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Preparing for variability and flexible processing are necessary for success.
Autologous cell therapies begin with collection of patient cells via apheresis. The properties and quality of these cells can vary significantly from patient to patient and as the result of different collection conditions/methods. Variability in cellular starting materials introduces significant challenges to standardization, and the variability in these materials may be compounded several-fold as downstream manipulations are performed for manufacturing of the final drug product.
“It is entirely possible,” comments Priya Baraniak, chief business officer of OrganaBio, “that a manufacturing process will work with a very high yield, meeting all critical quality attributes (CQAs) and release specifications for one patient’s cells and fail miserably for another. In the case of autologous therapies, this is a very high cost to bear for any one individual’s drug product. In addition, success vs. failure is quite literally a life-or-death situation; there are no additional chances for many of these patients.”
Some actions can be taken to minimize this variability, but not all of it can be controlled. Manufacturing processes therefore must be designed to accommodate the remaining raw material variability to ensure production of high-quality, safe, and efficacious cell therapies.
The level of variability for cellular raw materials used to produce cell therapies depends on several factors, including the target disease. The most obvious variability, according to Akihiro Ko, CEO and cofounder of Elixirgen Therapeutics, is the disease severity and the condition of each patient. “Genetic and epigenetic factors in addition to the general health of the patient, stage of the disease, underlying health of the patient including medications, medical conditions, and environmental factors contribute to the overall quality of autologous cell transplant,” agrees Cynthia Pussinen, CEO of Sernova.
Traditionally, chimeric antigen receptor (CAR) T autologous cell therapies have only been approved as last-line treatments. By the time a patient is enrolled in a trial or approved to receive a commercial product, their disease is well-advanced and they have already received prior treatments. “Prior treatment of the patient before apheresis can affect the quality, quantity, and functionality of the cells collected from the patient,” states Baraniak. “Chemotherapy, radiation, and/or administration of other drug substances such as immunotherapies as first-, second-, and/or third-line therapies have significant impacts on patient cells and their suitability for genetic modification and clinical-scale expansion,” she remarks.
The collection efficiency of leukapheresis products may be impacted by various patient-specific factors such as patient disease state, place in treatment protocol, age, pre-apheresis CD3+ cell counts, hematocrit level, and platelet level, adds Dalip Sethi, commercial leader for cell therapy technologies North America at Terumo Blood and Cell Technologies.
“Ultimately,” Ko concludes, “it is difficult to collect the same quantity and quality of cells from different patients.”
Patient-to-patient differences are, however, just the first source of variability. Both differences in material collection and therapy manufacturing processes can have significant impacts on cell counts and quality, observes David Smith, vice-president of development with BioCentriq. In fact, Pussinen contends, even the cells obtained from the same individual can vary if extracted at different time points (e.g., aging, disease progression) and different sources/locations.
Compounding this issue is the fact that not all apheresis protocols are the same and not all apheresis nurses receive the same type and level of training. “Hospitals and treatment centers have different processes for collecting, processing, and freezing cells for autologous use, which can contribute significantly to variability,” Pussinen says.
Different collection devices are used at different collection sites, according to Smith. Citrate-based anticoagulants are most common, but others are used that can have different effects on the collected material. The concentration of coagulant can also vary due to the need to halt collection due to problems with different patients, reducing the total collection volume from the typical 15 to 18 liters.
If incubation is required before transplant, then factors related to cell culture and storage will contribute to the variations, adds Pussinen. Cryopreservation media, freezing and thawing methods, and post-thaw recovery also feed into raw material variations. Quality-control factors also matter, she notes, because different facilities might have different regulatory or quality controls in place. Equally important, according to Smith, is the amount of time that passes from apheresis to manufacturing, which often differs depending on the locations of the collection and manufacturing sites.
Another complication noted by Smith and Baraniak is the use of consistent, healthy donor material during process development, as often cells from patients suffering from certain diseases are not available. “Risk is then incurred when moving to manufacturing using cells from unhealthy patients,” Baraniak says.
In addition, Baraniak notes that therapeutic developers have long had a bias for identifying donors/cells that work in their processes and sticking with those particular cells for all their process development and tech-transfer purposes. “Best practice,” she believes, “should include intentional introduction of donor/cellular starting material variability into processes in order to collect data sufficient for understanding which CQAs are truly indicative of manufacturing outcomes.”
Variability in cellular starting materials, if not controlled, can have implications for both upstream and downstream manufacturing operations. Impacts can be caused by differences in viability, growth, functionality during cell expansion, which all impact yield. More generally, manufactures can have difficulty reproducibly manufacturing their products and may fail to meet regulatory standards. Combined, therefore, cellular raw material variability can at a minimum increase the time and cost for product development, and in the worst case potentially influence safety and dosing and thus ultimately clinical outcomes.
For Pussinen, therefore, the “process is the product”. “If we change anything in the process, we run the risk—or benefit—of changing the resultant product into an effectively new and different product with a different analytical and quality profile,” she contends.
Some steps can be taken to introduce controls. Proper eligibility criteria ideally minimize the variability of patient-derived cellular raw materials, according to Ko. He adds that automation of collection processes can have a measurable impact, while consistency in the management of materials from collection to transport to processing must be an integral part of how these materials are handled.
Therapy developers can, Smith observes, put limitations on the patient populations they are targeting. They can also specify the use of a certain apheresis collection device to increase collection consistency and select specific shipping containers and logistic service providers to ensure control of material transport. Large biopharmaceutical companies have, in some cases, established specific protocols and provided training for apheresis nurses. This solution will not be tenable once large numbers of therapies are on the market, however, says Smith, as nurses cannot be expected to use highly specific protocols for each different therapy.
The best overall strategy, emphasizes Pussinen, is to use a risk-based approach that allows definition of the most critical starting materials and thus CQAs. In addition, to overcome lab-to-lab variability across processing sites, she would like to see standardized protocols implemented for cell collection, isolation, profiling, banking, quality control, and other processing, including cell expansion.
Sethi agrees that emphasis should be placed on standardized operator training across sites, better understanding of collection devices and methods involved, and optimization of collection methods to the specific target cell population. For instance, the collection process should be optimized to collect the required amount of target cells while reducing the amount of non-target cells such as granulocytes and platelets. Similarly, post-collection handling and logistics for transporting the raw material should be standardized as much as possible.
In the manufacturing environment, flexibility is needed, however, to accommodate incoming variable raw materials while still meeting good manufacturing practice (GMP) requirements, Sethi says. “In most cases, purification of specific cells is a requirement to generate the cell therapy product. For a cellular drug, the quality of drug products is always the top priority for therapy manufacturers. In addition to the purification and modification steps, the cell expansion platforms should allow enough flexibility to accommodate potentially different growth kinetics of variable raw materials,” he explains.
Flexibility can be challenging to achieve given the need to establish robust, tightly controlled processes, but Pussinen highlights a few strategies for incorporating flexibility into cellular therapy biomanufacturing processes. For instance, multiple cell sources could be used, such as isolating autologous mesenchymal cells from bone marrow and peripheral blood. Parallel processing in which multiple batches are run simultaneously can, according to Pussinen, reduce the impact of raw material fluctuations on overall production. In-process quality checks to support quicker and more-informed decision making can be valuable as well, as can modular process design with freezing of materials at various stages. Pussinen also points to the use of flexible and detailed standard operating procedures (SOPs) that include instructions on how to deal with different scenarios that might arise due to starting material variability as being beneficial.
In fact, cell therapy developers try to limit variability through stringent patient inclusion and exclusion criteria for clinical-trial participation and drug-product eligibility, according to Baraniak. They also, she says, try to minimize variability by setting as many specifications as reasonably possible around the collection, processing, manufacturing, and storage of these materials.
“Optimization for cell collection, standardization of training and post-collection methods, and flexible automated systems that are designed to accommodate variable growth kinetics can improve the overall cell therapy manufacturing process,” Sethi summarizes.
Process analytics play a critical role in the management of raw material variability. For instance, Ko highlights the measurement of cell count and viability as necessary for adjusting the dose of the final cell therapy product.
A multivariate, comprehensive approach to the development of in-process testing plans, assays, and the overall analytical testing strategy is necessary to understand the numerous important product characteristics, Pussinen observes. “Using a hybrid or analytical matrix/toolbox approach will help developers in more completely understanding their product,” she adds.
Specifically, Pussinen mentions the use of standard panels to reflect the typical range of values attributed to cell products, including viability, purity, potency, concentration, in-vitro functionality, and characterization/profiling. However, she emphasizes that several factors should be taken into account, such as the specific cell type, stage of the cell, stage of the manufacturing process, and the intended therapeutic application.
For some applications, those acceptable ranges for different values will be determined by preclinical or early phases of clinical trials, according to Ko. In addition, typically, there will be go/no-go criteria for many of these acceptable ranges. “If values fall outside of those ranges, it may not be possible to pursue the treatment,” he explains.
Process analytical technologies that provide real-time data can help achieve tighter process control through more timely feed rate adjustments and other modifications and thus avoid such situations, according to Pussinen. Process control with comprehensive data documentation, she believes, helps with identifying and tracking variability.
Developing standardized analytics can be challenging, however. “The cell and gene therapy industry is working diligently in the analytical development space on assay development and variability, assay controls, sample handling along with statistical hurdles associated with working on small numbers of manufacturing runs, patients, etc.,” says Pussinen.
One issue is lack of broadly accessible data, according to Baraniak. She suggests that “addition of data to a Biomedical Data Commons and using evolving data analysis techniques and software—perhaps even artificial intelligence (AI)—could really move the needle on better understanding patient variability and correlations to manufacturing success and clinical outcomes.”
While cell and gene therapy products may never be fully characterized, developers must have a solid understanding of their products and how they perform through the process. Consequently, the analytical and process development work along with the subsequent manufacturing process are crucial in defining the end product, according to Pussinen.
An effective manufacturing process can be viewed, according to Smith, as a funnel through which variability is reduced step by step, resulting in a therapeutic product with the desired quality attributes. It is essential, in fact, because many cell therapies represent the last chance of survival for the patients that receive them. “Manufacturers must be prepared to generate effective therapies from whatever patient material is collected,” Smith comments.
A typical first process step is to wash platelets from the apheresis material, as they are “sticky” and can cause problems in later steps. “Because washing can remove over 99% of platelets, the washing step removes any variability in platelet content,” Smith notes. Typically as long as there is a sufficient number of the desired cell type in the apheresis material, it doesn’t matter how many other cells are present or what types they are, as they will be removed in the cell selection step.
Issues that are more challenging to manage relate to the impact of the collection environment (anticoagulant type and concentration, for instance), storage and shipment conditions (time, temperature, shear, etc.), and early processing steps on cell quality, according to Smith. “Some cells are more sensitive to these issues. Monocytes, for instance, are quite finnicky and their quality can be impacted if they are manipulated too much, which can make them hard to select, grow, and differentiate,” he says.
Much depends on the protocol and the cell population being treated, adds Ko. “Less time can be spent on purification if you are able to treat many cells. It may be critical to do the purification process upfront, however, so that a relatively uniform cell population can be genetically modified,” he continues.
Given that process development typically proceeds with healthy donor cells and consistent apheresis material and focuses on minimizing process variability, it is inevitable that once a process is implemented using patient-derived raw materials, there will be process deviations. “The reality is that in autologous cell therapy manufacturing, deviations are expected,” Smith observes.
Any change from the standard operating procedure, such as adding a second wash step because a particular patient’s apheresis material contains ten times the normal quantity of platelets, must be written up as a deviation. Leaving cells that do not grow sufficiently in the allotted time for a few more days is another.
“Operators must leverage their past experience and assessment of the potential risks of any changes that must be made to accommodate raw material variation before making these types of decisions,” Smith adds. Some companies, he says, provide decision trees to help operators determine the appropriate actions to take based on what they are observing in a given process.
A key advance in autologous cell therapy manufacturing has been the introduction of automated processes, states Ko, as it has led to a drastic reduction in variability and therefore enabled the production of products with more uniform quality and efficacy. He points to automation of cell culture as an example. Ko also cites another benefit of automation as being the potential reduction of stress in the workplace for the technical staff handling precious patient cells.
Elixirgen Therapeutics found that automating its process for production of a cell therapy to treat patients with telomere biology disorders provided much greater consistency in both their process and product. For its Phase I/II clinical trial, cells were selected and treated with the company’s vector in a functionally closed tubing system using the CliniMACS Prodigy (Miltenyi Biotec).
“The elimination of manual manipulations meant elimination of the risk of contamination as well as the risk of human error. This system can also be leveraged for decentralized manufacturing solutions, which additionally eliminates time spent on transportation and risks associated with the freeze-thaw process (loss of cells and reduced cell health/viability), as well as other related issues,” Ko states.
The end goal of cell therapy development, whether autologous or allogeneic, is to get safe and effective treatments to patients. The numerous cell therapy products on the market were, despite the challenges posed by raw material variability, in many cases commercialized more rapidly than most biopharmaceutical products have been in the past, according to Smith.
That success can be attributed, in part, to the leeway regulators have granted cell therapy developers to manage that variability, insists Smith. “FDA has not reduced its expectations for safety, but has been accepting of deviations from set protocols as long as appropriate risk assessments are conducted and the changes shown to not affect safety,” he explains. “The regulators have recognized the need for flexibility given the variability associated with patient-derived materials. They have also seen the value in the funnel approach during manufacturing to remove that variability. They should be given more credit for making it possible to bring these novel and highly individualized therapies to patients in need,” Smith concludes.
It is vital to the long-term success of cell therapies, including driving down their cost to increase patient access, that manufacturing processes are robust enough to account for inherent variability in patient’s cellular starting materials. “The best approach to achieving this goal is to engineer failure in early; fail hard and fast, and apply those learnings to the next program,” states Baraniak.
Doing work upfront will pay off in the end, agrees Pussinen. “Expect variations in autologous transplants. Leverage risk-based assessments, AI, quality-by-design, and modeling. Gain an in-depth understanding of the variations in each specific product, implement quality controls, and apply flexible methods for cell processing. Understand the targeted conditions and patients, and learn what clinicians and commercial colleagues find meaningful and impactful.Meet early and often with the regulators. In essence, be as prepared as possible.”
Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.
Vol. 36, No. 12
When referring to this article, please cite it as Challener, C.A. Managing Raw Material Variability for Autologous Cell Therapies. BioPharm International 2023 36 (12).