Manufacture scale up and analytics are key challenges in AAV gene therapy development. Establishment of clear analytical strategies aligning with process & manufacture development from early translational development is critical for speed-to-clinical development. The presentation will discuss our integrated strategies and some key analytical development.

GeneWerk specializes in vector safety and integration site analysis for gene- and cell- therapy. We also offer a wide variety of custom-tailored services around safety and efficacy, such as analyses for gene-editing on/off-targets, TCR/BCR immune repertoire, or AAV impurities. Our goal is to assist clients in moving towards safer therapies.

AAV-based vectors used in Gene Therapy are new and complex modalities both in terms of structure and function. The mechanism of action of viral vectors is multifaceted, which requires matrixed approach for assessment of biological activity. This presentation will focus on stability-indicating methods for AAV-based vectors and will discuss challenges and opportunities for structure-activity relationship studies.

AAV gene therapy products have complex mechanisms of action that pose unique challenges to potency assay development. Determining the true biological activity often requires multiple assays (i.e. a matrix approach), and the strategy for implementing these assays may evolve through the product lifecycle. This presentation will discuss phase-appropriate development and qualification of different in vitro potency assays.

Vironova revolutionizes transmission electron microscopy (TEM) for nanoparticle characterization in the fields of gene therapy, vaccines, and drug delivery based on image analysis. The high-resolution images can provide essential information of morphology, packaging, integrity, and purity of the sample. Our in-house image analysis software and GMP-certified laboratory provide TEM services that answer questions which arise during the production process, helping in the decision-making in viral vector development.

The release testing package for plasmid DNA starting materials is more or less a platform set of assays while the amount (if any) of characterization testing is much less defined.  This presentation will discuss 4 different examples of nucleic characterization tools and how they can be used to enhance the release methods and  probe the quality of the starting material a little deeper than previously considered.

Virus-based gene therapies require complex analytical methods to assess key quality attributes during process development, GMP release and stability monitoring of clinical grade product. This presentation will discuss phase appropriate paradigms for analytical method development, qualification, and implementation into gene therapy programs as they transition from research into clinical stages. Some challenges with material and time limitations and proposed resolution strategies using platform-based methods will be explored as well.

As the gene therapy field continues to expand and companies increase the number of product batches manufactured per year, the burden on quality control for product release increases significantly. The presentation will showcase CGT Catapult’s advanced analytical platform for AAV product characterisation, which includes assay development for improving precision, assay automation and in-line product characterisation for closed processes.

T cells transduced with self-inactivating lentiviral vectors that co-express affinity-enhanced cancer antigen T cell receptors (TCRs) and T cell function modulators may demonstrate antitumor efficacy superior to TCRs alone. This work describes an analytical method which demonstrates the expression of transgenic T cell function modulators distinctly from their endogenous counterparts for characterization of lentiviral vectors and transduced T cell products.

In all biological products, distinguishing aggregated API from other particle types matters for understanding the root cause of instability. Until now, subvisible particle characterization methods have been unreliable, slow, and difficult to use across many workflows. Introducing the Aura, a 96-well, low-volume, high throughput aggregate and particle imaging system can rapidly size, count, and characterize biological particles and identify them as proteins, non-proteins, cellular aggregates, or other types of molecules.

For AAV gene therapy products, partial genomes and encapsidated residual DNAs impurities pose safety risk and compromise product efficacy. Their levels need to be monitored and controlled during product development. Next generation sequencing offers orthogonal approaches to traditional methods in characterizing such impurities, providing insights on genome integrity, DNA impurities levels, and size.

qPCR method is widely used to quantify genome titer and residual DNA in AAV gene therapy products. However, the standard method requires complicated sample treatment and is known to be variable. In this presentation, we will introduce a new method with optimization of AAV sample preparation and choice of qPCR standard, which not only simplify the method, shorten the assay time, but also improve overall assay performance.

Capsid titer can be measured using a variety of analytical techniques and is important for both early and late stage process development. Here, we compare methods for measuring AAV capsid titer and evaluate these measurements against other product quality attributes including genome titer and empty/full capsid ratios.

A growing number of gene therapy products are currently in preclinical or clinical trials to explore the potential in the treatment of multiple diseases. Among gene therapy vehicles, adeno-associated viral vector (AAV) is one of the most actively investigated deliver vectors with superior safety. GenScript ProBio helps solve the challenge of scalable and stable manufacturing for AAV from preclinical through IND application to clinical phases, bring excellent solutions to gene therapy.

Teknova is developing an optimized ion exchange (IEX) chromatography algorithm to purify full rAAV viral vectors that account for vector diversity. We rapidly assessed multiple parameters and created customized protocols and reagents for downstream processing. To date, we have used the formulated algorithm to develop optimal IEX protocols and ready RUO or GMP reagents for purifying multiple serotypes and payloads.

This talk will focus on advanced analytics of AAV with respect to critical quality attributes - vg titer, empty full AAV, aggregation, MS. In addition, this talk will show examples of orthogonal approaches to switch from traditional to advanced analytics for AAV.

The Cell & Gene Therapy industry is rapidly growing with more commercial product approvals anticipated each year. However, the lack of standardization and best practices around CMC programs creates hurdles to streamlined, cost-effective manufacture. Borrowing from the ‘A-Mab’ model in the monoclonal antibody field, the Alliance for Regenerative Medicine has produced a similar document titled A-Gene, a case study-based approach to integrating Quality by Design principles in gene therapy manufacturing.

Gene therapies possess substantial potential to be revolutionary curative treatments, but their broad implementation requires further progress in biomolecular understanding. High-resolution analytical approaches must be adopted in order to fully characterize these molecularly complex treatments and monitor the final drug product. AAV vectors have demonstrated their significant prospect for clinical impact. A multi-attribute mass spectrometry method was developed to enable AAV vector quality attribute monitoring and new peak detection, the roadmap for which should be widely applicable to other vector-based modalities.