The success of gene therapy owes greatly to the delivery vehicles used, such as adeno-associated viruses (AAV) and lipid nanoparticles (LNP). Quantifying quality attributes of gene vectors is important, as gene therapy products enter into the late development stages/QC environment. In this presentation, we demonstrate the use of three analytical techniques: batch dynamic light scattering (DLS), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and field-flow fractionation (FFF) with MALS.

Our cGMP production suites and single-use systems will allow the production of multiple products simultaneously at up to 3,000-liter bioreactor scale. We are developing reliable and robust production systems that can deliver higher amounts of AAV product per batch.

Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications and furthered interest in optimizing  manufacturing platforms. In this study, we describe our optimized protocol to produce rAAV9 by PEI-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the Herpes Simplex Virus (HSV). Using a newly developed purification protocol compatible with both upstream platforms, we generated rAAV9 stocks of high purity and assessed yields, quality and residual impurities. We found notable qualitative and quantitative differences between PEI- or HSV-mediated production, which will be presented.

Scalable production of AAV gene therapies remains challenging with respect to yield and removal of “empty” capsids that do not contain the therapeutic DNA payload. To begin to address these challenges an end to end AAV production process has been developed that enables a complete AAV purification solution. Here we show our process development steps and how to improve yield, with a particular emphasis on the depletion of empty capsids

There remains a significant need to improve adeno-associated virus (AAV) manufacturing platforms to achieve robust, high-yielding, scalable and cost-efficient processes. At Ultragenyx, we employ a HeLa producer cell line, helper-virus based infection process for AAV production which is reproducibly scalable to 2000L. To further improve on our batch process platform, we have developed an intensified process using perfusion for AAV production to increase cell density to achieve high volumetric AAV yield. The intensified upstream process along with an optimized downstream process, increases AAV productivity to =3E11 GC/mL , enabling delivery of a high purity AAV product suitable for clinical use.

Through the delivery of recombinant adeno-associated virus (rAAV) vectors, gene therapy has the potential to cure and/or treat many genetic disorders. To lower manufacturing costs and enable the treatment of larger populations, it is critical to improve process yield. In this work, we describe the approach taken to develop, optimize, and scale-up triple transfection bioreactor process to improve per-batch productivity.

O-Versatile™ platform enables flexible and high-quality process development and manufacturing solutions for a variety of gene and cell therapy products. The platform facilitates a high-titer, large-scale production capability and provides versatile development possibilities for various demands. The presentation will highlight research and process development of viral vector-based products with the application of novel manufacturing technologies.

We describe here a well-established platform process for LV production based on transient transfection of serum-free cells grown in suspension. Both upstream and downstream processes are highly optimized to achieve optimal vector yields and significant decrease in the impurities (host cell protein/DNA, plasmid DNA). The compatibility of this platform process has been evaluated with multiple CAR/TCR genes while the robustness is demonstrated in reproducible runs at pilot scale.

Umoja aims to transform cancer care by creating an off-the-shelf, direct injection lentiviral vector (LVV) drug product for in vivo CAR T cell generation and expansion. We will present our approach to reproducible and scalable manufacturing of high-quality LVV, with special focus on expediting process development with risk-based quality documentation and impurity clearance at key process unit operations to meet ambitious final specifications.

To streamline lentiviral vector (LV) manufacturing, we propose to use packaging cell lines in suspension cultures. The cells were designed using molecular switches to control the production of LV cytotoxic proteins. They can be used to generate LV through a one-plasmid transfection corresponding to the gene of interest, or to generate stable producer cell lines. Given the labile nature of the vector, we are evaluating the advantages of producing in perfusion mode, and the possibility of purifying in a semi-continuous way.

The traditional tools used for protein-based therapeutics often come short in case of viruses. This is particularly the case when the virus is to be used for oncolytic purposes rather than as a vaccine wherein the administered dosages are several-fold lower. Another major gap with viral vectors is in our knowledge of the biology and morphology of the therapeutic. The work presented will focus on our efforts in development of novel chromatographic purification techniques complimented with extensive characterization of our virus using a suite of analytics.

Lentiviruses have been widely used for cell therapy based applications. This presentation will showcase IDT Biologika’s platform based approach for Lentiviral Vector production. The strategies described here will enable a scalable and robust process that focuses on suspension transient transfection of serum free HEK cells.

There are a number of challenges drug developers face on the journey to the clinic, including high costs and competitive timelines. FDB has solutions that help our partners navigate the challenges of development and material supply for clinical trials as well as propel their life changing medicines to commercialization. Our solutions include a flexible AAV platform and a wealth of regulatory experience taking medicines to the market for our partners.

A platform-based approach for AAV purification enables rapid scale-up of high-quality vector, into manufacturing and the clinic. This presentation will showcase how Oxford Biomedica Solutions’ AAV platform was created by developing a deep understanding of chromatographic behavior, understanding the impact construct design and bioreactor performance have on purification, and focusing on process robustness throughout the development life cycle.

Recombinant adeno-associated virus (rAAV) has been broadly used as a vector for gene therapy applications. By combining the TECAN platform and light-scattering analytical methods, we demonstrated an integrated workflow to process clarified bioreactor material through affinity chromatography in a high-throughput manner, which greatly facilitated upstream process development, such as lead clones and production conditions screening. This methodology not only reduces material requirements and processing time, but also provides readouts of product quality attributes that enable rapid and efficient process development of rAAV products with various serotypes.

In recent years, significant resources have been invested into increasing the productivity of AAV manufacturing. Optimization of upstream processes has led to significant increases in AAV titer, and downstream purification strategies designed around lower-yielding production must be reevaluated to better accommodate the large increases in vector. This presentation highlights work done to modify downstream purification steps to handle a titer increase seen during the implementation of a next-generation upstream process.

Variable empty/full distribution coming out of the upstream unit ops coupled with the fact that empty particles co-purify with full particles remain a challenge in rAAV manufacturing. Modulating the mobile phase composition during the AEX load enables the flow-through or “partitioning” of empty capsids while selectively retaining full rAAV vectors. Minimizing empty capsid binding simplifies the elution strategy which can be a significant benefit during tech transfer and scale-up.

This talk will discuss transformative technologies in the production, filtration, and chromatography-based purification of AAV with real-life examples of the challenges that have been overcome. Some real-life examples will include the development of depth filtration for suspension AAV production, which increased capacity and recovery by 4x and 2x respectively, and a new strategy for ensuring viral clearance that resulted in >85% recovery.