In this presentation, development of a primary clarification protocol for separating lentiviral vectors (LV) from bioreactor-derived suspension cells using a scalable filter train will be discussed. Recent results demonstrating clearance of producer cells and debris from LVV with minimal to no loss of infectious titre will be shared.

There is an increasing requirement for generation of high quantity of potent rAAV vectors based on various serotypes. Baculoviruses and insect cells have a number of features that make them ideal for development of a robust system that can generate high quantities of rAAV. I will present our experience with baculoviruses/insect cell system in stirred tank bioreactor. Furthermore, I will discuss a comparison between the wave and STR bioreactors for rAAV production with BEVS and discuss the vector quality and potency generated by insect cell production.

At Ultragenyx Pharmaceutical, we have developed two high yielding suspension-based platforms that enable us to consistently approach our target of 10^14 vg/L. Our most significant advances have been made through cell substrate subcloning, parameter optimization, and media and feed development. Through standardizing our process development workflows and an iterative process improvement strategy, we have enabled rapid CMC development to meet the high dosing requirements of our rare disease programs.

Transiently transfected, suspension-based HEK293 cells is one of the most common platforms for producing recombinant AAV products, although scale-up challenges have been identified. The correlation between upstream process parameters, cell aggregation, and productivity were assessed through a series of lab-scale and pilot scale experiments. A novel method of quantifying cell aggregation rate was introduced to determine impact on process performance. Moreover, several offline/at-line analytical tools were also utilized to the production stage, to characterize the transfection complex.

Efficient AAV production requires an understanding of the complex interaction between traditional cell culture operation and the impact of transient transfection driven vector production. With real-time quality attribute assessment provided by integrated PAT, coupled with an increased understanding of production processes as a result of traditional offline omics evaluation, we identify strategies to improve future production approaches. Increased resolution within the design space can ultimately drive both process parameter range selection whilst increasing robustness.

The success of gene therapies has increased the number of companies that are entering this arena and seeking advice on how they should produce their product candidates. FUJIFILM is developing a platform for AAV production. Our platform will contain 1) a suspension HEK293 cell line 2) GMP grade Adenovirus helper 3) GMP rep/cap plasmid representing several AAV serotypes 4) a GOI plasmid for insertion of genes and 5) tools for in-process and release testing.

Recombinant adeno-associated virus (rAAV) has emerged as a dominant gene-delivery vector of choice. Despite numerous advances in the clinic, the efficiency and cost of producing rAAV drug product to meet a rapidly growing industry has significant room for improvement. AskBio has developed a model for advancing multiple clinical programs, with an emphasis on establishing early benchmarks in manufacturing feasibility, while focusing on continuous process development and improvements to large-scale manufacturing.

We report bioreactor performance and usability, cell distribution, pH and dissolved oxygen (DO) vessel monitoring, and adeno-associated virus type 5 (AAV5) transient transfection efficiency. These tests demonstrated that the iCELLis® 500+ bioreactor is a well-controlled system and the improved bioreactor provided homogeneous cell distribution and AAV5 transfection.

Virus-like particles and other extra cellular particles are a next generation of biopharmaceuticals. They can be produced by a wide variety of host cells. The challenge is the production of high titers and downstream processing. The particle of interest is contaminated with other particles with similar biophysical properties and therefore difficult to separate. Examples will be given for 3 different cell types.

The purification of adeno-associated virus has become an increasingly important topic in the field of biomanufacturing as the prevalence of AAV gene therapies increases. One of the major hurdles facing AAV purification is the separation of empty capsids from full capsids. This presentation will address Precision BioSciences’ advances in downstream chromatography for both capture and empty full separation steps.