Guest Column | January 31, 2025

New Reference Standards To Support Quality of AAV Raw And Starting Materials

By Anthony J. Blaszczyk, United States Pharmacopeia

DNA fragments samples-GettyImages-947584762

Gene therapy has become one of the fastest growing modalities in the biopharmaceutical industry. Recombinant AAV (adeno-associated virus) products have been growing in the number of products both on-market and in development. In 2023, the gene therapy market was estimated at over $9 billion and is expected to grow to nearly $24 billion by 2028.1 This rapid growth is driven largely by groundbreaking gene therapy treatments, which have demonstrated the ability to treat previously incurable genetic diseases. Although these drugs have great potential impact, they face many challenges, from the start of development through commercialization, including low yields, difficult scalability, high cost of materials, high levels of impurities, and analytical challenges.   

These challenges have resulted in extremely high costs of treatments that can limit patient access. The United States Pharmacopeia (USP) is attempting to ease some of these burdens through the generation of reference standards and analytical reference materials to support manufacturers from production to delivery.

USP is a nonprofit organization focused on building public trust in the supply of safe, quality medicines. USP Biologics is expanding standard development to support the quality testing of gene therapy throughout the totality of the biopharmaceutical product life cycle. Based on stakeholder feedback, USP has identified critical analytical challenges associated with AAV, including raw material qualification/validation, viral vector characterization, and quantitation of product and process impurities.

AAV is composed of a small non-enveloped capsid, which consists of three viral proteins: VP1, VP2, and VP3. The capsid contains a single-stranded DNA payload, which can be up to 4.8 kb in size.2,3 After administration, the AAV is transported to the target cell; this target-specific transportation is conducted by the serotype-specific capsid shell, specifically through the binding of the capsid to the cell receptors. The AAV subsequently enters the cell via endocytosis. After entering the cell, AAV releases its single-stranded DNA into the nucleus, where host cell machinery, such as DNA polymerase and RNA polymerase, catalyze the formation of double-stranded DNA and transcription. Next, the mRNA is transported from the nucleus to the cytoplasm, where the transgene is ultimately expressed. Due to its safety and efficacy, AAV vector technology has emerged as the frontrunner in gene therapy drug development.4,5

Even with its continued growth, AAV production and analytical testing remain challenging. Unlike more mature fields such as mAbs, there are no harmonized production, purification, or analytical testing procedures for AAV. Through the production of standards specifically designed to ease the biggest burdens associated with AAV, USP is trying to help manufacturers overcome these obstacles. Herein, we will present the variety of ways that USP is supporting the AAV field through the development of both physical reference material and documentary standards. These standards are designed to help ensure the quality of the raw and starting materials used during the production of viral vector products, such as AAV. In addition to a number of existing documentary standards, USP is positioned to offer a package of resources that can aid users from production through quality testing, which we discuss in a separate article.

Raw And Starting Materials

Gene therapy vector manufacturing is a complex process that presents unique challenges across the entirety of the production process. AAV production requires several discrete manufacturing activities, which involve growing cells, producing viral vectors in those cells, and purifying the AAV. Each activity has specific requirements and complex raw materials. One challenge is the lack of harmonization — differences exist between manufacturers for even the most general of steps, which increases the total production time and makes reference standard development challenging, as different analytical tests may be required for different production processes. For example, companies differ in cell line choice (typically either Sf9 or HEK293), plasmid selection, and purification strategy, which is usually either performed via chromatographic or ultracentrifugation separation.6,7 Nevertheless, there are commonalities between AAV productions, and USP is focusing on those areas for the development of standards to help ease manufacturing challenges. In fact, USP has numerous existing documentary standards already published that are relevant for evaluating AAV products. These include, but are not limited to, General Chapter <1047&GT Gene Therapy Products, General Chapter <1043&GT Ancillary Materials for Cell, Gene, and Tissue-Engineered Products, and General Chapter <1002&GT Filters and Membranes (currently in USP-PF).

Plasmid Reference Materials

Plasmids are critical starting materials for manufacturing AAVs as well as many other cell and gene therapy products. Because plasmids are composed of nucleic acids that are introduced into AAV drug products, either as a transgene or process impurity, thorough quality characterization is essential for product safety. Thus, it is imperative that AAV manufacturers have proper control strategies in place for characterization of plasmid DNA.

USP is taking multiple steps to aid cell and gene therapy manufacturers that use plasmid DNA as a critical starting material.7,8 USP formed an expert panel (EP) that was tasked with authoring General Chapter <1040&GT Quality Considerations of Plasmid DNA as a Starting Material for Cell and Gene Therapies. Moreover, six plasmid DNA analytical reference materials are being developed that can be used in the qualification (topology) of plasmid DNA starting material.

Because supercoiled plasmid DNA is the preferred isoform for manufacturers, accurately quantifying the percent of supercoiled plasmid is critical. In fact, supercoiled plasmid DNA is often considered the most efficient isoform for inducing gene expression as compared to the other forms, such as open circular and linear.9-11 To assist in topology assessment, which is a critical quality attribute for plasmid DNA, USP is developing a variety of plasmid DNA reference materials ranging from 4.2 kb to 12.3 kb. Each of these reference materials will be evaluated in a collaborative study using three of the most common methods for analyzing plasmid topology, capillary electrophoresis with laser-induced fluorescence (CE-LIF), agarose gel electrophoresis, and anion exchange chromatography. These reference materials can be used as a control to aid manufacturers in the qualification and/or validation of their in-house DNA topology method. Because of the extent of products being offered, as well as the thorough characterization using multiple topology methods, the USP reference materials should be suitable for nearly all users, regardless of the plasmid size or preferred testing method.  

Although plasmid DNA is widely recognized as a critical starting material for cell and gene therapy products, there is a lack of regulatory guidance for its use in manufacturing. USP General Chapter <1040&GT was drafted to provide best practices on sourcing, qualification, and testing of plasmid DNA. This general chapter will provide guidance where gaps are identified in existing guidance documents, specifically in the field of cell and gene therapy. This general chapter was published in USP Pharmacopeial Forum 496 on Nov. 1 and received more than 460 comments. The EP, which is made up of industrial and regulatory leaders, is currently working on addressing these comments. After the public comments have been addressed and are presented to the USP Expert Committee, and if the Expert Committee approves, the general chapter will be published in the USP NF and become official.

Endonuclease Reference Standards — Documentary and Physical

Another raw material used to manufacture AAVs is endonuclease, an enzyme that cleaves the phosphodiester bond on the backbone of DNA.12 In AAV production, endonuclease digests unincorporated DNA that is not in the AAV capsid. This critical step allows for the removal of unwanted DNA that, if present in the final product, has potential negative implications for patient safety. These impacts include the introduction of unwanted DNA into AAV capsids, unattended immune response, and/or production of unwanted biological products. To properly utilize endonuclease, enzyme activity must be determined. And, to properly calculate the activity of the endonuclease, an established method must be used to measure that activity. Although many laboratories have methods in place to test this activity, there is no single harmonized method being used; thus, the activity values generated can differ depending on the method employed. As is true with many enzymatic activity assays, relatively small method variations can result in significant differences in the reportable data.

To address this issue, USP is developing both a documentary standard and a physical standard. The documentary standard will be a below-1000 procedural general chapter that provides a compendial method to determine endonuclease activity. USP has also developed a physical endonuclease reference standard. The compendial method will provide a single test procedure that can be adopted by any lab, and the reference standard provides well-characterized material that can be used to assess system suitability of the method. USP envisions many benefits to the industry upon release of these standards, including increased consistency between vendors, more defined control strategies, time and money savings from adopting a validated method, and testing harmonization.

Conclusion

The quality of raw and starting materials in the production of viral vectors is critical for the quality and safety of the final drug product. The use of reference standards to assess quality can help manufacturers ensure the quality of these materials. As described herein, USP is releasing reference material that will aid manufacturers in performing their characterization. Because of the complexity and vast number of raw and starting materials, additional standards, many of which USP is currently developing, will be needed for the CGT community to have a full toolkit of reference materials.

References:

  1. MarketsandMarkets (2023). Gene Therapy Market: Growth, Size, Share, and Trends.
  2. Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983;45(2):555-564.
  3. Samulski RJ, Muzyczka N. AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu Rev Virol. 2014;1(1):427-451. 
  4. FDA. Approved Cellular and Gene Therapy Products. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products Updated August 02, 2024. Accessed September 30, 2024.
  5. Burdett T, Nuseibeh S. Changing trends in the development of AAV-based gene therapies: a meta-analysis of past and present therapies. Gene Ther. 2023;30(3-4):323-335.
  6. Liu S, Li J, Peraramelli S, et al. Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells. Mol Ther. 2024;32(1):74-83.
  7. Srivastava A, Mallela KMG, Deorkar N, Brophy G. Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors [published correction appears in J Pharm Sci. 2021 Sep;110(9):3324. 
  8. Jiang Z, Dalby PA. Challenges in scaling up AAV-based gene therapy manufacturing. Trends Biotechnol. 2023;41(10):1268-1281.
  9. Sousa A, Sousa F, Queiroz JA. Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(30):3507-3515.
  10. Levy MS, Lotfian P, O'Kennedy R, Lo-Yim MY, Shamlou PA. Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method. Nucleic Acids Res. 2000;28(12):E57.
  11. Bilal AS, Parker SN, Murray VB, et al. Optimization of Large-Scale Adeno-Associated Virus (AAV) Production. Curr Protoc. 2023;3(5):e757
  12. Benedik MJ, Strych U. Serratia marcescens and its extracellular nuclease. FEMS Microbiol Lett. 1998;165(1):1-13. 

About The Author:

Anthony Blaszczyk is in the Pipeline Development group within USP’s Global Biologics department. At USP, he works with scientific experts and stakeholders to develop new standards to support biopharmaceutical quality assessment and development. Prior to USP, Anthony worked at Catalent Cell and Gene Therapy, where he managed an analytical development team responsible for the development, qualification, and transfer of methods. He obtained his Ph.D. in biochemistry from Penn State University in 2018.