Synthetic nucleic acids can be formulated as virus-like particles (VLPs) for vaccines or gene therapeutic delivery vectors. These VLPs can be used to display variable copy numbers and types of peptide and protein antigens, as well as sugars and small molecules for programmable immune cell targeting and stimulation. I will present our work on design and fabrication of DNA-based VLPs for application to subunit vaccines for COVID-19 and AIDS.

Sickle cell disease (SCD) is an inherited genetic disease of the blood with no known cure. Stem cell gene therapy is an emerging experimental therapy for SCD with the potential for lifelong cure, but it is an expensive multi-step treatment regimen. We developed a quantitative systems pharmacology model to predict how varying treatment will affect post-treatment hemoglobin and red blood cell dynamics after autologous stem cell gene therapy.

Adeno-associated viral vectors present key challenges in formulation development, including high molecule complexity and limited material for development. This talk will discuss several high-throughput strategies to overcome those challenges. Results will include case studies for engineered AAV capsids and common drug-product impurities.

Gene Therapy fill-finish operations present control strategy challenges related to the small volumes of solution being processed. Requirements tied to specific routes of administration of the gene therapy drug product may impose additional challenges. Formulation and drug delivery case studies will be shared that highlight these challenges. These examples show the importance of an integrated, end-to-end approach when developing a gene therapy drug product manufacturing process.

AAV has shown great potential as a gene delivery vehicle; however, the lack of rapid, high-throughput analytical platforms to assess sample purity of in terms of full and empty capsids creates a major bottleneck in the large-scale manufacturing of AAV. We propose a novel high-throughput methodology, which integrates a microfluidic electrophoresis platform with a mathematical model for the rapid assessment of AAV samples. This research was funded by Perkin Elmer.

Recombinant Adeno-Associated Viruses (rAAVs) deliver therapeutic DNA for gene therapy. However, rAAV manufacturing is imperfect, producing mostly defective capsids without the complete therapeutic gene. Here, we used microfluidic resonators for biophysical measurements of both rAAV and rAAV-producing cells. We juxtapose our approach with ddPCR measurements, and modelling efforts to identify correlations between biophysical measurements and genome and functional titers, towards rapid quality control of rAAV-based gene therapies.

The smaller manufacturing scales of AAV-based products make it particularly challenging to develop methods with very limited sample availability. Use of traditional methods for the separation and quantitation of high molecular weight (HMW) species is challenging given their larger size. This talk focuses on enumerating existing methods for HMW and particle characterization and their limitations. In addition, emerging methods and their advantages over existing ones will be described.

Mass photometry is a novel, easy-to-use bioanalytical technology that measures the empty-full AAV capsid ratio in minutes using minimal sample amounts and without the need of sample preparation. Circumventing the requirement of large capital expense and skilled operators, it can be employed throughout the manufacturing process. We present a novel mass photometry instrument dedicated to the challenges of AAV characterization. 

With the importance of biophysical methods such as SV-AUC in the gene therapy space, standardization of the experimental design, analysis, software and the inclusion of reference material is critical. SV-AUC is the gold standard in the determination of the empty, intermediate/partial, and full capsids of rAAV. In this talk, the implementation of standard procedures in the QC environment (SV-AUC data collection, software, and data analysis) will be discussed.

LC-MS-based characterization assays have been increasingly applied to support AAV-based gene therapy programs. Under denaturing conditions, the constituent capsid proteins can be analyzed to achieve serotype identification, global PTM characterization, and stoichiometry assessment. Under near-native conditions, the intact capsid ensemble can be analyzed to quantify empty, partial, and full capsids. In this presentation, we will discuss some emerging LC-MS techniques for AAV attribute characterization. 

Because of its sensitivity, dynamic range, selectivity, and speed, mass spectrometry (MS) is the gold standard for biomolecule analysis. However, the ~1 megadalton (MDa) mass limit of conventional instruments limits applications to larger species. Simultaneous measurement of m/z and z by charge detection (CD) MS enables analysis of gene therapies (revealing full and empty capsids, multimers, aggregates, intact and partial genomes) and other large assemblies (e.g., viruses, exosomes, nanoparticles).

Adeno-associated virus is a leading vector for gene therapy, but significant gaps remain in understanding AAV degradation and stability. We studied degradation of an engineered AAV serotype at physiological pH and ionic strength. Solutions with mixed full and empty AAV were held between 30-53oC, with molecular weight changes monitored versus time by total light scattering intensity. Results demonstrate fundamental differences in degradation behavior between full and empty AAV, revealing key interactions between viral capsids and their cargo that should be understood when designing a storage strategy of an AAV drug product.

Innovative analytical tools from Bio-Techne can support gene therapy workflows from discovery to quality control and address certain critical quality attributes of your therapeutic. Today’s presentation will explore the many ways viral vector analysis is streamlined with ProteinSimple instruments and how our products improve line-of-sight across the development process. 

Recombinant adeno-associated virus (AAV) mediated gene therapy is a promising technology in biotechnology industry. AAV is comprised of three viral proteins, VP1, VP2, and VP3, with conserved molecular weights at a distinct. These conserved profiles can be used to distinguish product-specific capsid proteins from unwanted process impurities, and the variation in these capsid profiles and ratios can provide a specific capsid “fingerprint” to determine capsid serotype. In this presentation, we present the work on developing and establishing a high throughput CE-SDS platform to screen high volumes of samples from process development and manufacturing control for AAV capsid purity profiles as well as capsid ratios for several different capsid serotypes.

The drastic advancements of human cell and gene therapy in recent years has opened the door to new treatments, paving the way towards cures of diseases once considered incurable. However, this development is not without pitfalls. This talk will address common oversights sponsors make leading up to an IND submission, which can lead to product delays, a clinical hold, and treatment delays, and key factors to consider as you are preparing your IND submission, such as key timelines, why selecting the right partner is critical to success, understanding key inputs from your CDMO before submission, and pharmacology and toxicology considerations 

Complete and precise characterization of AAV capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The method can be used for detailed AAV characterization, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsulated DNA molar mass, and total capsid and vector genome titer. These applications make this a powerful tool for gene therapy product development and process analytics.