Published March 25, 2022
The following article originally appeared on March 10, 2022 in Cell & Gene here.
With an increased focus in the pharmaceutical industry on addressing unmet medical needs, areas of the market like cell therapy are growing rapidly. The ability to use a patient’s own cells to treat and even cure disease is revolutionizing the future of modern medicine, leading to exciting discoveries and novel treatments. However, the specificities of bringing these products to market are creating significant challenges for drug manufacturers that must be overcome in order to realize the true potential of cell therapies. One approach that can aid in overcoming these bottlenecks is leveraging the experience of those already tackling the complexities of cell therapy manufacturing.
Dr. Carolyn Wrightson, Chief Technical Officer of Cellipont Bioservices (formerly Performance Cell Manufacturing), and her team pioneered the historical development of an autologous canine mesenchymal stem-cell (MSC) product as well as human mesenchymal stem-cell drug product manufacture. These milestones ultimately facilitated in early 2020 Cellipont Bioservices’ ability to rapidly develop and manufacture an allogeneic human MSC product (now entering Phase II Clinical Trials) for the treatment of COVID-19 patients suffering from acute respiratory distress syndrome. To help guide the pharma community toward a new era of patient care, Dr. Wrightson shares some of the valuable lessons and considerations she and her team learned about stem cell therapy scale-up and optimization.
A Journey To GMP
Cellipont Bioservices traces its roots back to 2002 when its team was part of VetStem Biopharma. Initially focused on improving veterinary care through regenerative medicine, the Cellipont Bioservices team has worked with 31 different species, including humans, and more than 2,500 primary cell cultures, generating data that has helped provide a roadmap for large-scale manufacture of stem cell therapy. In the very beginning, the company’s breakthrough was to safely derive the stromal vascular fraction (SVF) from adipose cells and use that as a direct stem cell therapy product for treatments in companion animals. The Cellipont Bioservices team helped advance VetStem’s pipeline, including three active investigational new drug (IND) applications. One of those INDs involves an off-the shelf allogeneic cell therapy product that would lower the cell therapy treatment entry-point for senior dogs with arthritis that are not good candidates for surgery.
As the FDA began to enforce regulation for similar cell therapy treatments for humans in 2018, VetStem recognized that its in-house expertise was of great value for human cell therapy development. The company refocused staff and GMP manufacturing resources on human cellular therapeutics manufacturing. Cellipont Bioservices (previously known as Performance Cell Manufacturing), was formed to provide GMP cell manufacturing services in its GMP cell therapy manufacturing facility. VetStem also launched a human-focused sister company, Personalized Stem Cells (PSC). Currently, PSC is supporting three investigational new drug applications for human autologous and allogeneic MSCs (adipose non-cultured and cultured) and last year filed an IND application for a Cellipont Bioservices-manufactured drug for acute respiratory distress therapy for COVID-19 patients. Cellipont Bioservices experts have worked with companies and research institutions preparing cell therapeutics for Phase I and Phase II clinical trial(s). The culmination of these efforts is a range of experience in both human and animal cell therapy manufacturing, from which Cellipont Bioservices has established a rich library of lessons learned and best practices.
In September of 2021, a healthcare-focused private equity firm, Great Point Partners, acquired Performance Cell Manufacturing. The company now operates as Cellipont Bioservices, an independent Contract Development and Manufacturing Organization (CDMO). Cellipont Bioservices’ focus is to leverage its decades of cellular therapy development and manufacturing expertise to serve new emerging cell therapy companies as they seek to advance their innovations. Cellipont Bioservices continues to expand manufacturing capacity and in-house expertise which now includes manufacture of stem cells, CAR-T, iNK, Dendritic cell therapies, and plasma derived therapy products.
Stem Cell Manufacturing Process
As with any cell therapy product, the stem cell manufacturing process begins with the receipt of donor tissue (or blood products) from a donor that has been pre-screened per applicable regulations as a source for donor primary cells or products. This process must include the documented handling of the tissue collection from the donor to the site of manufacture. For example, the tissue must be shipped in a validated temperature-controlled shipper and received to the manufacturing facility within the specified time limit and specified temperature range. The incoming tissue must be inspected for intact packaging, temperature, identity, and time of collection before it is accepted and moved into the manufacturing process, whereupon meeting the acceptance criterion is now considered a GMP raw material. A unique donor identifier is assigned with a corresponding quality released batch record confirming that the materials are from a qualified donor and the record is maintained throughout the entire manufacture process.
Creating a Master Cell Bank
To create a master cell bank, the primary cells are formulated in standard inoculum densities and grown in a monitored and controlled chamber where cell growth and appearance are regularly monitored to ensure the raw material remains within specification. Any data and observations are noted in the batch record. The cells are then passaged one or more times at 75% to 95% confluence and frozen in aliquots suitable for expansion in small or large batches. This is one of many purification steps in the isolation of the drug active ingredient.
It is important to keep in mind that the master cell bank is for GMP manufacture, so there are specific regulatory criteria that must be met this early in the process as this material will be the same used for the commercial manufacturing product. This process also requires the creation of a sampling plan with quality control and assurance departments to establish handling requirements for the master cell bank sampling program. For example, cells that have been stored in dimethyl sulfoxide are not amenable for use in a polymerase chain reaction (PCR) assays, and because several PCR tests are required for the master cell bank, it is important to establish a sampling plan in advance of storing the master cell bank. The required test input requirements must be understood and accommodated in the sampling plan.
Other developmental goals that must be accomplished with donor cells before being ready to begin manufacturing include:
Scale-up of cell therapies goes beyond considerations of size and/or quantity of the culture vessels required. Time and motion studies for the manufacturing process are a necessary element as well as establishing acceptable parameters for material hold times. For example, when harvesting four T-225 flasks, it is possible for cells to be counted and back into a flask after only an hour. However, if you scale-up to 30 10-layer Corning® CellSTACK® culture chambers, there are increments of time added together before the cells are re-seeded and returned to incubation or proceed on to cryopreservation. The scale-up process must take not only time into consideration, but also the media and temperature for the drug substance as it is being held. Therefore, it is critical to understand the potential time and motion to set allowable hold times and in-process hold specifications. Failure to do so can result increasing the development timeline or only allowing smaller batch sizes which significantly increases manufacturing and release testing costs. The regulatory agency will want to see that these stress tests have been conducted and hold times have been developed for the steps in the manufacturing process.
It is also important to determine a path to scale to reach the yield required for the drug product batch size. This must take into consideration not only the Phase I/II clinical needs, but also quality sampling required for drug product release, stability in storage and during clinical delivery, development material for drug characterization tests, and regulatory retain requirements. As the manufacturer, the entire quantity of drug required to support the clinical, quality, and pharmaceutical development must be taken into consideration in advance of manufacturing the drug product to ensure that a sufficient quantity of the manufactured drug product is made. Also keep in mind there are often changes in clinical trial demand or product losses which may be incurred during shipments. Development validation and suitability for release testing, which must be done before the drug product can be released, is frequently underestimated. As early as possible, generate material from the engineering batches to the development group so assay development can begin in parallel to scale-up of manufacture. This ensures the time for development of assays prior to validation required for releases of the drug and timely execution of stability studies with the final product.
When the cell therapy drug is frozen for short- or long-term storage, the delivered dose once it is thawed must also be considered, as a frozen product is not necessarily the yield of the thawed product. Therefore, from the drug design specification, project back to the fill and formulation required to deliver the dose. This is often conducted in a series of controlled validation studies for formulation, fill, and cryopreservation in the final container closure during the pharmaceutical development phase.
Once the number of cells required to achieve the required pre-formulation yield is known, determine the footprint necessary to reach that goal. The table below provides vessel and incubator estimates to meet scale-up requirements as you transition from research and development to manufacture to support the clinical development of a new investigational cell therapy drug.
Research and Development that does not take into consideration the at scale requirements for space and operators required within the developed hold times may delay the ability to move into sizeable scale increase required to manufacture the material needed for the clinical batches. A process driven toward a larger number of vessels and equipment can drive manufacture risk toward operator error, equipment failures, and failure to maintain an aseptic process. Facility design specifications are required and ensure there is a space large enough to scale-up to the manufacture levels required. The facility is a critical component of the drug manufacture process, so part of the product design is making sure there is safe and suitable space for manufacturing. This includes designating areas that are large enough to move equipment in and out of as well as space for personnel gowning and materials controls.
Another consideration of scale-up is that cell growth is also impacted by the amount of time it takes for material to warm up in the incubator. This may not be issue when working in smaller T-flasks, but it would for example when transitioning to growth in a Corning HYPERStack® cell culture vessel, because of the density of the plastic, volume of culture media, and the total mass being loaded into an incubator at full scale. It is not uncommon, especially in the last passage, to need an extra day from what was initially projected in the at scale growth plan, because of the added amount of time needed for the seed required to be harvested, quantitated, characterized, seeded to the flasks and then for the seeded flasks to re-equilibrate in the incubator and begin the propagation process. In these larger vessels, there are also losses due to dead space in the flasks, however this loss is by far outweighed by the reduction in the number of flasks handled by operators and handling time.
Operator time can also be reduced by transferring basal media, wash solution, and detachment media into quick connect or weldable bags using Corning Easy-Grip Polystyrene Storage Bottles fitted with a 0.2 um PTFE filter and MPC. These media transfers can be performed prior to working with cells allowing for the team’s focus on the day of harvest to be cell detachment and characterization. Tools such as this can create flexibility in the process by allowing one team to simultaneously make final media formulation while another is detaching from vessels, characterizing, and preparing for re-seeding. When validated, the bulk of basal media can be placed into an incubator to warm up in advance along with the HYPERStack cell culture vessels, with separate incubators being used for cell growth. As each Corning HYPERStack cell culture vessel is filled and seeded, they should be immediately returned to begin incubation. With each stack weighing 15 to 20 pounds, it is important to ensure the incubator shelves are strong enough to withstand the pressure, as any dips can impact uniformity of seeding.
Establish In-Process Controls
Implementing in-process controls ensures product quality and mitigates contamination risks during process. Some areas to consider for in-process controls include:
Determine the drug quality release specifications for the cell therapy product. This typically includes fill volume, identity, appearance, container closure, the total viable cell count (to ensure it meets the dose specification), sterility (bacteria, fungi, and mycoplasma), endotoxin, and potency. Early development of an appropriate potency assay will also support the validation of the in-process controls for manufacture and storage of the drug product.
Achieving commercial scale manufacture in a consistent manner will likely demand scale of manufacture that would exceed that which is capable in a flask-based manufacture process. After evaluating multiple bioreactors compatible with adherent cell manufacturing, Cellipont Bioservices is in the process of evaluating the Corning Ascent™ FBR (fixed bed bioreactor) as an emerging technology capable of meeting the need of scalable biomanufacturing.
Overall, the chemistry manufacturing controls are essential and critical for an IND application. You must have all drug development and manufacturing information documented in a clear and understandable way and be able to reproducibly demonstrate the manufacture of the drug substance, from your finished engineering batches to clinical batches and, finally, for the commercial drug product manufacture.