Streamline the AAV Manufacturing Process with High-Grade Starting Materials
Authors:
Rachael Hardison, Ph.D.,
Manager, Technical Sales, Forge Biologics
Don Belcher,
Plasmid DNA is one of the most common starting points for manufacturing recombinant adeno-associated virus (rAAV) vectors. Gene therapy developers need a depth of knowledge around the critical quality attributes of both plasmid DNA and the finished rAAV gene therapy product. Forge offers clients a seamless plasmid-to-AAV manufacturing workflow under one roof, governed by the same quality structure and policies. This vertical integration allows for continuity in manufacturing, better control of project timelines, and streamlined, efficient communication with clients on manufacturing status.
The use of plasmid DNA in the pharmaceutical industry originated with the production of recombinant proteins; however, the expansion of viral vector-based gene therapies has placed a renewed need for the high-grade, efficient production of plasmid DNA. As regulatory oversight for cell and gene therapies continues to evolve, starting with high-grade plasmid material, such as Forge’s GMP-Pathway plasmids, will remain essential in the production of genetic medicines like rAAV.
High-grade plasmid DNA production is currently an industry bottleneck. Long lead times for plasmid DNA represent a risk for clinical pipelines. In addition, many CDMOs that provide cGMP plasmid DNA produce DNA for several applications, from starting materials for cell and gene therapies to vaccines. There is a global need to increase plasmid DNA manufacturing capacity for rAAV and other applications such as mRNA. Forge Biologics recently announced the addition of plasmid DNA manufacturing services that will be made available specifically for AAV manufacturing clients. In-house plasmid production allows Forge to control better delivery timelines and grade of AAV manufacturing for our clients.
Plasmid DNA Quality Characteristics to Consider in AAV Manufacturing
Triple transient transfection using plasmid DNA is a standard route of rAAV manufacturing. Triple transfection consists of the delivery of three plasmid DNA constructs:
- a gene of interest (GOI) plasmid that contains a genetic payload flanked by two inverted terminal repeats (ITRs),
- a rep/cap plasmid that contains a sequence for AAV capsid proteins (VP1, VP2, VP3) and replication proteins, and
- an adenovirus helper (Ad helper) plasmid that contains genes that are required to produce rAAV in HEK293 cells.
These plasmids, when combined with a transfection reagent, form nanoparticles that can interact with the host cell membrane and enter the cell. To maintain the efficacy of the triple transfection method in producing the desired AAV product, there are several plasmid quality characteristics to consider, including (1) ensuring low residuals and high purity, (2) plasmid homogeneity (e.g., high percentage of supercoiled DNA), and (3) ITR sequence integrity. The high GC content and palindromic nature of ITRs makes GOI plasmids susceptible to mutations in these regions during plasmid production. Disruption of ITRs can lead to reduced efficacy in viral packaging; therefore, it is imperative to confirm the sequence of ITR regions in GOI plasmids prior to transfection. Forge offers Next Generation Sequencing (NGS) to verify the sequence identity and the integrity of ITRs in GOI plasmids. Restriction digests are also included in release testing to demonstrate plasmid identity in parallel with plasmid sequencing.
Another key aspect of plasmid material quality is the absence of cross-contamination from other plasmid constructs. Low-cost research-grade plasmids are often made with multi-use materials or in shared laboratory spaces. Forge takes the extra precaution to mitigate the risk of cross-contamination by manufacturing client plasmids using single-use materials in controlled plasmid manufacturing areas. This includes single-use components across upstream and downstream plasmid production workflows. For example, single-use fermenters (SUFs) are used for upstream plasmid production to allow for scalability without compromising quality or yield. Similarly, when producing plasmids, product-specific, chromatography columns allow downstream plasmid purification to desired specifications. This workflow reduces any risk of plasmid product cross-contamination during chromatographic operations.
Navigating the Path from Discovery to BLA with a Phased-Approach to Plasmid DNA
In plasmid manufacturing, there are several quality grades available. While the brand names for these materials may differ, there are three broad categories used for classification: (1) Research-Grade, (2) Intermediate Grade and (3) cGMP-Grade.
In addition to research-grade plasmids, Forge offers GMP-Pathway plasmid manufacturing for AAV clients. Each grade is defined by increasing levels of product quality, documentation, and facility controls. For example, several plasmid manufacturers offer intermediate-grade plasmids as an alternative to cGMP plasmids for Phase I/II AAV manufacturing. Intermediate-grade, such as Forge’s GMP-Pathway, refers to a plasmid typically not produced to cGMP-grade specifications yet is of higher quality than a research-grade plasmid. The intermediate grade of plasmid arose following the European Medicines Agency (EMA) guidance in 2005 that required plasmids, when used as starting materials for viral vector production, to be “high-grade.” Still, a definition for the standards that plasmids should meet has not been explicitly stated. cGMP-grade or a high-grade intermediate-grade of plasmids are required to produce any viral vector that is to be used in humans in Phase I/II clinical trials.
Using research-grade plasmid DNA during the initial stages of development can provide a financial reprieve for gene therapy developers in place of costly cGMP plasmids. Research-grade plasmid DNA is often used for the pre-clinical discovery stage, including lead candidate selection, but may be used throughout the development of a process for AAV manufacturing. Research-grade plasmid DNA may also be used to manufacture a client's toxicology-grade AAV vector for in-vivo animal model studies.
High-grade intermediate-grade plasmids such as Forge's GMP-Pathway plasmids are appropriate for clients to use in Phase I/II AAV manufacturing. Forge's GMP-Pathway production process incorporates single-use systems and multi-modal chromatography for exceptional purification and recovery of supercoiled plasmid DNA. In addition, Forge's GMP-Pathway plasmids include a final fill and finish within a cGMP suite. Quality oversight of plasmid production is an added benefit of some manufacturers' intermediate-grade plasmid production. Forge's GMP-Pathway plasmid production quality oversight includes QA review of executed batch and analytical testing records.
cGMP plasmid DNA is needed for the manufacturing of Phase III and commercial viral vector products. Plasmid manufactured to cGMP specifications, while of increased quality, often requires a significant time and cost investment. Understanding which grade of plasmid DNA is required for various stages of the viral vector manufacturing process saves time and reduces cost while ensuring production of high-grade clinical trial materials (e.g., AAV viral vectors). Forge offers flexibility in its approach to consulting with clients to determine a phase- and cost-appropriate plan for plasmid production for the client's specific program.
Analytical Considerations When Generating Plasmid DNA for AAV Gene Therapies
Regulatory agencies have different testing and characterization requirements for plasmids used as a direct injectable versus a plasmid used as a starting material for vector manufacturing. Four critical elements of GMP-Pathway and/or cGMP plasmid quality for use in AAV manufacturing include:
- Plasmid identity (e.g., sequence demonstrates the absence of mutations or inverted terminal repeat (ITR) truncations).
- The absence of cross-contamination between plasmids or products.
- Traceability to demonstrate animal-free components.
- Documentation.
In 2020, the FDA released updated guidance for cell and gene therapy manufacturers to establish regulatory considerations for gene therapy plasmid manufacturing(1). Forge offers analytical testing suites for GMP-Pathway and cGMP plasmids that follow regulatory guidance for the manufacturing of plasmid DNA. Forge’s GMP-Pathway and GMP plasmids (coming in 2023) are derived from established & characterized bacterial master cell banks (MCBs). Recommended testing for bacterial cell banks used to manufacture plasmid DNA includes bacterial host strain identity, plasmid retention, bacterial cell count, bacterial host strain purity, plasmid identity, and full plasmid sequencing. In addition to performing these recommended tests, Forge also confirms the absence of any lytic bacteriophage in the client’s MCB.
Forge’s suite of release testing for purified GMP-Pathway plasmid DNA includes identity (restriction digest and sequencing), sterility, endotoxin, residuals (DNA, RNA, protein), and a report of the plasmid homogeneity (supercoiled) percentage. Forge provides clients with a CoT that lists the testing and specifications required for the MCB and the plasmid grade produced, as well as a TSE/BSE statement that plasmids have not been produced with any reagents of animal origin.
Finally, Forge performs GMP-Pathway and GMP plasmid and master cell bank stability studies to demonstrate the gene therapy starting materials' lasting quality and stability. Like AAV gene therapy products, stability studies for MCB are performed following the ICH Final Guideline on Stability Testing of Biotechnological/Biological Products.
Starting with the End in Mind
As a gene therapy developer, carefully considering the manufacture and testing of plasmids is prudent for laying the groundwork for AAV manufacturing. Leaning into a phased approach for plasmid grades and partnering with Forge to produce high-grade DNA and AAV under the same roof can provide both early and established gene therapy developers with a cost-effective jumpstart to process development and clinical manufacturing.
References:
- Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications. 2020