INTRODUCTION

Cell-based advanced therapies are intrinsically different to the more traditional small molecule and biologics-based therapies. As such, they come with specific manufacturing and quality challenges.

For example, the inherent complexity and dynamic nature of cells means that they may adapt according to their in vitro and in vivo environment. Further, the heterogeneity of source cells and the potential of contamination with adventitious agents result in additional challenges.

Excluding cord blood products, as of April 2020, there are six cell therapy products approved in the US, and seven in the EU. These include genetically modified cell therapies such as Kymriah and Strimvelis, somatic cell therapies such as Alofisel, and tissue engineered products such as Holoclar. This article covers CMC considerations in the EU and US which are applicable to all advanced cell therapy medicinal products, focusing on aspects where they differ most significantly from small molecules and biologics. Due to the diverse nature advanced therapies, both the EMA and FDA advocate a risk-based approach whereby the specific risks of a product and manufacturing process are identified and control/mitigation measures are put in place to address the potential risks.

SOURCE CELLS

Source cells can either be autologous or allogeneic primary cells, or established cell lines. EC Directive 2006/17/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells describes the testing requirements in the EU. In the US, FDA Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue- Based Products (HCT/Ps) describes the testing requirements for compliance with 21 CFR part 1271. Appropriately characterised Master Cell Banks (MCB) and Working Cell Banks (WCB) should be used where the cell lines are used as the cell source. The requirements for characterisation and testing of these cell banks are described in ICH guideline Q5D.

In the US, the FDA guidance does not apply to cells and tissues for autologous use, as laid out in 21 CFR 1271.90. However, testing should be considered from a manufacturability perspective (i.e. potential segregation of manufacturing for seropositive patients). In the EU, the minimum set of biological testing requirements apply to both autologous and allogeneic donations. However, with autologous cells and tissues, positive test results don’t necessarily prevent their use if appropriate storage facilities are available to ensure no risk of cross-contamination with other grafts, no risk of contamination with adventitious agents, or mix-ups. Validated assays capable of detecting human infectious agents with appropriate sensitivity should be used, taking into consideration medium components such as antibiotics that may interfere with the assays. Furthermore in Europe, if blood or a blood component is to be used as the starting material in a cell therapy, then the collection and testing requirements laid out in the EC Directive 2002/98/EC setting standards of quality and safety for the collection, testing, processing, storage and distribution of human blood and blood components and amending Directive 2001/83/EC must be complied with.

All establishments and personnel involved in cell procurement and testing of the apheresis material must be qualified by a competent authority for the purpose of those activities.  This includes the use of FDA-licensed kits in the US, and the use of CE-marked kits in Europe for respective donor testing. In the US, compliance with Federal and State laws related to donor procurement should be followed and sites must have appropriate licenses. A risk-based approach to donor site selection and site qualification can be taken by the applicant. Compliance with internationally recognised standards such as FACT-JACIE International Standards for Hematopoietic Cellular Therapy Product Collection, Processing, and Administration by sites provides an additional level of assurance on the ability of the sites to procure material (e.g. will have a Quality Management System [QMS] in place).

To ensure traceability, unique patient identifiers are required. In the EU, the Single European Code (SEC) system for coding of donated cells and tissues is used. As described in Commission Directive (EU) 2015/565 amending Directive 2006/86/EC as regards certain technical requirements for the coding of human tissues and cells, the SEC has a specific format and with associated data, should be entered onto the EU Coding Platform. In the US, 21 CFR 1271.290 provides the requirements for tracking cell-based products.

QC RELEASE TESTING

Release testing of cell therapies should always include microbiological testing, and assessments of identity, purity, potency, viability, and total cell number, unless otherwise justified. Microbiological testing includes sterility (bacterial and fungal testing), mycoplasma and adventitious viral agent testing. Like most other biological medicinal products, cell therapies cannot be terminally sterilised, which can make microbiological control challenging. Therefore, an emphasis is placed on an aseptic manufacturing process. As described in the EC Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products, this includes the use of clean areas of appropriate environmental cleanliness level, and sterilisation of materials, equipment and other articles that are introduced in a clean area, so as to avoid introducing contamination.

In the US, the FDA recommend the use of sterility tests described in 21 CFR 610.12 and USP <71>, although USP <1071>, which covers rapid sterility methods, was finalised in December 2019. Additionally, the FDA recommends that the validity of the sterility assay is assessed using the bacteriostasis and fungistasis testing as described in USP <71>, in order to ensure that any antibiotics present in the product do not interfere with the results of sterility testing. In the EU, Ph Eur 2.6.27 describes alternative sterility tests which can be used for cell-based products. These include a 7-day growth promotion test as well as rapid direct detection tests.

Identity tests are required to distinguish the product from other products being processed in the same facility. Depending on the cell population, phenotypic and/or genotypic assays can be utilised for identity testing. For example, flow cytometry for phenotypic identification of cell surface markers or PCR-based methods for genotypic characterisation may be used. Purity tests should identify any potential process-related or product-related impurities. Product-related impurities may include non-viable cells, or cells of different lineages or different stages of differentiation.

Process-related impurities would encompass raw materials used in the manufacturing process, for example residual cytokines. As well as identification of potential process-related impurities, a dilution factor calculation should be provided to demonstrate that there are sufficient washing steps to dilute them to an acceptable level. 

As described in ICH 6QB, potency is the quantitative measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties. A suitable potency assay, based on the intended biological effect which should ideally be related to the clinical response, should be in place prior to starting clinical trials. Although in vivo or in vitro assays may be used, due to the often limited time between end of manufacturing and release to administration to the patients, and the lack of reliable animal models, in vitro assays such as flow cytometry and Elispot and frequently used to measure potency of cell-based products. In the US, FDA Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products provides guidance on the development of potency tests for advanced therapy products. Although it is specific to immunotherapy products, the EMA guideline on potency testing of cell based immunotherapy medicinal products for the treatment of cancer (EMA/CHMP/BWP/271475/2006) also provides useful guidance which may also be relevant to other types of cell therapy products.

During early product development (pre-clinical, Phase 1 and early Phase 2 studies) it may be appropriate to have wider acceptance ranges, as characterisation data and an understanding of the product’s biological properties are obtained. Thus, the emphasis during early phase development should be on identification and control of raw materials. As development progresses, it is generally expected that acceptance criteria will change as product knowledge increases. Donor variability may be a potential issue with defining specifications and acceptance criteria with cell therapies, particularly with autologous therapies. It is expected that some parameters may be subject to more donor variability that others. As a result, it is important to understand the product’s critical quality attributes (CQA), and how they are impacted during manufacture. It should also be noted that the EC GMP and GCP guidelines specific to ATMPs include provision for the administration of ‘out of specification’ cell-based products in exceptional circumstances. Due to the serious and life-threatening nature of conditions which are often targeted with cell-based therapies, there may be circumstances where administration of ‘out of specification’ products is justified, taking into account the alternative options for the patient and the consequences of not receiving the product.

COMPARABILITY

It is anticipated that manufacturing changes will be made throughout the development program. These may include introduction of additional manufacturing sites as scale of production increases, changes in equipment, raw materials and critical starting materials, and changes in the manufacturing process. A suitable comparability plan is required to demonstrate that the changes to not impact the safety or efficacy of the product, and that the post-change product is comparable to the pre-change product. 

As described in the EMA Q&A document, “Comparability considerations for Advanced Therapy Medicinal Products (ATMP)”, due to the inherent complexity and variability of cell-based therapies, they are outside the scope of the ICH Q5E guideline on comparability of biological/biotechnological medicinal products. However, many of the general principles of ICH Q5E do also apply to advanced therapies. A risk assessment should be performed to assess the potential impact of the change to the CQAs of the product, and the product’s safety and efficacy. As development progresses, the risk of introducing manufacturing changes increases, and thus a more comprehensive comparability program may be required. Thus, the stage of development together with the extent of the manufacturing change should be considered when assessing the risk and determining the appropriate amount of data required to demonstrate comparability. Analytical methods should be selected which are capable of detecting changes to the product’s CQAs. These should include as a minimum, the methods used for QC release testing. In addition, extended characterisation and methods related to the functional and biological characteristics of the product are recommended. Although the analytical methods do not need to be fully validated, especially at an early stage of development, the methods should be robust and sensitive enough to detect differences. Furthermore, EMA recommends that where the intrinsic variability of the methods is low and precision and sensitivity is high, a smaller number of batches may be tested in order to demonstrate comparability. 

A side-by-side comparability approach is recommended, especially with autologous products, where there is likely to be significant donor variability. However, this may not always be possible, as particularly with autologous cell therapies, obtaining an adequate quantity of source material to manufacture two full scale batches can be a challenge. Where a side-by-side comparability study is not possible, it is acceptable to compare post-change data to historical pre-change batch data. Acceptance criteria should also be defined and justified based on available batch data, taking into account the intended clinical use of the product, as well as knowledge of the product, manufacturing process, control strategy, and risks that are relevant to the proposed change.

As stated in the FDA draft Guidance for Industry: Comparability Protocols for Human Drugs and Biologics: Chemistry, Manufacturing, and Controls Information, the acceptance criteria can allow for differences in product attributes if justification is provided based on assessment of the effects of the change on safety and effectiveness. This may be especially relevant to cell therapies due to the inherent variability in products. 

Stability studies are not mandatory for a comparability exercise. As cell therapies are generally cryopreserved and have a relatively long shelf-life, full real-time stability studies are not usually required. However, EMA recommend performing stability studies under accelerated or stress conditions, in order to identify possible changes in the degradation profile of the product. In-use stability studies are recommended to identify any potential changes in the recovery of the cells following storage.

CONCLUSION

Every cell therapy product varies in its complexities and applications, and has its own characteristics which should be considered when preparing a CMC package for regulatory submission.

The factors discussed in this article may not be applicable to every cell-based therapy, and therefore it is crucial that each Sponsor identifies the specific risks and risk factors associated with their product and uses a risk-based approach to determine the level of CMC data required. Examples of potential risk factors associated with cell therapies include microbiological contamination, the capacity of the cells to proliferate and differentiate and possible tumourigenicity, and the ability of the cells to initiate an immune response.

Extensive characterisation early in development helps to understand the potential risks as well as provide information to demonstrate risk mitigation. It is also highly recommended that Sponsors consult with the relevant regulatory agencies on the specifics of their product at an early stage in development.