The One Process for Small Volumes in Biopharma

From aliquoting and filtration to freeze and thaw, our integrated single-use solutions support efficient handling of small-volume biopharmaceutical products

Why is this process the one for you?

The Accurate One

Fill and freeze your products with the highest accuracy. With a filling accuracy of less than +/- 1 mL and reproducible freezing recipes for your optimal result.

The Flexible One

Independent from type, manufacturer and size of your single-use bag, the modular platforms and single-use technologies will meet your requirements.

The Secure One

Automated aseptic filling at highest accuracy minimizes both operational human errors and overfilling of single-use bags. Controlled plate-based freezing and thawing of single-use bags prevents loss of product quality due to cryoconcentration and reproducible freezing and thawing.

The Trusted One

With more than 300.000 RoSS® shells sold and 400 platform systems commissioned, Single Use Support is a trusted pioneer in biopharma for optimizing drug substance handling. 

Process diagram illustrating small-volume drug substance handling in biopharma manufacturing

Robust storage & shipping

RoSS technologies provide process flexibility for smallest volumes with highest accuracy:

  • Closed, robust and tamper-evident protection of your single-use bag for highly valuable drug substances
  • Vendor-agnostic and hence compatible to other systems during senstive freezing applications
  • Process flexibility for safe handling regardless of type and size of bag manufacturer without being trapped in siloed biopharma process solutions 
Preview of Single Use Support's ebook about advanced atmp fluid management

Advanced ATMP Fluid Management - eBook

The manufacturing and logistics processes for small volumes of ATMPs are complex, and there are still weaknesses, particularly in the areas of inter-site transportation, aseptic aliquotation and cryopreservation. 

In order to move ATMPs, which are typically packaged in single-use bags, from point A to point B in the most effective and efficient manner, there is an urgent need for reliable equipment that covers the steps of filling, freezing, shipping, thawing and draining. All of these process steps require robustness, ease of use, sterility and fast handling. Single Use Support helps address the various challenges of low-volume fluid and cold chain management with innovative end-to-end process solutions.

Find guidance and download the new eBook.

Advanced ATMP Fluid Management - eBook
 

Downloads

Preview of Single Use Support's solution overview about handling small volumes

Solution Overview

Small Volumes - Solution overview

Small Volumes - Solution overview
 
Preview of Single Use Support's application note about controlled filling and freezing of cells

App Note

"Bestcellers": Controlled Filling & Freezing of Cells - App Note

"Bestcellers": Controlled Filling & Freezing of Cells - App Note
 
Preview of a guide about viral vector manufacturing

Guide

Challenges in viral vector manufacturing - Guide

Challenges in viral vector manufacturing - Guide
 
  • Automated aliquoting system - for small & large volumes

    Automated aliquoting systems are becoming more and more popular all over the life sciences sector, as well as in biopharmaceutical production. Here, precision is a fundamental requirement in fluid management. From aliquoting to liquid handling, achieving accuracy and efficiency is a continuous challenge. Automated systems provide a feasible solution, streamlining workflows and enhancing assays. In this article, we will explore the impact of laboratory automation, particularly how automated liquid aliquoting solutions optimize reagent handling and throughput. Automated filling with Single Use Support   What is an automated liquid handling system? An automated liquid handling system is a sophisticated instrument utilized not only in lab automation but also in the biopharmaceutical industry. Its primary function is to optimize the process of liquid transfer and dispensing, ensuring precision and efficiency. These systems are integral to a wide array of scientific and research applications, providing a high level of accuracy in fluid handling. Automated liquid handling systems are indispensable for a variety of assays and tasks, where the precise and reproducible manipulation of reagents is of paramount importance. They excel at automating repetitive and time-consuming tasks, thereby reducing the potential for human error. Equipped with solutions like liquid handlers and other labware, these systems are not confined to laboratory settings alone; they are also extensively employed in the biopharmaceutical industry. In both research laboratories and biopharmaceutical manufacturing facilities, automated liquid handling systems contribute to enhanced efficiency and consistency, ensuring the reliability and reproducibility of critical processes. From homogenizers to automated aliquotation systems: Applications of liquid handlers Automated aliquoting systems are dynamic solutions that find application across a multitude of fields, offering precision and efficiency in liquid handling tasks. Here is a glimpse into several fields where these systems play a pivotal role: Vaccine production: Automated aliquoting systems are critical in the precise dispensing of liquids for vaccine manufacturing into single-use containers, ensuring consistency and adherence to Good Manufacturing Practice (GMP) standards. Cell and gene therapy production: The smaller the volume, the more important is accuracy. Systems for CGT applications are essential for accurately handling critical components like viral vectors, cell cultures, and gene therapies in cell and gene therapy production, meeting the stringent requirements of low-volume batch sizes. Next generation sequencing (NGS): In genomics, automated aliquoting systems enable the precise distribution of nucleic acid samples and reagents, a critical step in NGS workflows, e.g. DNA normalization, Clinical diagnostics: Automated aliquoting systems are employed in clinical laboratories for tasks like sample preparation on microplates and well plates, assay development and labeling, ensuring reliable and accurate results, e.g. in PCR testing. Drug discovery: In pharmaceutical research, these systems facilitate the distribution of compounds, reagents, and samples in high-throughput screening processes, expediting drug discovery. Biobanking: Biobanks like cell banks use automated aliquoting systems to precisely aliquot and store biological samples, ensuring the integrity and traceability of specimens. Challenges in pharmaceutical aliquoting – why automation is key In pharmaceutical aliquoting, several challenges can impede the efficiency and precision of critical liquid handling processes. Automation has emerged as a pivotal solution to address these challenges, offering a range of benefits in pharmaceutical manufacturing and research. Pharmaceutical production often involves diverse batch sizes, from small-scale research to large-scale manufacturing. Automated aliquoting systems provide the flexibility to handle varying batch volumes efficiently, ensuring consistent and precise liquid distribution regardless of the scale. Compatibility with different systems, containers, and formats is equally essential in pharmaceutical operations. Automated systems are designed to seamlessly integrate with a variety of packaging options, workstations, and configurations, reducing integration complexities and enhancing adaptability. Cross-contamination is a critical concern in life sciences and clinical applications. Automated aliquoting systems incorporate features that minimize the risk of contamination, such as automated sample preparation and validation, ensuring the integrity of samples and data. Manual aliquoting processes can be resource-intensive and susceptible to human error. Automation and solutions like robotic arms or pipetting devices streamline the process, reducing the need for manual intervention. This not only enhances efficiency but also optimizes resource allocation and labor utilization, leading to cost-effective operations. Automated aliquoting systems also offer a high level of reproducibility, ensuring that each aliquot matches the intended specifications. This precision is essential for maintaining consistency and data integrity, particularly in experiments like ELISA assays and other critical applications. Automated aliquoting with Single Use Support Single Use Support offers a comprehensive range of automated aliquoting solutions within the RoSS.FILL product line. These innovative systems are designed to address the diverse liquid handling needs in various industries. Let's take a closer look at each RoSS.FILL variant: RoSS.FILL Lab Scale: small volumes: 1ml to 1000ml RoSS.FILL CGT (Cell and Gene Therapy): small volumes: 1ml to 1000ml RoSS.FILL Bag: 1L to 1000L+ RoSS.FILL Base: 1L to 1000L+ RoSS.FILL Bottle: simultaneous filling of up to 20 bottles (up to 5L) Fill & Filtration product range Automated aliquoting system for small volumes The precise distribution of small volumes of liquids is a critical step in various fields, from scientific research to pharmaceutical production. Single Use Support offers two exceptional solutions under the RoSS.FILL product line: RoSS.FILL CGT and RoSS.FILL Lab Scale, each designed to streamline automated aliquoting of small batches with precision and efficiency. RoSS.FILL CGT is a fully automated aseptic filling system tailored for the specific requirements of cell and gene therapy production. It is particularly well-suited for applications with low-volume batch sizes. This advanced system enables the simultaneous filling of multiple small single-use bags, making it an ideal choice for handling volumes ranging from 1mL to 1000mL. RoSS.FILL CGT offers a high level of accuracy and efficiency, and it provides the option to attach more racks for enhanced throughput. RoSS.FILL Lab Scale is a versatile automated filling platform designed for laboratory settings. With the capability to handle volumes from 1mL to 1000mL, it is adaptable to a wide range of applications in research and development. This system boasts a small footprint and state-of-the-art technology, ensuring precise and efficient liquid handling. RoSS.FILL Lab Scale is an excellent choice for labs and research facilities that require automated sample aliquoting with consistent results. Both RoSS.FILL CGT and RoSS.FILL Lab Scale offer solutions that enhance the accuracy, efficiency, and reliability of automated small volume aliquoting, making them valuable tools in various industries, including pharmaceuticals, biotechnology, and research laboratories. With features like an automated stepper function as well as an integrated automated sealing function and labeling function, these systems contribute to the precision and consistency required for successful experiments and manufacturing processes. Automated aliquoting system for large volumes In liquid handling, achieving efficient, fast, and safe bulk filling processes is paramount. To do so, Single Use Support's RoSS.FILL product line includes three essential variants – RoSS.FILL Base, RoSS.FILL Bag, and RoSS.FILL Bottle –, which excel in these aspects, catering to a wide range of applications. RoSS.FILL Bag is a versatile automated single-use aseptic bag filling machine, accommodating a wide variety of sterile connection and disconnection options. This system is capable of filling volumes up to 400L per batch with exceptional accuracy. RoSS.FILL Base serves as a flexible platform for bulk filtration and dispensing drug substances into single-use bags. It offers limitless batch volume possibilities, with the capacity to fill several 2D single-use bags up to 500L. For even larger quantities, the system can attach a 3D single-use bag, allowing bulk filling of up to 1000L. It is the go-to choice for applications requiring high-throughput operations and exact calibration. RoSS.FILL Bottle is a fully automated aseptic single-use bottle filling and filtration system that is designed for the precise handling of both drug and non-drug substances. Its high filling accuracy ensures consistency in every operation, making it an excellent choice for applications like drug discovery, where precision is of the essence. The system can simultaneously fill up to dozens of bottles, operating as a closed system to guarantee the safety and integrity of the filling process. [[download-1-email-detailed]] These RoSS.FILL variants offer high-throughput solutions, making them suitable for applications in drug discovery, nucleic acid research, and more. They can elevate liquid handling efficiency, ensuring precise and safe bulk filling processes in a variety of fields. Advantages of automated liquid handling with Single Use Support Single Use Support's automated liquid handling solutions offer numerous advantages that cater to the evolving needs of various industries. One key advantage is scalability, enabling these solutions to adapt to changing production requirements seamlessly. Whether you need to process small or large volumes, these systems can efficiently accommodate your demands, making them cost-effective in the long run. Interoperability is another strength of these solutions. They can seamlessly integrate with different systems, packaging solutions and other consumables, promoting a cohesive and efficient workflow including real-time monitoring. This level of variability is particularly valuable in industries with diverse liquid handling needs, as it ensures a high level of flexibility and operational compatibility. Moreover, Single Use Support's use of single-use technologies adds an extra layer of flexibility and economy to these automated solutions. By incorporating single-use components, they minimize the risk of cross-contamination and reduce the need for extensive cleaning processes. This not only enhances safety but also contributes to resource efficiency. Scalability to handle varying production volumes, interoperability with different systems, and the utilization of single-use technologies for enhanced flexibility, safety, and resource efficiency: The advantages of Single Use Support's automated liquid handling solutions make these systems indispensable in industries where adaptability, precision, and economy are paramount. [[download-2-email-detailed]]  

    READ MORE
  • Small volumes of ATMPs: Safe handling of single-use bags

    In recent years cell and gene therapies (CGT) have proven to be promising approaches to personalized medicine. Patients needing a cell and gene therapy as an advanced therapy medicinal product (ATMP) rely on their treatment being ready as soon as possible. For example, there are CAR T-cell therapies that have been approved for for very aggressive and hard-to-treat types of cancer. Moreover, CGTs yield a small volume of individualized product for each single patient. Loss of the shipment or damage to the transported goods have serious consequences for patients. Not only a safe transport, but also speed, accuracy and process flexibility play an important role in manufacturing and patient safety. Therefore, reliable, safe, and scalable solutions are needed. RoSS.KSET satisfies all those requirements. It is an integrated solution to protect and safely transport single-use bags for small volumes at low-temperature refrigeration. The secondary packaging also enables advanced fluid management and cold chain logistics by accessing modular aliquoting of small volumes into single-use containers and plate-based freezing for the best freezing performance.   More about RoSS KSET Single-use bag protection for ATMP During autologous cell therapies, each patient receives an individually manufactured product. Therefore, the drug substance is of unusually high value. Having a safe and standardized logistics process in place is essential for success. But also allogeneic cell therapies or viral vector production for gene therapies require a safe handling of small volumes. In this case even in different scales. In most cases, the volume of ATMP remains small. But either bag sizes or bag quantities can have an influence on manufacturing facilities.  RoSS.KSET is a solution to safely protect small volume single use bags of all types used in cell and gene therapies. It is designed to be used effortlessly by medical and biotechnological personnel: robust and closed construction, extra space for safe storage of satellite samples and vials.  Read more: How to avoid single-use bag leakages in bioprocessing Meeting high standards in cell and gene therapy Meeting the high standards in cell and gene therapy requires a huge effort in process and logistics optimization and standardizing. With highly personalized drug substances, e.g. CAR T-cells, strict requirements for the associated logistics and supply-chains have to be met. These parts of the CGT approach are essential to treat patients effectively and economically. Here, loss of drug substance is a major issue. Beyond implementing cGMP production into manufacturing facilities, automated solutions help fulfil 21 CFR Part 11 requirements. Reported audit trails and monitoring support the way to a more efficient manufacturing process of advanced therapies. Small scale solutions It all starts small. Therefore small scale solutions are required in various areas of pharma applications. Here is an except of where advanced solutions for small volumes are required Clinical studies from Phase I to Phase III Clinical trials Cell Banking  Autologous and allogeneic cell therapies Gene Therapies, including viral vector production Non-viral vector production, such as LNPs. Plasmid DNA It all involves the processing of small volumes of very highly valuable substances. It is essential to minimize any risk of failure, be it from inconsistencies and deviations while handling or damage of single use bags during the transport. To make ATMPs manufacturing work on a world-wide scale, innovative automated solutions to all these issues are needed. CGT bag protection system - read more   Secure protection of small volume bags The handling of highly valuable small volume single-use bags is challenging. At the cold temperatures needed to keep the drug substance from disintegrating, single-use bags become prone to breakages from impacts or vibrations during transportation. Even a tiny leak has serious consequences for the seriously ill prospective recipient of the CAR T-cell therapy. But also overfilling through manual filling can be a reason for leakages in single-use bags. Single Use Support’s RoSS.KSET was specifically designed to secure single use bags while being in the supply-chain. Their 3D foam lining offers protection against shocks and vibrations, and the robust construction offers a safe shell against impacts. The materials were chosen for their thermal conductance to ensure optimal behaviour during freezing and thawing. The compact design allows for efficient storage in cryoconditions. Fast and flexible transportation of small volume bags Cell and gene therapies require the handling of miniscule amounts of liquids, sometimes less than 5 mL. Standardization and automation of such processes are needed to reduce error rates and contamination risks. Single Use Support solved these challenges with a series of platform systems: RoSS.FILL is an automated aseptic filling platform with unprecedented filling accuracy. Its version for small volumes can fill single-use bags and medical devices with less than 5mL and unbeaten precision. It allows the automated processing of liquids with high flexibility. RoSS.pFTU and RoSS.LN2F are freezing and thawing platforms that are scalable and compatible with single use bags of all sizes and customized types. Both enable controlled freezing liquids in single-use bags.  Together with the protective shell RoSS.KSET, Single Use Support's filling and freezing platform solutions are an ideal part of the logistics side of personalized medicine. More about RoSS-pFTU platforms How Single Use Support's RoSS KSET helps to improve cell and gene therapies Cell and Gene therapies, such as CAR T-cell therapy, have proven their potential to become standard treatments to hard-to-treat diseases. Among the obstacles to their commercialization are the handling and logistics of small volumes of highly valuable and irrecoverable individualized drug substances. Single Use Support has developed end-to-end solution in transporting small but valuable therapies safely. As response to various day-to-day challenges of manufacturers, the platforms RoSS.FILL and RoSS.pFTU enable a safe and monitored aliquoting, freezing and thawing of small volumes into single use bags. The complementary shell RoSS.KSET protects the products from damage during storage and transportation. Single Use Support strives to further improve the ATMP manufacturing and make advanced therapies an affordable treatment option for patients all over the world. Learn more about RoSS.KSET

    Read more
  • Stressing Cells: The Role of Cryoprotectants in ATMP Cryopreservation

    In cell and tissue therapies, living cells are used to treat previously chronic diseases, like cancer forms, arthritis, or certain autoimmune disorders. To be effective, the living therapeutic product requires administration with a sufficient cell number at a high cell viability and functionality. Freezing of cells during cryopreservation attempts to target those quality requirements by simultaneously ensuring a prolonged shelf life of the starting material as well as the final therapeutic product. This provides greater flexibility for patients and during the manufacturing process. Cryopreservation accompanies the entire manufacturing and logistics process of Advanced therapy medicinal products (ATMPs) and thus has an immense impact on the therapeutic success and safety of the product. [[1]] Cells and tissue therapies that are currently approved, originate from a variety of cell types like dendritic cells, chimeric antigen receptor T (CAR-T) cells, haematopoietic stem cells, fibroblasts, chondrocytes, limbal stem cells and adipose-derived stem cells. [[1]] Due to the variety of cell types as starting material and their different optimal conditions during bio-cryopreservation, each cell and tissue therapeutical product requires its own specific and coordinated cryopreservation workflow to maintain the viable recovery and therapeutical efficacy. [[2]] Fluid and Cold Chain Management of ATMPs Unlike centralized manufacturing of large identical batches, autologous ATMPs are patient-specific with one batch of cell product, and distant manufacturing sites [[3]] making them incompatible with typical supply chains. Starting material and the final product forming a cryogenic cold chain. This chain ensures the integrity of materials during transportation, storage, and thawing, completing the autologous ATMP supply chain. The process involves patient assessments, cell collection, cryopreservation, transportation to the manufacturing site, manufacturing and manipulation, transportation, and final thawing at the clinical site, before final administration. [[2]]  Cryopreservation offers scheduling flexibility, minimizes logistical risks, and allows for timing that best suits the patient. Additionally, it provides benefits such as accommodating delays and eliminating time constraints for initiating manufacturing. All that mentioned therapeutic advantages can be attributed to an increased shelf life of the therapeutic product. [[4]] Increasing ATMPs shelf life At the very early stage in the ATMPs supply chain, as soon as cells are removed from their natural environment, they start to lose function. Keeping somatic cells outside the body viable and functional active for an extended period can be carried out by different approaches.  One strategy is to mimic the cells natural condition within a controlled aseptic system to enable all cell relevant functions (metabolism, expression, signalling & transport). Most cell types (except cancer cells) can be ex-vivo cultured for a limited number of passages until the cells loses proliferative potential and accumulate mutations.   Keeping Cells Alive Outside Their Comfort Zone Cryopreservation follows an opposite approach and attempts to shut down metabolic activity completely, at temperatures below -130°C. Compared to cells undergoing cryopreservation, cells in the cell culturing setting live a quite comfy life; with steady temperatures at around 37°C degree, sufficient oxygen, continuous fresh media, and even their waste gets removed. On the opposite, cryopreserved cells must deal with extreme conditions like ice crystals that are going to punctuate their cell membrane, severe dehydration due to freezing induced osmosis, and cell toxic cryoprotectants (especially at ambient temperature). Exposing cells to such conditions might seem counterintuitive on the first glance, thereby cryopreservation must be seen under the light of arresting biological degradation and conserving cellular functionality by immobilizing water through freezing. The objective of an optimal cryopreservation strategy is to deactivate (put on hold) degenerative cellular pathways and preserve proliferative potential by reducing the temperature below –130°C, this halts molecular transport, allowing cells to enter a state of "suspended animation" without compromising the quantity, quality, viability, and recovery of cells. [[2]] "Cryopreservation must be seen under the light of arresting biological degradation and conserving cellular functionality by immobilizing water through freezing."Khalil Essani Biophysics of freezing cell therapeutics In order to understand cryopreservation and the role of cryoprotectants, it is necessary to look at the biophysical behaviour of water in a cellular context. Water molecules have a dipole nature due to their OH-group, that is engaged in braking and reforming of weak bonds. As water is cooled, molecules get closer to each other, and the breaking of bonds diminishes. Upon reaching a critical point in local thermal conditions, known as the phase transition, hydrogen and oxygen molecules start forming sufficient H-bonds (4 hydrogen bonds), reorganize in a more space demanding hexagonal structure and initiating the creation of an 'ice embryo' through a process known as ice nucleation. [[5]] The formed ice crystals lead to a reduction in the free water available for cellular processes. Water molecules that normally are involved in the solvation of (salts, proteins, lipids, sugars) molecules, getting removed from the hydration shells of the solute molecules, while joining the formation of ice crystals. Consequently, the solutes within the solution become more concentrated as water is locked in the ice lattice. This results in water efflux (transport out of the cell) to establish osmotic equilibrium within the cell. Especially during slow freezing more time for water removal out of the cell is provided, resulting in increased cell dehydration (cell shrinkage). During cryopreservation, the drop in temperature typically induces extracellular ice formation, except in cases of rapid freezing. In a rapid freeze protocol, a cell struggles to establish equilibrium with the external environment due to the rapid formation of extracellular ice, which limits the continuous water transport from inside the cell to the extracellular environment. Consequently, the cytoplasm becomes increasingly super-cooled (higher solute concentration, decreases freezing point), elevating the likelihood of nucleation and subsequent intracellular ice formation. [[5]] An effective cryopreservation strategy aims to prevent intracellular ice formation during the transition from the aqueous phase to the ice phase. Success in cryopreservation hinges on achieving the glass transition temperature (-123°C), wherein the liquid transforms into a solid state, without compromising the quantity, quality, viability, and recovery of cells.   The role of cryoprotectants in cell freezing Cryopreservation has the potential to induce various cellular injuries (f.e freezing induced increase in osmolarity, physical punctuation), which may result in adverse alterations in cell morphology, characteristics, proliferation ability, and function. To reduce the effects of ice crystal injuries, and osmotic injuries, cryopreservation protocols usually incorporate cryoprotective agents (CPAs). These agents operate through diverse mechanisms, such as lowering electrolyte concentration and hindering ice formation by forming hydrogen bonds with water molecules, thereby preventing their association with ice crystals.  The general chemical structure of a CPA contains a polar group, by which it interacts with the water molecules through forming hydrogen bonds. The hydrogen bonds between the cryoprotective agent and the water molecule are stronger than the water-water molecule interaction (hydrogen bonds). In other words, the cryoprotective agent attracts water molecules and thereby removes free water molecules that otherwise would be accessible to form tight water-water molecule interaction, that are needed for critical sites for crystal nucleation. [[6]] Read more: Evaluating freeze-thaw processes in antibody production Permeating agents vs. Non-permeating agents Permeating cryopreservation agents are highly water soluble at low temperatures, contain a polar group that facilitates interaction with the OH-group of water, are of amphiphilic and have a small size (100 da) to diffuse through the cell membrane. The permeating feature enables the CPA to remove water from the intercellular space, and thereby supressing the ice formation inside the cell. Furthermore, a good permeating cryoprotectant, should balance osmotic imbalances and should be low toxic to the cell. The most common permeating CPA include, DMSO, ethylene glycol, propanediol, glycerol. All of them are of small size and contain a hydrophilic as well as hydrophobic feature (giving them an amphiphilic property) that enables easy transport along the amphiphilic cell membrane. [[6]] Non-permeating CPA are macro molecular cryoprotectants that include sugars, polymers, and proteins which are excluded from transportation across the cell-membrane, either due to their large size or their polarity nature and the lack of an adequate transport system. [[7]] There function is also based on attraction of water through highly polar groups. They can be dimers, trimers, or polymers. Some common non-permeating CPA are polyethylenglycocol (PEG), polyvinylpyrrolidone, raffinose, sucrose, trehalose. There are approaches, that for non-permeating agents, like trehalose, the cell is genetically modified to express a trehalose transporter to allow transport across the membrane.   More about types of CPAs Challenges in Adding Cryoprotectants Cryoprotectants are critical in facilitating successful cryopreservation techniques. Nevertheless, they impose two principal risks for biological samples:  Cytotoxicity and  osmotic shock associated with their introduction or removal Non-penetrating CPA are usually less toxic than penetrating CPA, but also less effective. In practice, usually a combination of both is applied, to control water efflux and attract water molecules. Though penetrating CPA are quite small, they still require a certain time to diffuse inside the cell and establish a chemical equilibrium. To allow sufficient internalization of intracellular CPA, the cells are typically incubated with the CPA for a couple of minutes, depending on the specific cell type. Notably, in the case of dimethyl sulfoxide (DMSO), cytotoxicity exhibits a temperature-dependent behaviour, necessitating its incremental addition to a pre-chilled sample. [[7]] The setup of a cooling and homogenizing cells prior to aliquoting and freezing helps to extend the window to prevent damages to cells. RoSS.PADL and RoSS.FILL CGT are such examples where cells are cooled and kneaded simultaneously to facilitate a cell aliquotation with consistent cell counts from bag to bag (see image below). Furthermore, the time between addition of cryoprotectant and cryopreservation should be minimised to limit any cytotoxic effects, but long enough to allow CPA internalization. The same has to be considered during thawing, in which the contact time between the CPA and the cells should be reduced during ambient temperature to a minimum. Following cryopreservation, apoptosis and necrosis typically manifest within 6 to 24 hours during post-thaw culture. This leads to a substantial decline in cell viability and compromised cellular function due to the cryopreservation process. The dysregulation of biochemical pathways after cryopreservation varies among different cell types, underscoring the importance of adopting a "cell-type dependent" approach. Existing literature indicates that T-cells, for instance, experience apoptosis after cryopreservation, with approximately 40% of cells undergoing apoptosis 8 hours post-thaw, followed by extensive cell death. [[8]] Regulatory Challenges with Croyprotectants in ATMP Cryopreservation Enhancing ATMP Cryopreservation - An Outlook The past has shown that there is the continuous attempt for updating and harmonization of guidelines and standards. In the future a focus on stability studies to determine the shelf life in an adequate and meaningful way will be introduced and aligned. Such actions can significantly reduce the cost of stability studies without compromising safety as well as to support the comparison of data from manufacturing and especially cryopreservation. From a material aspect, attempts to substitute cell toxic CPA - produced under GMP conditions and approved for therapy - with less toxic ones or certain mixes are under investigation. Less toxic CPA would allow to increase their concentration, and subsequently enables to apply a faster freezing scenario.  Implementation of safe integrated block chain solutions within the supply chain, in combination with Industry 4.0 and the Internet of Things, will enable a fully and detailed tracking - from verification of the origin and quality of materials to the unique batch-record of the therapeutic product inside the administered cryo-bag and its bag-history (temperature records). Furthermore, it enables the interoperability between different stakeholders in the supply chain, such as single-use equipment supplier, filling and freezing unit supplier, manufacturer, transport company, clinical centre. This allows seamless data sharing and collaboration while maintaining data security. This will secure therapeutic safety and efficacy but also eases to fulfil the regulations regarding record-keeping and documentation.  The overall challenge in cryopreservation for ATMP is the dependence on manual processes during the research phase and its transition to a commercial, automated, closed cryopreservation workflow, where entire processes need to occur within very tight parameter limits. These would be filling time, incubation time and temperature for CPA incubation, freezing rate inside the bag. [[download-1]] References Iglesias-Lopez C., et al.: Current landscape of clinical development and approval of advanced therapies, DOI: 10.1016/j.omtm.2021.11.003. Available at: Current landscape of clinical development and approval of advanced therapies - PubMed (nih.gov) Hawkins, B, et al:  Biopreservation Best Practices for regenerative medicine GMP manufacturing & focus on optimized biopreservation media. Cell Gene Therapy Insights 2017; 3(5), 345-358. doi: 10.18609/cgti.2017.035Available at: insights.bio/cell-and-gene-therapy-insights/journal/article/410/Biopreservation-Best-Practices-for-regenerative-medicine-GMP-manufacturing-focus-on-optimized-biopreservation-media  Jandova M. et al.: The role of cryopreservation techniques in manufacturing, transport, and storage of Car-T therapy products. Cryo Letters. 2023 May-Jun;44(3):123-133. Available at: https://pubmed.ncbi.nlm.nih.gov/37883165/  Coopman, K., Medcalf, N.: From production to patient: challenges and approaches for delivering cell therapies. In: StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute; 2008. 2014 Mar 31. Available at: https://pubmed.ncbi.nlm.nih.gov/24945057/ Murray, K., Gibson, M.: Chemical approaches to cryopreservation. Nat Rev Chem. 2022;6(8):579-593. doi: 10.1038/s41570-022-00407-4. Epub 2022 Jul 18. Available at: https://pubmed.ncbi.nlm.nih.gov/35875681/ Whaley, D. et al.: Cryopreservation: An Overview of Principles and Cell-Specific Considerations, Chemical approaches to cryopreservation, Cell Transplant. 2021 Jan-Dec; 30: 0963689721999617. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995302/ Murray, K., Gibson, M.: Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants, DOI:10.1021/acs.biomac.0c00591, Available at: https://www.researchgate.net/publication/341957118_Post-Thaw_Culture_and_Measurement_of_Total_Cell_Recovery_Is_Crucial_in_the_Evaluation_of_New_Macromolecular_Cryoprotectants Sarkar S. et al.: Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing. Cryobiology. 2003;47:44–58. doi: 10.1016/S0011-2240(03)00068-3. Available at: https://pubmed.ncbi.nlm.nih.gov/12963412/

    READ MORE

Send a request