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by Bio-Process Systems Alliance Cell and Gene Therapy Committee

The Bio-Process Systems Alliance (BPSA) was formed in 2005 as an industry-led international industry association dedicated to encouraging and accelerating the adoption of single-use manufacturing technologies used in the production of biopharmaceuticals and vaccines. Corporate members include plastic-equipment suppliers, service providers, and users in the biopharmaceutical industry who share this mission. A key focus of BPSA’s core activities is to educate its members and others through sharing of information and development of best practice guides that help suppliers, users, and regulators to safeguard the quality of drugs produced with single-use technologies (SUT).

This article is designed to provide guidance on cell and gene therapy (CGT) manufacturing, regulations, and best practices regarding implementation of single-use components. It is largely based on experience gathered from the use of these products in the blood processing and biologics manufacturing spaces. Differences between those areas and cell therapies are highlighted throughout this article. This full series will be published and available online at www.bioprocessint.com.

Background
Successful commercialization of cell therapies relies on the development of a scalable manufacturing process that can produce products of appropriate quality
routinely and cost-effectively. Although the challenges associated with establishing these robust manufacturing processes can be therapy specific, a few common themes arise from the fact that the product being manufactured is of significantly higher complexity than standard biologics, such as monoclonal antibodies or recombinant proteins.

Many cell therapies have shown significant promise in curing or alleviating a variety of diseases in clinical studies. Momentum in the field is growing, as demonstrated by an investment of over $50B—$12B since the beginning of 2016 (Alliance for Regenerative Medicine [ARM])—and FDA approval of two therapies in 2017 (Kymriah and Yescarta). The pipeline also is very strong with a further 93 therapies currently in late-stage clinical trials (ARM data, Reference 1).

Cell therapy is defined as the administration of cells to a patient to treat disease. Cell therapies can use either a patient’s own cells as starting material (autologous cell therapy) or a donor cell that is expanded and used to treat several patients (allogeneic cell therapy). In many cases, cell therapies can involve genetically modifying cells. For example, a genetic modification is made to a patient’s cells ex vivo and then administered back to the patient. One example of these types of therapies is chimeric antigen receptor T cell (CAR-T cell) therapies. These therapies have been successfully applied to treat various forms of cancer and work by modifying a patient’s T cells ex vivo to attack and kill cancer cells after administration back into the patient.

SUT, also known as disposable technologies, have gained extensive use and acceptance in the manufacture of monoclonal antibodies and recombinant proteins because they allow for greater flexibility, speed, and safety in the development of these therapeutics. Because of their widespread use in this area, a significant amount of effort has gone into development of standards involving their implementation and use in manufacturing.

Currently, the use of SUT in cell therapy is standard practice, owing in part to the fact that the field is industrializing. Single-use technology is perfect to facilitate this industrialization by leveraging existing bioprocessing capabilities of closed systems and eliminating cross-contamination. This is particularly advantageous for autologous therapies, because SUT eliminates the risk of cross-contamination between patient samples.

Figure 1: CAR-T manufacturing workflow

With more cell therapies approaching commercialization, it is important that therapy developers have a clear understanding of both the opportunities and the challenges associated with developing an entirely single-use manufacturing process. This article uses the CAR-T cell manufacturing workflow (Figure 1) as an example, but the contents can be generalized to all cell and gene therapies.

Manufacturing Process Summary
CGT products demand additional requirements on manufacturing equipment. Because CGT products are biological in nature, noting that cell-based therapies cannot be terminally sterilized by filtration, and cells that have direct contact with manufacturing vessels are the final form of drug products, the general requirements on common manufacturing equipment systems become even more stringent. As discussed in this article, requirements on sterility, biocompatibility, extractables/leachables, and particulates must be carefully met to ensure final CGT products’ quality, safety, and effectiveness. Because CGT products are often produced in small batches — or in the case of autologous CGT products where one patient’s own cells are processed in a single batch for a patient’s own use — SUT have become a common manufacturing platform. They often use plastic or other materials that are economically suitable and easy for operation. With such materials used in production, not only is sterility critical, but stability, physical integrity, and strength of SUT also are vital to ensure safety and effectiveness of CGT products. With autologous CGT products, defects in SUT leading to manufacturing failure will put patients at significant risk, even leading to death.

Another unique aspect of CGT production, particularly for autologous CGT, is to ensure traceability of drug products throughout the supply chain and prevent any mixups. Chain of custody must be implemented from the start of cell sample collection to finish when CGT products are administered into designated patients. SUT by nature suits this purpose nicely when mechanisms are in place throughout the supply chain to ensure chain of custody.

Aseptic Considerations: Losing valuable patient samples due to contamination caused by faulty aseptic procedures must be avoided at all costs. Understanding what to look for in your single-use systems is vital to ensuring that your patient gets a safe and unadulterated treatment. In this section, we will explore how to choose the right solution and what you should be looking for from your suppliers to ensure success.

Consideration of Suitability and Adoption of Bioprocess Equipment for CGT Manufacturing: Strong clinical responses have laid the groundwork for propelling cell- and gene-based therapies toward commercialization. In 2017, the industry saw two major announcements by the FDA for unanimous approval of CAR-T cell therapies for Novartis and Gilead. Many of these types of therapies are being fast-tracked through clinical trials under orphan status to help advance their evaluation and development. Because of these accelerated timelines, unlike the traditional timelines of monoclonal antibodies, the industry has leveraged equipment and single-use products traditionally designed and reserved for bioprocessing. CGT manufacturers have found that these products are not ideally suited for the required purpose.

There are many unique differences between traditional bioprocess manufacturing and CGT manufacturing. Before we press on, it is critical to understand these differences to ensure the development of industry standards for single-use systems specifically designed for CGT requirements. Bioprocess manufacturing consists of stable cell lines, such as Chinese hamster ovary (CHO) cells that have been stably transfected with a gene of interest that allows for monoclonal or biologics production. Processes like these have been established for several decades. They use well-established upstream workflows, and downstream processes remove impurities that may have been introduced into the final product. Critical quality attributes of the final product are also well known, and impacts of specific materials that encounter the product are well observed and documented. Unlike monoclonal antibody or biologics production, where the cell is not the final product, cell therapies are the final product, which adds complexity. Upstream and downstream processes do not follow a standardized template, and any material that is introduced to the process is likely to affect the patient, even at residual levels. Another large hurdle for CGT is that critical quality attributes are still being explored.

Table 1: Common requirements for bioprocesses and cell and gene therapies (CGT) (product defined by monoclonal or CGT material)

Although the bioprocessing industry has laid much of the groundwork for addressing the challenges and necessary changes to SUT standards, it is vital that CGT not only build on them, but further develop them. Bioprocess and CGT are distinct from one another, and thus their end-user requirements are not identical. Some of the most common requirements for bioprocesses and CGT are listed in Table 1.

Research and Clinical Processes with SUT: Apart from comparing the differences between traditional bioprocesses and CGT processes, there are many important considerations among CGT research and commercial participants. Many people in research and development are not well versed in the nuances and requirements of SUT systems for CGT because their focus is primarily on driving products to clinic, not necessarily improving the processes. There is no doubt that R&D groups, along with academic medical centers (AMCs), are critical for CGT. Partnerships between researchers and large pharma (e.g., University of Pennsylvania with Novartis, City of Hope, and Gilead Sciences) have helped propel the industry forward. That said, all SUT systems may not be exactly suited to CGT manufacturing. Because of the nature of this industry where AMCs drive clinical pipelines, open communication between groups will help drive understanding and the need for new standards.

Applying SUT to CGT: The CGT industry is responsible for understanding and implementing SUT standards and ensuring that correct actions are taken to ultimately provide the safest products to patients. Although vendors and developers have distinct responsibilities, it is quite important that we use the knowledge and experience of all people operating in this field. Furthermore, it is even more pressing that, as the field begins to develop SUT products specific for CGT applications, we ensure the education of all operators working with these consumables. It is vital to understand how specific processing steps that use SUT can affect the final product. For example, testing materials with your cells/process is critical to ensure that the SUT selected does not have a negative impact on the process, cell growth, yield, cell efficacy, cell proliferation, and so on. Not all common bioprocess materials are suitable for use in cell therapy applications.

Equipment Requirements: SUT has been typically manufactured using flexible film-based materials. The expansion of SUT into CGT applications has led to the introduction of rigid-walled containers and enclosures. However, the roles of both types of containers are effectively the same: to protect the integrity of the process and the safety of the operators. Flexible-walled containers, more commonly referred to as single-use bags, can be manufactured from a range of different polymers, the more common ones listed below

• Ethylene-vinyl acetate (EVA)
• Gamma-irradiation stable fluoropolymers
• Polyvinylidene fluoride, also called polyvinylidene difluoride, as the product contact layer in a multilayer laminate film, and commonly referred to as PVDF
• Polyethylene formats, including but not limited to those below. Depending on the manufacturer, these differ in the types of polyethylene (PE) in contact with the product, the number of layers of materials used, and different types of outer layers used to enhance physical strength and improve puncture resistance: LDPE = low-density PE, LLDPE = linear low-density PE, ULDPE = ultralow-density PE.

For rigid-walled containers, products already on the market are manufactured from materials, including polycarbonates and polysulfone. For all SUT, regardless of material, supplier, or composition, critical performance attributes contribute to the process. However, there is currently no unified standardized process for the testing that is performed to determine suitability of a product against each parameter.

Most manufacturers test critical attributes of SUT using a standard process. However, for many critical attributes being tested, there are multiple valid test methods. The BPSA Single-Use Manufacturing Component Quality Test Matrices lists many of these, including the American Society for Testing and Materials (ASTM) and the International Standards Organization (ISO), that can be used to provide data. SUT manufacturers can select the test method that they want, usually based on familiarity with a testing body, relying on previous experience of that specific process, or even selecting the test method that shows their products in the best light. That makes it very difficult, if not impossible, for an end user to effectively compare performance data from two or more suppliers when the test data presented are performed using different test methods, and therefore have different levels of impact on a final product.

Table 2: Tests and test methods for critical attributes

Table 2 lists some of the different tests and associated test methods that can be selected by a manufacturer for testing the identified critical attributes. This is not designed to be an exhaustive list, but is more to guide readers to understand the testing that may be performed and the different testing bodies involved.

Manufacturers can also undertake and report data on tests such as determination of the secant or Young’s modulus (ASTM D822), density (ASTM 792), haze and clarity (ASTM D1003), glass transition temperature (ASTM E1640), puncture strength (FTMS 101B), dart drop test (ASTM D1709), and so on.

Other compliance requirements for SUT include certification that the materials of construction of a device are free from the causative agents of bovine spongiform encephalopathy (BSE) and transmissible spongiform encephalopathies (TSE).

In CGT applications where the patient’s cells are both the starting raw material and the end product, it is not possible to use a filtration step to remove extraneous particles from the finished product. It is therefore critical to assess the manufacturer’s claims and testing processes to ensure that both visible and subvisible particles are either not present or are not generated during the process, as through spallation during any pumping step using flexible tubing. As mentioned earlier, once a CGT process is exposed to particles, they cannot be removed easily, and the risk associated with infusion into the patient must be assessed.

Material manufactured using injection molding or extrusion techniques can have slip agents or extrusion agents added to the process or to the raw material composition. These agents aid in the processing efficiency of the operation but can extract into fluids that contact the surface postprocessing. It is important that manufacturers identify and test for these agents as part of the determination of extractables in their validation documentation.

A full description of the impact and importance of the determination of extractables and leachables from SUT has been detailed in previous BPSA publications (www.bpsalliance.org) and is the subject of a separate BPSA paper directly related to the CGT market and applications. It is recommended to perform a risk assessment based on the intended use of the component with respect to the chemical nature of process fluid, process conditions, and opportunity for clearance of any potential extractables from the drug substance. Extractables characterization is recommended for high-risk SUT components.

It is worth taking time to consider that the above requirements apply not only to the product-contact surface in a multilayer film, but to all layers of construction. It has been shown that over time, materials from outer layers in a film can leach through the product-contact layer of the film into the product. ISO11137/11135 advises on sterility sampling plan methodology of SUT according to lot size. ISO is typically implemented for medical devices.

Sterilization Methods of Choice for SUT: Typically, SUT used for cell therapy manufacturing are constructed of either a laminated, flexible polymer film or a rigid plastic. Choice of sterilization method depends on the component materials and the physical design of the product. Gamma irradiation is often the method of choice when available. Typically, such techniques are qualified through the well-defined methods in sterility validation compliant to ANSI/AAMI/ISO 11137 VD_max25 (2). This is the use of the radioisotope cobalt 60, which offers high penetration allowing sealed single-use products to be sterilized with ease. This method kills bacteria by breaking the covalent bonds in their DNA. Typical exposure doses of between 25 and 40 kGy are used. As part of the in-process validation of each gamma-irradiation cycle, the gamma dosage is measured at multiple locations within the irradiation chamber during the exposure to ensure that the minimum gamma dose required has been achieved in all locations. Depending on the single-use system manufacturer, gamma-sterilized SUT are tested poststerilization using a combination of real-time and accelerated-aging test methods. This provides end users with a validated shelf life, below which end users can be assured that the products will remain sterile. The provision to this statement is that the packaging of the SUT must remain intact and not be compromised. The validated sterile shelf life of a specific SUT can vary widely depending on the materials of construction, complexity of the assembly, and so on, but a shelf life in the range of 12–36 months is typical.

Certain materials are not gamma irradiation compatible and/or are made of a material that casts a “shadow” and prevents full exposure to irradiation. Also, materials in the product can be adversely affected by gamma irradiation (e.g., change in appearance, unacceptable extractable and leachable profile, or deterioration of desirable physical properties). Gamma irradiation can also become very expensive when done in small lots.

Training: Single-use products are not a panacea if used improperly. Training users in aseptic techniques and the correct unpacking, installation, handling, and disposal procedures are all vital requirements to ensure the proper handling of SUT to obtain optimal performance. Training must encompass everything from receipt, storage, opening, use in laboratories, and final disposal. Standard operating procedures (SOPs) that are clear and provide guidance will go a long way to ensure that successful new products hit the market.

Disposal: In the production of cell therapies using SUT, significant amounts of single-use material will require careful consideration to ensure both safe and environmentally friendly disposal. BPSA has published a Guide to Disposal of Single-Use Bioprocess Systems. The principles and methods detailed therein apply equally to the cell therapy market.

Looking Ahead
Part 2 of this three-part series will provide a regulatory overview for single-use technologies for cell and gene therapies, including discussions of classifications and compendial tests.

Reference
1 Alliance for Regenerative Medicine (ARM) State of the Industry Mid-Year Update: https://alliancerm.org/wp- content/uploads/2018/09/State_of_Industry_Update_CGTSept2018.pdf.

2 Method VD_max 25 kGy as a Sterilization Dose:Method VD_max. AAMI Technical Report TIR33-2005: Sterilization of Health Care Products, Radiation, Substantiation of a Selected Sterilization Dose. Association for the Advancement of Medical Instrumentation, 2005.

For more information, contact Kevin Ott (ottk@socma.com). This article is published in extended form with permission from Bio-Process Systems Alliance (BPSA), 1400 Crystal Drive Arlington, VA 22202; www.bpsalliance.org. The white paper is available at http://bpsalliance.org/cell-and-gene-therapy-resources.