"Hello and welcome to CELLINK's webinar titled Cool Cells, Cooler Results, Innovative Preservation Solutions for Cell Therapy presented by Arium Mini Laddie. Please allow me to introduce myself. My name is Shan Yao Xiang, and I'm a Market Entrance Strategy Manager at Sartorius, and I will be your moderator for today.

So now let me introduce our speaker for today, Rukmini Laddie.

Rukmini is an external collaboration manager at Sartorius where she leads the formation of partnerships for immune and stem cell therapies through scientific collaborations. So she holds two master degrees in pharmaceutical science and has over seven years experience as a bioprocess development engineer. Before joining Sartorius, Rukmini worked in R and D and process development in the field of cell therapies. So let's get started. We hope you will enjoy today's webinar about cell preservation. Bugbini, please start when you are ready.

All right. Thank you, Shania, for the introduction.

All right, greetings everybody. Welcome to today's webinar on Innovative Preservation Solutions for Cell Therapy. I'm delighted to have all of you as we explore several key aspects of cell therapy preservation.

In today's webinar, we'll address the challenges of preserving cell therapy products and highlight the key benefits of our all in one solutions.

Then we'll explore the suitability of cold storage for cell therapy, as well as discuss how custom solutions with recombinant human albumin can improve your post op cell viability.

Lastly, we'll conclude our webinar by summarizing the key insights gained from our today's presentation.

Now before we begin, let's take a moment to understand the current landscape of cell therapy drug development and see why is it important to preserve cells.

As you can see from the graph, the landscape of drug development, it's flourishing. With over three thousand five hundred drugs currently under development, these diverse cell therapies are across CAR T cells from NK cells and many more.

However, we should know that the success of these therapies is heavily dependent upon the preservation of the cells and maintaining their viability.

This preservation process is utmost important as the cell therapy products when given to the patients are safe, they are efficacious, and stable throughout the treatment process.

Now with that high level overview, let's dive deeper into understanding some of the challenges in cell therapy process.

Now, as we all know, the workflow of cell therapy, it's intricate.

It all begins with the initial harvest of the starting material at the clinic, which is then transported with utmost care and efficiency to the processing facility.

At this crucial stage, the cells undergo a series of meticulously designed processes in order to achieve the desired characteristics within the cells.

Now, as we move forward in this chain, the next challenge lies in the reliable transport of these processed cells back to the clinic for patient administration.

So overall, this complex and interconnected supply chain of maintaining the essential elements of the cells, it becomes of absolute critical importance when given to the patients.

And now let's focus on addressing some of the key building blocks of this supply chain and try to understand how we can overcome them.

So let's take for an example, starting with the freezing medium.

This is the step at which most of the cell therapy developers, they possess the challenge of selecting the optimal formulation that could cater to diverse cell types and therapies. Now this crucial step sets the foundation for a successful cryopreservation.

Moving on to the cooling method, here our challenge lies in achieving controlled cooling rates while delicately avoiding any cellular damage.

And now once the cells are frozen, the challenge now shifts to the storage temperature.

Here, we must ensure appropriate storage temperature for long term usage.

And lastly, as we progress to the thawing and the post thawing stages, our focus now shifts in implementing uniform thawing techniques in order to optimize the cell functionality upon revival.

So let's take a moment to understand on a very high level overview what happens during the cryopreservation process.

So now as the cells cool down, they face the challenge of extracellular ice crystal formation that disrupts the balance within the cells causing them dehydrated.

In some cases, cells may even super cool below the equilibrium freezing point, which can cause formation of intracellular ice.

Now when it comes to time when it comes the time to warm the cells, the ice crystallization can cause a mechanical damage resulting in cell lysis.

And if by now the process is not fully optimized and controlled, it can induce apoptosis, which can eventually cause delayed cell death post thawing.

Therefore, cryoprotective agents are typically added in the formulation to overcome some of these challenges. And amongst them, DMSO stands out as the popular choice because of its effectiveness protecting, effectiveness in protecting cells during thawing as well as the feeding steps.

Although many cell therapy developers, they still rely on homebrew cryosolutions.

These include a salt and a DMSO combination, but the problem is the suboptimal performance of this formulation.

There is a potential contamination risks. There are loss of process standardization and regulatory compliance.

Thinking of all these factors and to address the issue and streamlining the care preservation process, I'm pleased to introduce our innovative solutions that have been developed to effectively address some of these challenges at hand.

With our all in one ready to use, we provide a comprehensive approach to self preservation. In today's webinar, I will be highlighting some of our flagship products, NutraFees D5, NutraFees D10, and NutriStore.

And I'll also speak about how these products can benefit your preservation needs.

Additionally, to complement your custom preservation needs, we offer recombumin elite and recombumin prime, which are the superior choice when compared to human serum albumin.

And now let's start with a very high level overview with one of our flagship products that is NutraFease D10.

NutraFease D10 is the all in one ready to use solution that's completely free of animal components, serum, and proteins.

It's medium based and is defined with a ten percent DMSO concentration and is chemically defined.

It's particularly useful for sensitive cells and is designed for safe freezing at ultra low temperatures, ensuring the long term storage of cells.

It has been shown to have proven efficacy in preserving cell quality attributes across diverse cell types.

Similarly, let's also take a look at the Neutrophies D5.

Neutrophies D5 is a salt based solution. It is also all in one, ready to use solution, and is chemically defined with a five percent DMSO concentration. It's also completely free of animal components, serum, and proteins.

It also enables safe freezing at ultra low temperatures and is suitable for clinical applications.

And lastly, it has been tested across a wide range of cell types, ensuring high cell viability and recovery post thaw.

Okay. Now let's take a closer look at each of these products.

Starting with NutraFreeze D5, let's dive into some of the case studies and explore the data to understand its performance.

Okay, so here is a representative study design involving human mesenchymal stem cells.

MSCs were sourced were sourced from various tissues, such as bone marrow, adipose tissue, and were cryopreserved using neutrophils D10 for up to a week. Now following thawing, the cells were cultured in MSC NutriStem medium, and they underwent quality control checks.

Additionally, in a separate long term study, MSEs were cryopreserved for a period of up to five years, followed with post saw evaluation.

And now let's peek at the data to gather some insights.

In the short term cryopreservation studies, as we can see from the slide, graph A demonstrates the high cell viability above ninety five percent post thaw, which is equivalent to the other serum free solutions that are seen in the market.

While to the right on the slide, you can see graph b, which exhibits comparative data on the fold expansion of the cells, revealing more than seven fold increase in after expansion by day three post thaw.

Now overall, this highlights the superior viability and the proliferation of rate of the cells compared to the other streamed feed solutions in the market.

Graph C and D, they display the comparative data for immediate post thaw as well as viability after three days of recovery. And in both the conditions, we can see that the cells show very high viability, which is equivalent to or above ninety one percent.

The cells also maintain over ninety five percent expression of multipotency markers indicating the preserved functional characteristics of the cells after freezing.

Now also let's take a look at the long term data.

Looking at the long term preservation data in graph a, we are observing the MSCs derived from bone marrow. It maintained a remarkable cell viability of ninety one percent and above, even after three years of storage.

Similarly, in graph b, MSCs derived from the adipose tissue, they exhibited a viability of ninety four percent and above after five years of cryopreservation.

Now all of these findings, they highlight the long term performance of neutrophil's d five, regardless of the tissue source from which the cells were cultured.

So now building upon these compelling results, I would now be sharing some case studies highlighting the D10 applications.

So here is a representative case study. This was conducted at the Ottawa Hospital Research Institute. It compares the neutrophils D10 with a homebrew and a competitor trial solution.

As you can see from the experimental design, MSCs were cultured from healthy donor bone marrow in ten layer hyperflasks. The cells were then cryopreserved at different concentrations, followed by a post hoc culture and quality assessment.

Now the data from the graph labeled A and B, they show the comparative viability of the cells measured from trypan blue and annexin dyne, while the graph C on the top right corner, it represents a cell morphology after six days of culture.

As you can see from the data, the graph clearly demonstrates MSC Scry Preserved and Eutrophies D10. They had the highest post op viability compared to the other cryosolutions.

Now let's turn our attention to an external validation assessment.

In this study, Eutrophies D10 was evaluated for cryopreserving pluripotent stem cells in comparison to the wire cell standard cryo media.

From the experimental design, THORT samples underwent evaluations for expansion at various passages.

They were analyzed for phenotype, and karyotype analysis was performed at the end of the study.

The graph E and B on the slide in the middle, they demonstrate the D5 results. They maintained a comparable viable cell density as compared to the wire cells control during the first and the second passages. Moreover, the cells in this time, they retain ninety five percent marker expression for the undifferentiated cells.

Additionally, the karyotype analysis revealed no clonal abnormalities.

So overall, in summary, neutrophil's d ten had successfully met all the quality requirements for preserving the integrity as well as the quality of the cells by the end of the study.

Okay. So now coming to this next flagship product, which is the neutrophil d five.

So let's take an example. Here is a cryopreservation study design for again using a human mesenchymal stem cells with neutrophil d five.

In this study, MSCs from various tissues were cryopreserved in d5 for up to a week. Subsequently, the cells were thawed, and they were cultured in MSC nutristem xenophree media followed by quality control checks.

Now let's dive into the data.

The graph A and B on the slide, they demonstrate the viability of MSCs obtained from various tissues. And as we can see from the data, the results remained comparably consistent, exceeding ninety six percent viability expression, both prior as well as after thawing.

Additionally, graph c provides a summary of the representative images after three days of culture, demonstrating a robust proliferation rate and healthy cell morphology from the two different tissue sources.

So in conclusion, D5 demonstrated a consistently high viability across diverse tissue sources, while it also promoted the enhanced cell proliferation.

Furthermore, exploring the phenotypic data, neutrophil d five effectively preserved the essential surface markers for the MSCs while it also kept the hematopoietic contamination to the minimum.

Now moving forward to the impact of d five on the differentiation potential.

The images on this slide over here, they serve as a visual representation of the successful differentiation of MSCs into adipotrinic, chondrogenic, and osteogenic lineages.

Furthermore, it's important to note that the karyotic analysis of all the tested samples, it revealed a normal genetic profile.

So these findings indicate that the preserved cells, they not only had the potential of a good viability maintaining good fold expansion, as well as also had the differentiation potential and the genetic stability of the cells was sustained after cryopreservation with the neutrophils d five.

Okay. Now to further uncover valuable insights, let's assess some case studies in which both D5 and D10 cryopreservation solutions, they were studied side by side. It's important to note here that the comparison of these solutions was done in order to have a comprehensive assessment to identify the effectiveness of these solutions in different cell types.

So here's another another example.

In this slide, cryopreservation of patients' CAR T cells was performed with two donors using both the solutions T5 and T10. In the study, twenty five million CAR T cells were cryopreserved for three days, and then they were thawed and expanded in NuTriStem media for up to ten days.

Graph a on the slide, it showcases the impressive performance of both the solutions, consistently maintaining cell viability above ninety two percent and a percent yield exceeding fifty three percent.

Additionally, to the right, we can see from graph b, it displays the fold expansion results of both the donors demonstrating a comparable outcome.

So this indicates that the consistent and reliable performance of both the solutions were seen in irrespective of the donor as well as irrespective of the performance of the cells.

Furthermore, the study also reveals that the CAR expression in these cells, it remained consistently high, ranging from sixty two percent to seventy eight percent in both the solutions.

Graph b to the right, it confirms that both the solutions effectively also maintained the desired distribution of CD four and CD eight T cell subset, supporting the functionality and the therapeutic potential retention of the cryopreserved CAR T cells.

Overall, these findings highlight the successful preservation of CAR T cells, ensuring high cell viability, percent yield, and maintaining the desired T cell subsets.

Okay. Now before we wrap up with the case studies, let's review one last study.

In this study with embryonic stem cells, cells were cultured as single cells using NutriStem PSC media, and then the cells were cryopreserved in each of the solutions for eleven days.

Post thawing the cells, the growth recovery and the characteristics of the cells were evaluated.

From the data, as we observe the first column in this graph, we can see that in both conditions, the cell viability was above eighty five percent.

And this number only kept increasing as we compared two different data sets from day three and day four.

Similarly, the flow cytometry data further complemented its performance as the average expression of the desired pluripotency markers was close to ninety seven percent.

So in a nutshell, the key takeaways from the slide are both the solutions apart from the features that we just discussed, they are manufactured under CGMP conditions.

They have been tested in diverse cell types. It's also important to note that D5 being a salt based solution with a lower concentration of DMSO is specifically designed for clinical application.

All right. Now let's switch gears and talk about an alternative approach to cryopreservation, the method of cold storage.

So as I mentioned earlier, the traditional cryopreservation approach for cell preservation, it involves a complex multi step workflow, starting from harvesting cells to cryopreserving them and eventually throwing them for infusion.

However, as we all know, this process is time consuming, it's logistically complex, and is expensive.

To this end, NutriStore, a cold storage medium, offers an alternative solution for storing cells at two to eight degrees Celsius over a short period of time.

How would this help? This approach would offer simplicity and convenience for storage and transport. It would bypass the traditional freeze thaw steps.

Overall, it will provide a convenient solution for cell therapies in order to preserve their cells for the short term period of time. Now let's review the product details.

NutriStor Cold Storage Solution is also ready to use. It's animal component free, it's serum free, and protein free, suitable for cold storage at two to eight degrees Celsius.

Its drug master file will be available soon.

It ensures high cell viability and recovery rates post cold storage and is suitable for clinical applications.

Now based on the study is conducted so far, let's look at some examples to evaluate its performance compared to the current market competitors.

Alright. So in this study, fresh PBMCs from two donors, they were stored in NutriStore and a competitor solution for four days, followed by seeding and culturing for eight days.

Graph b graph a, sorry, on the slide, it showcases d five microscopic images, revealing a typical and healthy morphology of the PBMCs stored in UT store compared to the competitor solution.

Also to the right on graph b, it demonstrates a higher fold expansion and comparable viability in PBMCs stored in our NutriStore solution.

Thus overall, it highlighted a superior performance in preserving cell viability and promoting cell growth.

Let's take another representative study here. In this study, MSCs were stored in NutriStoRE and accommodated the solution for four days, and subsequent post storage evaluations were conducted.

From the graph in the middle containing the percent cell viability and recovery rates, we can see that the NutriStor cold storage solution outperformed the leading competitor.

After four days of storage, cells stored in NutriStone showed significantly high recovery rates while maintaining excellent viability.

Lastly, NutriStone maintained the trilinear differentiation potential and genomic stability of the cells, also consistently outperforming the leading competitor and ensuring the optimal cell functionality for therapeutic applications.

Okay.

Now the second part of this today's webinar, we'll switch gears again and explore the application of recombinant human albumin in cell therapy preservation.

Let's start with a brief background. As we all know, human serum albumin is the most abundant protein in plasma and is essential for maintaining blood's colloidal stability.

Its properties include it's highly soluble and stable and also acts as a carrier for transporting a wide range of substances.

Now because of its low immunogenicity, it is compatible for various applications.

However, its application in cell therapy production, it poses significant challenges.

One such significant drawback is the lot to lot inconsistency.

Variation in the human serum albumin in the batches can greatly affect the quality and the performance of the cells during cryopreservation process.

Another concern is the risk of pathogen contamination. HAC from human blood carries the risk of transmitting infectious agents, raising the safety concerns for cell therapy.

Furthermore, we are talking about regulatory hurdles adding complexity, and also the demand of HSA can sometimes outpace the availability and supply.

So with these disadvantages, let's talk about some alternative solutions.

As an alternative solution, I'm pleased to introduce the recombinant human albumin as it offers several key advantages.

It is ready to use, it's animal component free, it is chemically defined, and it provides optimal conditions for not only cell cryopreservation, but cell proliferation, cell viability, and post thaw cell recovery.

It effectively overcomes the challenges related to the batch to batch inconsistency, ensuring a high purity and maintaining a safe manufacturing process.

Furthermore, the reuse of recombinant albumin, it enables customization and scalability and addresses the limitations of issues associated with a human serum albumin.

Lastly, it can also be the choice of your supplement for cell culture media, wash buffers, formulation buffers, and other applications.

Now let's explore some case studies highlighting its application in cryopreservation.

Here is a representative study design with mesenchymal stem cells. In this study, two freezing conditions were compared. DPBS with a ten percent DMSO and two percent recombium and a DPBS condition with ten percent DMSO and two percent human serum albumin. On the day of thawing, cells were rapidly thawed in water bath, diluted with HSC or recombium in plasma light, and the DMSO was then washed off by centrifugation.

Lastly, each of the condition was assessed for viability and potency and other assays.

Alright. So the graph displayed on this slide, it summarizes the results of three GMP grade cell lines indicating a higher cell viability with recombumin compared to HSA over seventy two hours post op.

As we can see, recombumin consistently maintain a viability above seventy percent while the human serum albumin condition experienced a significant drop.

So this represents a significant increase of the product shelf life as compared to the standard conditions using HSA.

Let's continue with the data from this study. The study also investigated the apoptotic state of the cryopreserved cells. So this was assessed by assessing the number of live cells that were in early or late stage, number of cells that were in the early or the late stage apoptosis. Apoptosis.

So the graph on the left, it showed no significant differences between recombumin and HSE on the total apoptotic cells. However, if you take a closer look at the graphs on the right, the recombumin preserves the cells in the early apoptotic stage, prolonging their shelf life.

In addition, the phenotypic characterization of the MSCs at zero to seventy two hours, it preserved its identity, and in both the conditions had the positive desired positive, which is a CD seventy three, CD ninety, CD one zero five, and the absence of the markers of CD thirty one, CD forty five. Also, the low level of HLA Doctor expression indicated the maintenance of the MSC phenotype during cryopreservation.

Lastly, taking a look at the in vitro differentiation assays, it confirmed the multipotentiality of the cells and the ability to differentiate into fat, bone, and cartilage lineages.

Now moving to another cell type, let's take example of induced pluripotent stem cells.

So here, iPSCs were cultured as single cells to eighty percent confluency, and they were frozen in one million cells per ml in different concentrations of recombium with standard cryosolutions. And also a commercial IPC cryosolution was used in this for comparison.

So here the cells underwent slow ramp freezing followed by storage in liquid nitrogen and subsequently the cells were thawed for post thaw evaluation.

In order to evaluate its performance post thaw, two hundred thousand cells were cultured for several days and the number of colonies formed were counted.

So I will be giving a representative example in this slide.

So in this slide, as you can see from the graph, recombumin demonstrated a significant improvement in colony formation post cryopreservation that also outperformed not only the standard cryopreservation solutions, but also outperformed the commercial IPC cryopreservation solutions.

Moreover, there was no observed change in blood potency of the cells.

Additionally, these findings underscore the effectiveness of recombumin in enhancing the post cryopreservation outcomes for colony formation in the iPSC stem cells.

Likewise, let's take another example with iPSCs again. In this study, the cells were cultured as single cells to ADP C confluency, and they were frozen in one million cells per ml in three different cryosolutions with and without the addition of recombumin.

To the right, we are looking at the data from this study. The graph on the right clearly illustrates that the presence of recombumin is significantly improved the colony formation, not only the cryopreservation one and two, but also in the commercial IPA C cryosolution.

Also, it's important to highlight here that there was no observed change in the pluripotency of the cells.

Now moving forward to T cells. I'll cover some cryo studies from here onwards with T cells. So starting in this study, here the T cells were isolated, they were expanded, crybreserved at a concentration of ten million cells per ml. Crybreservation was done using an isotonic solution with a ten percent DMSO, and they were supplemented with different recombinant albumin or HSA.

The third cells were washed. They were resuspended in the same solution with an albumin or HSA and then kept at four degree Celsius for testing.

Here is the data of the live cell numbers measured over time.

The teal colored box on the graph, it highlights the recombium exhibited superior performance post to top of the cells with the live cell numbers up to forty eight hours being higher compared to the other conditions, including the rice derived albumin.

I'll continue with the T cells case study. Here is another example of cryopreserved T cells. When the T cells were isolated, they were expanded and a concentration and were cryopreserved at a concentration of ten million cells per ml.

In this study, the cells were evaluated for early apoptosis with and without recombium at various time points post to thawing.

And here we see the inclusion of recombium in the cryopreserved samples resulted in fewer apoptotic cells compared to the samples with the HSA.

Moreover, the recombium samples, they maintained reduced apoptosis throughout the whole time, indicating the potential to extend the viability window for the cells, especially during the patient administration phases.

Keeping the momentum on T cells. Here is another example of T cells where the cells were cryopreserved in plasma light at now different concentrations of the DMSO along with recombumin or HSA to see if the difference of the DMSO makes any difference.

After rapid thawing, cell viability was measured using Trap and Glu exclusion assay.

Taking a look at the data, recombium in the cryopreservation medium, it enabled the preservation of the live cell numbers even with the reduced levels of DMSO.

So this finding is crucial as the viability of the cells was not compromised, ensuring their optimal functionality and therapeutic potential.

Okay. Here is another example case study. Now this is slightly different as this study focused on enhancing the hypothermic storage of MSCs at room temperature without cryopreservation.

The cells were stored in plasmidolide supplemented with recombumin albumin or HSA, and sampling was performed every twenty four hours to gather the data on the viable cell counts and the surface markers.

Now taking a look at the data, as we can see from the graph, recombumin, it improved the cell stability post expansion to allow flexibility in shipments.

Moreover, there was no observed change in the multipotency of the cells. Taking a look at the marker expression to the right hand side of the slide, the cells retained the versatility and the differentiation potential.

Also, these findings suggest that simplified storage and transportation of MSCs, they improve the potential of improving the accessibility as well as the availability of the MSC based therapies.

Okay.

So to sum up the key benefits of recombumin amongst many others in cryopreservation, it has shown to improve the cell viability. Recombumin, it helps to maintain high cell viability during the freeze thaw as well as the storage steps.

It enhances stability by preserving the cell structure and functionality. Indeed, it retains, as seen from the data, it retains the desired surface characteristics and also maintains the surface markers and protein expression and supports the preservation of cell functionality by protecting the vital components through and through.

As a key takeaway from today's presentation, our innovative all in one chemically defined ready to use solutions for cryopreservation and storage include NutriFreeze solutions, D5 and D10 and NutriStor, and they enhance the viability and simplify the process.

Recombumin, it serves as a superior alternative to human serum albumin, offering superior performance, supporting the regulatory compliance for faster market access.

Now I would say please scan the QR code or type in the web address to request your free samples today.

Finally, we are proud to present our recent accusations, which highlight the Sartorius' commitment to continuously invest in our media and critical raw materials portfolio.

With all the combined years of expertise and global manufacturing and distribution network, we are here to partner with you in addressing any challenges that you may face in the supply of raw materials.

Thank you.

Thank you, Rubini, for the interesting presentation. So if you audience, if you have a question, please type in the question box in the control panel and we'll go through it. And we have received some questions already from the audience.

So, our first question is, can I order custom formulations?

So if there is a strategic and a commercial value to this, we are more than happy to help you all with the custom formulations and also help with the packaging. So the answer in short is yes.

Okay. The second question is, can I keep cells in minus twenty or minus eighty for longer periods time?

Okay. So with any freezing solution, it is not ideally recommended for storing cells if having a cryoprotectant such as DMSO for long term storage at minus twenty to minus eighty degrees Celsius.

For the Digifix and d Digifix d five and d ten, we recommend having the storage at ultra low temperatures. That's minus one hundred eighty six ideally.

Also, it's suitable if the cells are sensitive. So it's preferred that you start with the minus eighty probably with the mister Frosty for, like, twenty four hours, but then immediately go to the liquid nitrogen tank first storing the cells.

Sounds good.

The next question is, did you try to use Nutristore as formulation buffer for CAR T cells based shock problems? He is asking what is injectable?

Okay.

NutriStore an ancillary material. So it can be a part of your cell therapy process, but it cannot be a part of your final product.

Unless the customer wants to do the due diligence, we are here to support for regulatory purposes, but it's not injectable.

Thank you. Next question is, how do you validate UTFE? Yes, how do you validate it?

Sure.

So D5 and D10 have been tested on diverse cell types, but the assays performed are for, you know, the quality control checks are for pH, sterility, mycoplasma is there, appearance, osmolality of the solutions. So along with the different testing with different cell types, also perform the basic UC for these solutions.

Okay.

The next question is: Do you plan for long term stability studies such as three, six, twelve month storage and recovery? I believe it's referring to neutrophil freeze.

Oh, yes, absolutely. As I've shown from the data on the earlier slides. Yes, we do encourage the customers to try out these solutions not only but for short term storages, but also long term storages.

The only criteria here depends upon the application of the cell type and the maintenance of the freezing temperatures through and through this process. So that has to be completely controlled by the customer. But"