Tam Duong, Scientist II, Cell and Gene Therapy Research and Development 
Tam Duong

Scientist II, Cell and Gene Therapy Research and Development

Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery to treat a variety of human diseases. A non-enveloped virus, rAAV can be engineered as a vector to deliver a therapeutic gene directly into the patient. Its use has grown as increasing numbers of gene therapy innovators have discovered the advantages of rAAV-mediated gene transfer, which include high biosafety, low immunogenicity, a broad range of infectivity, and stable expression (i.e., by providing long-term gene expression in vivo).

Sustaining the growth of rAAV gene therapy products requires development and optimization of accurate and robust analytical methods to ensure safe and appropriate therapeutic dosages selected for treatment or clinical applications. At Lonza, we are actively engaged in actualizing such advances, as a means to improve upon current analytical approaches.

Addressing the limitations of current rAAV analytical methods

The characteristics of rAAV present unique challenges to the development of analytical methods and the identification of critical quality attributes (CQAs). One of the most pressing challenges is inefficient packaging of the viral genome into the capsid, a phenomenon that can present the following complications:

  • Empty or partial viral genome in the final AAV gene therapy product: In a field in which twenty to thirty percent recovery of manufactured product is widely considered the industry standard, this can be a formidable problem.
  • Compromised efficacy: Capsids containing incomplete genomes can affect product efficacy through the inability to transduce target cells or through delivery of an incomplete or non-functional therapeutic genome.
  • Higher dosing requirements: A low percentage of full-length capsid may require a higher total capsid dose, which may trigger undesired immune responses from the patient.

Consequently, gene therapy scientists are working to develop methods to distinguish between full, partial, and empty AAV product. The ability to robustly analyze an AAV product at any level of fullness can help to optimize viral vector upstream and downstream processes, a benefit that may facilitate production of a full-length, high-quality product.

Lonza’s approach to advancing innovation in rAAV analytical methods

Our team is developing an analytical toolbox that covers multiple angles of characterization of an AAV product, including viral vector integrity, infectivity, and quantification of full vs. partial vs. empty virus particles. The toolbox includes:

  • Platform assays to determine viral vector infectious titer, an historically challenging attribute to detect due to high variability between tested samples.
  • High-precision assays designed to yield accurate titer quantitation with additional information on:
    • Viral vector genome quality (e.g., integrity, full vs. empty)
    • Viral vector impurity (e.g., presence of residual host cell DNA, host cell protein)

A particularly promising area of focus is on an optimized TCID50 assay (50% Tissue Culture Infectious Dose), an in vitro cell-based method widely used to determine AAV infectivity. This assay may improve upon current infectious titer detection methods, as a more accurate and more robust assay may facilitate early identification of out-of-specification samples during rAAV process development and manufacturing. Additionally, its use will facilitate transfer into a regulated Good Manufacturing Practice/quality control (GMP/QC) environment. The optimized TCID50 assay appears to have great potential for enabling future client projects by providing high-precision, high-throughput analysis of viral vector samples, a benefit that may accelerate upstream and downstream research while also expediting regulatory submission processes. A manuscript describing the development of the optimized TCID50 assay is currently under revision.

Fueling future innovation in rAAV gene therapy development

Lonza’s status as a global company operating across five continents confers some key advantages in rAAV analysis.

  • Our global presence allows us to make rAAV projects a truly collaborative effort involving several different departments including:
    • Research and Development at Lonza Houston (US)
    • Process Development/Bioassay Services (PD-BAS) at Lonza Houston (US)
    • Applied Statistics at Lonza Portsmouth (US)
    • Digital Transformation Group Operations at Lonza Basel (CH)
    • Drug Product Services at Lonza Basel (CH)
  • Utilization of multiple resources and different areas of expertise helps us to overcome obstacles, and facilitates tailoring of our research approach to meet client needs.
    • Those advantages place Lonza in a unique position to pioneer novel methods to characterize viral vectors beyond the genomic titer. By offering the benefits of enhanced data precision and consistency, reduced assay workload (i.e., by eliminating the need for standard curve preparation), and implementation of automation, we aim to improve rAAV analytical tools to meet the surging demand for in vivo gene therapies to treat human diseases.

Additional Information and Disclaimer

Lonza Group Ltd has its headquarters in Basel, Switzerland, and is listed on the SIX Swiss Exchange. It has a secondary listing on the Singapore Exchange Securities Trading Limited (“SGX-ST”). Lonza Group Ltd is not subject to the SGX-ST’s continuing listing requirements but remains subject to Rules 217 and 751 of the SGX-ST Listing Manual.

Certain matters discussed in these articles may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. Investors are cautioned that all forward-looking statements involve risks and uncertainty and are qualified in their entirety. The actual results may differ materially in the future from the forward-looking statements included in these articles due to various factors. Furthermore, except as otherwise required by law, Lonza Group Ltd disclaims any intention or obligation to update the statements contained in these articles.

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