Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. We present the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full AAV6.2 capsid percentages, using the CIMac™ AAV Analytical Column that can detect as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples. This research was developed with funding from the Defense Advanced Research Projects Agency (DARPA).

• NEW TALK - This Presentation Will be Given for the First Time

Viral-based gene therapy products are complicated molecules, containing nucleic acid, lipid, and protein elements that require extensive characterization to ensure product consistency, efficacy, and safety. Analytical methods play a key role in process development, process control, and characterization of drug substance or products. An LC-MS method was developed to assess viral protein ratio, post-translational modifications, virus- and cell-associated proteins of viral vector products and help establish key structure-function approaches to broadening the understanding of how these viruses interact with cells.

Given the complexity of Adeno-associated Virus it poses a very big challenge for the analytics. In this presentation I will cover some of the innovative approaches we have taken to overcome these challenges using a variety of techniques including Mass Spectrometry and Analytical Ultra Centrifugation.

Gene therapy can induce an unwanted immune response that can have an influence on the efficacy and potency of the treatment. Additionally, pre-existing immunity towards AAV and CRISPR can also neutralize the therapeutic effect. In vitro assays such as T cell proliferation assays can be used to assess this unwanted immunogenicity in an early phase. Innate immune assays can be used for the evaluation of potential impurities and innate response inducing contaminants. Sensitive T and B cell Fluorospot assays can be used to monitor patient' specific immunogenicity.

Getting a quick answer on AAV titer, empty/full ratio, and aggregation state is critical to most decisions in the lab. Stunner’s dye-free, label-free, and standard-free AAV Quant delivers all that data using just 2 µL of sample, in less than a minute - way faster than AUC, ELISA and ddPCR. Learn how Stunner’s AAV characterization delivers hassle-free answers.

An important PQA of AAVs is the percentage of full particles versus empty particles lacking the genome. A large percentage of empty particles decreases the transduction efficiency, increases the therapeutic dose, and poses a safety risk. Analytical ultracentrifugation is the standard for measuring percent full, but it is time-consuming and requires a large amount of material. To overcome this, we developed a high-throughput cIEF method that can identify DNA-containing peaks for quantifying the percentages of full and empty particles and compared these results to other established techniques.

• ​NEW TALK - This Presentation Will be Given for the First Time

Cell-based products tend to be novel and more complex compared to other biologics, presenting significant analytical challenges. For example, product understanding is essential as therapies progress through clinical trials; however, the development of bioassays to identify CQAs and evaluate potency and comparability can be limiting. Moreover, some cell-based products have a short shelf-life or small-batch sizes that necessitate alternative testing approaches. These and other analytical development considerations will be discussed.

Quantitative potency assays were developed to demonstrate correction of b-thalassemia and sickle cell disease properties in an in-vitro cell culture system. Potency was found to be specific to the beta-globin lentiviral vector and dependent on transduction efficiency of the autologous gene therapy drug product, demonstrating ability to reject sub-functional drug products. Considerations for assay development, qualification results, and redundancy to transduction efficiency methods will be discussed.

The analytical demands of cell and gene therapies can be extremely challenging throughout the development phases. The use of bioassays facilitates the different developmental phases as they can be used to determine key parameters including: vector potency, product identity or determination/titration of neutralizing antibodies. Here, we present different strategies for the development of reliable bioassays for specific or more global use by showcasing a selection of our engineered iLite® cells.

• physical and thermal stability 
• size and titer
• surface characteristics such as charge
• payload/ transgene load

Cell/gene therapies have shown to be the most effective treatment for unmet medical needs, such as for hematological malignancies and inherited diseases. Cell/gene therapies are novel modalities which are expected to have complex bioanalytical strategy for assessing pharmacokinetics and Immunogenicity in clinical studies. The challenges lie in the complex critical questions-driven bioanalytical strategy, unconventional methodologies used for bioanalytical methods, and lack of regulatory guidance on “fit for purpose” method validation. This presentation will provide considerations in developing and implementing bioanalytical solutions to enable advancement of cell/gene therapy platforms in clinical studies.  

A growing number of clinical trials and marketing authorizations of rAAV-based therapeutics underlines their value in therapy. Therefore, scalable production, purification methods and reliable analytical methods are mandatory. Here, we present novel chromatography-based purification strategies, which are explored as scalable alternatives to established gradient ultracentrifugation-based protocols. Different analytical options for rAAVs will also be presented and compared. Finally, an outlook on stabilization of rAAVs in different formulations will be given.

• NEW TALK - This Presentation Will be Given for the First Time

Antibodies play a crucial role in the analysis of CAR-T cells. Particularly of interest is the class of anti-idiotypic antibodies that are directed against the CAR since these enable the specific detection of the modified T cells using flow cytometry. Here we present the generation of specific anti-CAR-T cell antibodies and demonstrate how CAR-T analysis benefits from our modular antibody assembly technology using SpyTag-SpyCatcher protein ligation.