The proven efficacy of approved cell and gene therapies (CGTs) has led to an explosion in the number of CGT products being developed in the European Union (EU) and the United States (US)¹. However, with complex formulations containing recombinant nucleic acids or engineered cells and/or tissues, CGTs often require extensive and complicated preclinical and clinical development processes before they are suitable for human use¹. In this article, we discuss the importance of reagent quality control (QC), especially endotoxin testing, during CGT development and explore how the selection of reagents and suppliers helps ensure the safety of innovative new CGT products.
 
Start with the highest quality reagents possible
Although the raw materials that go into pharmaceutical products are not specifically regulated, the raw materials used in CGT development must be safe and suitable for their intended purpose. While all parties involved in the CGT development chain are obliged to document their actions in compliance with established good manufacturing processes (GMP)², ultimate responsibility for QC remains with the manufacturer of the final drug product³. In the US, CGT manufacturers need to follow 21 CFR Part 211 [21 Code of Federal Regulations 211.84(d)(1)], which requires full identity testing for all incoming lots of drug product components³.

While sterility testing rules out the presence of living microorganisms, there are other forms of contamination, like endotoxins, to consider. Endotoxins are lipid A components from the cell wall of Gram-negative bacteria, typically released after cell death or lysis. In addition to negative effects on tissue cultures, endotoxins can cause mild to severe immune responses in animal models and humans. The significant risk posed by these contaminants requires reagent and buffer manufacturers to specify the endotoxin content of these products and to ensure the endotoxin levels remain within strict limits.

By knowing the exact endotoxin level in buffers and reagents, manufacturers of CGTs can have additional confidence in their final drug products. The consequences of endotoxin contamination in clinical trials or production lots of parenteral therapeutics can be severe, even leading to death⁴.
 
Robust quality control includes endotoxin testing, even at early stages of CGT development
So, how does one avoid such complications and ensure endotoxin levels remain within approved limits throughout the development and production process? The use of high-quality, endotoxin-tested reagents at the beginning, middle, and end of development and manufacture of CGTs is essential to ensure high quality, reproducibility, and uniformity across the process. Therefore, it is important that organizations developing CGTs select reagent suppliers capable of providing stringently manufactured and tested products that have undergone rigorous QC.
 
For organizations developing CGTs to be confident that they are starting with the highest quality reagent possible, their reagent suppliers should be able to demonstrate that their materials comply with specific endotoxin requirements and that endotoxin levels in final products do not exceed the overall limit specified by the appropriate regulator. The quality of raw materials is a key concern for suppliers of buffers, solutions, and reagents destined for use in pharmaceuticals, and a good supplier will have robust testing procedures in place to ensure that the endotoxins present in their products fall below levels that could cause endotoxin-induced toxicity in the final therapeutic drug.
 
In the US, the FDA has set the acceptable limit of endotoxins in medical devices to be 0.5 endotoxin units/mL. This limit could be lower depending on how or where the device will be used in patients⁵. Reagent suppliers should also be able to define this value to meet the needs of a particular application⁴.

The United States Pharmacopeia (USP) provides strict testing guidelines to ensure that all pharmacological products, from input and processing reagents to pharmaceuticals, adhere to high quality standards¹. The bacterial endotoxins test (BET) is described in USP Chapter 85 (USP ). A good reagent supplier should be able to demonstrate that the BET is implemented at every step of its manufacturing processes to ensure endotoxins are not inadvertently introduced or concentrated.
 
Another crucial consideration is the method used for endotoxin detection. The Limulusamebocyte lysate (LAL) test is required for compliance with USP . There are multiple types of LAL assays (i.e., gel-clotting, turbidimetric, and chromogenic). The chromogenic method—measured kinetically—has the highest sensitivity, with a limit of detection of 0.005 EU/mL (EU = one international unit of endotoxin).
 
A supplier should follow the USP guidelines strictly and employ methods to manage endotoxin masking. Certain substances (e.g., some proteins, cations, excipients, or even some types of plastics and glass) can lead to test interference—either enhancement or inhibition. Including a positive product control for every sample helps alleviate these challenges and should be part of assay qualification.
 
Fundamentally, anorganization developing CGTs must choose a reagent supplier that operates a sophisticated quality management system and ensures robust product QC. The supplier should be able to perform endotoxin testing and quantify endotoxins throughout the entire manufacturing process, e.g., in the water for production, pre-filtration, and final products.
 
Selecting a reagent supplier for the development of cell and gene therapies
Compliance with QC standards is a basic requirement for products manufactured under GMP conditions. As discussed, reagent suppliers should be able to quantify endotoxin content of products through rigorous testing. However, when developing CGTs, it is also important to consider overall supplier manufacturing standards used throughout the development process, including early-stage, research use only (RUO) products.
 
A company that manufactures RUO products to the higher medical device standard, ISO 13485⁶, instead of ISO 9001⁷, is highly desirable. ISO 13485 specifies the requirements for a quality management system (QMS) for the design and manufacture of medical devices (including pharmaceuticals). It requires established processes, policies, and procedures, which ultimately support creating products that are fit for purpose. Contamination control and product cleanliness are also taken into consideration.
 
A reagent supplier should provide lot-to-lot consistency by performing the quality control and analytical tests necessary to ensure product specifications are met. In addition, valued reagent suppliers will employ staff with a technical background in design and development processes and, for adaptability and flexibility, be capable of making large numbers of different buffers and reagents packaged in a variety of container types.
 

To meet safety testing needs for CGTs, it is essential that quality is built into the manufacturing process. A supplier of buffers and reagents that can meet a higher-quality manufacturing standard (ideally capable of both GMP and ISO 13485 processes for RUO product manufacturing) and demonstrate rigorous endotoxin testing, will provide greater reassurance that the final CGT product are consistently safe and compliant. In essence, the use of strict quality management systems by reagent suppliers both helps to ensure higher quality products and to facilitate the transition to GMP, smoothing the journey from development to commercialization of therapeutics.
 
Conclusions
Testing of products at the beginning, middle, and end of development and manufacture of CGTs is essential to ensure quality, safety, and uniformity across the process.
 
A good supplier of high-quality buffers and reagents should be capable of demonstrating that their products have undergone robust QC throughout their entire manufacturing process, including specifying endotoxin content.
 
It is also advisable to partner with suppliers that can support all stages of the CGT development pipeline, from R&D to GMP manufacturing with the highest possible manufacturing standards, QC processes, and regulatory knowledge. This helps ensure a smoother path to the clinic as well as safe, pure, and effective CGT products.
 
References
1.    Iglesias-Lopez C, Agustí A, Obach M, Vallano A. Regulatory Framework for Advanced Therapy Medicinal Products in Europe and United States. Front. Pharmacol. 2019.
 
2.    WHO. Control and safe trade of starting materials for pharmaceutical products. www.who.int/medicines/publications/qa_starter/en/index1.htmlaccessed November 10, 2020. 
 
3.    Peters R C. Locking Fraudulent Materials Out of the Supply Chain. 2019. Volume 43, Issue 4. Page Number: 60–62.
4.    Gorbet M B, Sefton M V. Review: Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. The Biomaterials: Silver Jubilee Compendium, 2004: Chapter 6.2 Endotoxin
 
5.    FDA. Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers www.fda.gov/media/83477/downloadaccessed November 10, 2020.
6.    ISO. ISO 13485:2016—Medical Devices—Quality Management Systems—Requirements for Regulatory Purposes, www.iso.org/standard/59752.html, March 2016.
 
7.    ISO. ISO 9000 Family—Quality Management, www.iso.org/iso-9001-quality-management.html, accessed November 10, 2020.
 
 

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Garrett Perry is a Technical Services Specialists at Teknova, an ISO 13485-certified and GMP reagent manufacturer. www.teknova.com.