Within the talk novel approaches for the characterization of particulate impurities in biologics will be presented, and compared to state-of-the-art technologies for submicron and subvisible particle analysis. Besides new analytical technologies, the integration of machine learning for data analysis will be presented.

Gene therapies offer tremendous promise to address human disease, but their complexity and diversity present unique challenges to those seeking to standardize materials and methods. Establishing relevant and applicable standards that apply to gene therapy development is essential in maintaining safe and effective therapeutics. Alignment on best practices can also provide clarity and consistency for developers of these therapeutics, especially as gene therapies progress into the late stages of development. Setting standards is especially important in the gene therapy space, where changes to manufacturing processes are frequent, and the development is in orphan indications, limiting the possibilities for comparison with other manufacturing processes. Therefore, the USP is working with stakeholders to respond to the needs in the field by developing both documentary and physical reference standards to help harmonize and standardize methods and practices. This presentation will summarize existing documentary standards that apply to gene therapies and provide an update on new standards under development to the support quality of raw materials like plasmid DNA and the manufacturing and testing of AAV-based gene therapies. Development of reference standards to support analytical testing of critical quality attributes will also be discussed.

Demonstrating Analytical Comparability in Gene Therapy:

Biophysical characterizations of AAV vectors are necessary for understanding the quality of purified AAV vectors. I will introduce characterization methods that can be performed with small amount of AAV vectors and will contribute to the improvement of the manufacturing process.

Multi-wavelength analytical ultracentrifugation offers two highly precise and orthogonal characterization methods in one experiment to identify the ratio of any loading state of AAVs, ranging between empty capsids to overfilled capsids. Furthermore, our method can detect contaminants such as protein aggregates and free nucleic acids. The method can be performed in sedimentation velocity mode, providing ultimate resolution and contaminant identification through an extended molar mass dynamic range, while obtaining highly accurate molar ratios for protein and DNA, present in each analyte. The second method is an analytical buoyant density equilibrium (ABDE) experiment, which offers higher throughput and sensitivity, and much lower sample requirements. For both methods, multi-wavelength decomposition of the hydrodynamic dimension additionally provides an orthogonal characterization dimension, further improving the resolution of this approach. In this talk, I will present examples, and discuss the experimental approaches used to characterize viral vectors and their nucleic acid cargo load by multi-wavelength analytical ultracentrifugation.

We are performing extensive MS method development to characterize critical quality attributes in gene and cell therapy products, not only proteins but lipids and oligonucleotides as well. Utilizing new MS technologies for protein analysis, we are gaining a better understanding of post-translational modifications on gene and cell therapy products as well as investigating critical structure/function relationships.

Process development for adeno-associated virus (AAV) manufacturing faces unique challenges given the complex nature of the molecule. Biomarin employs multiple biochemical and biophysical techniques to overcome these challenges in order to optimize process development efforts with the ultimate goal of innovating in the field of AAV production for the benefit of our patients.

Many challenges faced by CGT products impact the strategy taken to design, characterize, and validate manufacturing processes. Specifically, the complexity of these modalities coupled with scarcity of material for use in knowledge building studies makes it hard to reduce studies needed for launch. Current regulatory requirements such as justifying scale-down models and/or surrogates used pose additional challenges. This presentation will explore such challenges and approaches used in CGT process validation. A few case studies will be presented.

Challenges of AAV downstream processing – including high levels of product variants and complex characterization – are exacerbated by pressures for early implementation of process innovations. We show high-throughput case studies for early assessment of platform fit, process optimization, and process sensitivity. Together these generate optimization and risk assessments early in development, reducing risks of process changes in large-scale manufacturing.

Inspired by the intrinsic nature of microRNA-mediated mRNA cleavage, we developed a microRNA-targeting mRNA as a switch platform called mRNA bridge mimetics to regulate translocation of proteins. Combinatorial treatment with cisplatin and miR-21-EZH2 axis-targeting CRISPR Self Check-In improved sensitivity to chemotherapeutic drugs. Using the endogenous mRNA decay mechanism, our platform is able to remodel a cell's natural biology to allow the entry of precise drugs into the nucleus, devoid of non-specific translocation. This strategy is promising for applications in which the reaction must be controlled via intracellular stimuli and modulates Cas9 proteins to ensure safe genome modification in diseased conditions.

We are building a high-performance computing-powered bioengineering platform to mine, design, model, and test novel viral biologics. Better viral delivery methods will expand the available biologic tool kit to produce better medicines for intractable and incurable diseases. A new software platform specifically tailored to mine, design, model, and test the human virome (and others) for new therapeutic modalities will enable exploration of this therapeutic space in a safe, rapid, and cost-effective manner.