting the position that with proper, well managed collection, processing and cryopreservation frozen leukopaks can replace fresh for cell therapy manufacturing.
One common thread running through all these publications is that post-thaw viability is only as good as your pre-freeze viability, therefore optimizing handling and processing immediately prior to cryopreservation is critical for post-thaw recovery. In the next blog in this series, we will look at the critical factors that can affect the post-thaw recovery and viability of immune cells from cryogenic leukopaks and how the ability to address these factors leads to the success stories outlined above.
Figure 2. Discovery’ expedited processing workflow for cryopreserved products. Workflow 1 shows a simple leukopak cryopreservation protocol, with LPs frozen within 2 hours of extraction from the patient or donor. Workflow 2 shows some of the alternative flow paths incorporating a selection of the downstream processing and services offered by Discovery. All housed within a single, state-of-the-art facility to maximize recovery and viability.
Not all cryopreserved leukopaks are created equal
Handling living cells is extremely complex and requires deep knowledge, experience, and cutting-edge technology to be successful. Discovery’ proprietary closed-system cryopreservation protocols developed and refined over many years represent a step forward in the handling and processing of leukapheresis products. Couple this with a modern, purpose-built research facility designed around the needs of the living cells that we work with and you can see why Discovery is relied upon by some of the world’s leading cell therapy manufacturers to supply starting materials for their autologous and allogeneic products and research programs.
Discovery supplies cryopreserved or fresh leukopaks in either GMP compliant or RUO formats as required to support early-stage research all the way through to full-scale commercial manufacturing. With a wide range of in-house services such as cell sorting, and advanced characterization techniques available to customize and characterize cell and tissue samples, including our proprietary hyper-annotated® feature giving researchers the clearest picture possible of their cell populations.
If you missed the first two blogs in this series, you can read part 1 and part 2 here.
