CombiGene is developing a Gene Therapy (GT) for the treatment of drug-resistant focal epilepsy. The lead candidate drug CG01 uses an adeno-associated virus (AAV) to administer a combination of neuropeptide Y (NPY) and its receptor Y2 genes directly to the part of the brain where the epileptic attack starts. In this presentation I will discuss the CMC aspects for a GT, some pre-clinical data and assay developments will be shared.

Manufacturing processes for gene therapy products are generally low yielding compared to production processes for other biologics. The material requirements to support analytical, comparability, and stability activities at all stages of development are likely to exceed the requirements for clinical dosing. This talk will highlight some of the key material requirements at different stages of development and discuss options for maximizing use of available material.

Adenovirus-based COVID-19 vaccines will pave the way for the approval of other biological products, such as adenovirus-vectored oncolytic viruses. Even though there is common ground in manufacture between adenovirus-based products and other virus-based products, there are some critical CMC challenges about oncolytic viruses that encompass process development, product scale-up, and analytical methods.

Transmission electron microscopy (TEM) image analysis is used to identify structural parameters correlated to important biological functions for quality control, efficacy and safety. We will present how GMP-certified TEM analyses can be highly valuable in the development of gene therapy products and what makes cryoTEM methods unique for AAV full/empty/intermediate characterization.

Immunoassays are commonly used for viral vector titer and impurity analysis during bioprocess development, drawbacks in plate-based methods prevent their full implementation. Immunoassay data for AAV multi-serotype and lentiviral vector titer and process-related impurities analysis, using Gyrolab® microfluidic immunoassay platform demonstrate significant improvements in analysis speed, dynamic range, and sample volume consumption. Data supports the implementation of Gyrolab technology in quality control and during bioprocess development of cell and gene therapies.

As a CDMO specialized in rAAV vector production, in Viralgen we are continuously improving the quality control processes to ensure the best product quality. The Gyrolab platform and associated AAVx titer and HEK293 HCP detection kits have allowed us to improve the quality of our analytical results within a standardized process, obtaining accurate data in less time and reducing the volume of reagents and samples required for each test.

Gene therapies based on rAAV are a rapidly developing therapeutic area in the pharmaceutical industry. With this rapid development, it is therefore necessary to cover a broad analytical portfolio. In this context, electrophoresis methods have continued to evolve, and capillary electrophoresis (CE) has established itself as a high-throughput method for process monitoring. With CE methods, both AAV and pDNA production can be easily monitored. In this talk, an overview of implemented CE methods will be given and compared with established methods such as gel electrophoresis, UPLC, ELISA, and AUC.

Sensorion is a biotech company dedicated to the development of therapies for genetic forms of hearing loss. Two novel gene therapy programs include deafness due to Otoferlin deficiency as well as Usher Syndrome type 1. Since the Otoferlin gene is large and exceeds the AAV packaging capacity, two AAV vectors have been developed. The product characterization of the dual vectors will be presented.

The Aura GT, with its ability to count, size, and ID particles, is a powerful tool for the assessment of AAV stability and aggregation. Combining proprietary Backgrounded Membrane Imaging with Fluorescent Membrane Microscopy provides further insight into particle identification including the presence of protein and DNA aggregates. Built around USP788 guidelines, Aura GT provides the tools necessary to quantitate particles in the subvisible range in a high throughput, low volume assay.

Recombinant adeno-associated virus (AAV) are critical components of gene and cell therapies, which show incredible promise for the treatment of disease. Accordingly, the need for large-scale manufacture of safe and effective viral vectors has never been greater.

The improvement of upstream processes for the delivery of high amounts of AAV Vectors is achieved through the smooth interplay of high throughput screening in microbioreactors combined with highly automated high throughput testing with minimal sample amounts. Short turnaround times enable rapid investigations of different parameters in the upstream processes and the impact on the viral vector yield. The presented case studies show the variation of different upstream conditions resulting in optimized upstream process layout and the verification of the findings in lab scale.

Agile and robust manufacturing processes are essential to the fast-paced gene therapy and biotechnology industry. To support the progress of pipeline candidates and deliver different drug product strengths for clinical studies, manufacturing platforms and their delivery strategies need to be flexible to prevent delays to clinical studies. A robust and flexible manufacturing strategy enables early phase clinical studies to be fast-tracked and further product specific process optimisation can be later in the development cycle. Data will be presented that demonstrates comparable process performance across different transgenes and serotypes.

Oxford Biomedica (OXB) is a pioneer in developing products based on viral vectors. To meet the forecast vector demand for gene and cell therapies, OXB has evolved strategies to develop the next generation manufacturing processes to yield higher vector quantities, suitable product quality attributes and acceptable cost of goods in order to advance development of a diverse product portfolio including Lenti, Adeno and AAV products which currently present significant challenges.

The presentation focuses on cell line generation from two different packaging cells (GRPG and GPRTG) for lentivirus production with focus on cell line development and selection strategy for clone screening. We have compared GPRG versus GPRTG packaging cell lines using stable transfection of concatemer encoding a selectable marker and the gene of interest. The presentation also covers process optimization strategies for high virus titer yield with adherent bioreactors (perfusion process).

Manufacturing AAV gene therapies presents interesting challenges for process development. In uniQure, we are working towards developing a new producer cell line with the following characteristics: i) able to grow in suspension culture, ii) able to grow without FBS, iii) with AAV production genes stably integrated in the genome, iv) robust and able to growth at relevant process conditions. This new cell line will be the backbone of our new processes that will be easier to scale up, robust, and able to hit the market in shorter timelines.

VIVEbiotech is a CDMO fully specialized in the development and manufacturing of lentiviral vectors. VIVEbiotech operates according to the regulations of both the FDA and the EMA manufacturing lentivectors that are distributed worldwide. VIVEbiotech has a strong USP department which focuses its activity on improving the relevant upstream steps of the production process. Much of their activity is focused on the fine-tuning of the transient transfection process using PEIpro from Polyplus.

Reliable and robust manufacturing improvements are needed to meet the full potential of gene therapy demand.  Two case studies for AAV and Lentivirus illustrate the benefits of intensification and simplification of production and clarification processes for optimal and cost-effective viral vector manufacturing.

KrosFlo TFDF filtration technology was found to increase total yield of adeno-associated virus serotype 8 (AAV8) and lentivirus by 3-fold and 2-fold, respectively, compared to a batch bioreactor process.

Gene therapies offer exciting avenues as curative therapies for unmet patient needs. Often these therapies are expected to advance rapidly through clinical stages. Moving from lab to clinic in a timely, predictable way requires companies to develop robust, scalable, and reproducible processes. This talk will discuss how leveraging a manufacturing platform can expedite the critical path from clinical to commercialization, focusing on FUJIFILM Diosynth’s AAV platform and supporting phase-appropriate analytical methods.