Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells

Abstract

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Process Improvement of Adeno-Associated Virus Production

“…In addition, calcium and lipofection have been used to deliver plasmids for adherent AAV production (Negrini et al, 2020). A developed dynamic mathematic model has indicated that the coordination between ssDNA replication and capsid synthesis is a key parameter to produce full capsid AAV (Nguyen et al, 2021). Despite these achievements, the low robustness of production process, high variation of titer and productivity and low biological activity of end product remain the major challenges in AAV production in research laboratory (Wright, 2008;Nguyen et al, 2021).…”

Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors

“…To this end, the innovation and optimization of transfection‐based AAV vector manufacturing remain an active area of research. Computational work modeling plasmid uptake, expression, and vector assembly kinetics in HEK‐293 have demonstrated utility in identifying molecular bottlenecks to improve vector yield and quality (Nguyen et al, 2021). Design of experiment approaches to optimize process parameters have also recently been shown to be effective in increasing AAV vector yields across a wide range of serotypes (Zhao et al, 2020).…”

“…coprecipitate heterogeneous plasmid mixtures into larger complexes for transit across the cell membrane, and so in theory, all successfully transfected cells should contain the genetic elements necessary to produce AAV (Cardarelli et al, 2016; Erbacher et al, 2004; Fus‐Kujawa et al, 2021). Reported transfection efficiencies (measured by the expression of a fluorescent marker) for well‐optimized processes range from 40% to 60% (Chahal et al, 2014; Nguyen et al, 2021). However, the degree to which transfection efficiency corresponds with the proportion of productive cells in the culture remains largely untested.…”

Mechanistic Modeling of Viral Particle Production

“…Scaling up and optimization of viral system manufacturing processes are currently active areas of research. Some of the challenges are generally applicable to most viral systems, e.g., inefficient downstream separation and purification [10], while others are system specific, e.g., low yields in recombinant AAV (rAAV) production due to inefficient filling of capsids with the desired genetic material [11]. These challenges have motivated significant research activity throughout the manufacturing and development pipeline e.g., engineering and optimizing cell lines [12,13], engineering the viral system itself to facilitate production [14,15], bioreactor process development and intensification [12,16], and improving the performance of downstream separation processes [10,17].…”

“…The use of modeling strategies in biotechnology is well-established (a cursory search of the literature will reveal a plethora of studies and reviews on the topic e.g., [18,19]) and has significantly contributed to the development of the field. Incorporating modeling into the process research and development workflow can contribute several benefits such as providing deeper process insights into the dynamics and multivariable interactions present in the system [11,20], enabling the deployment of advanced process control and monitoring strategies [20,21], enhance process optimization methodologies [22], and guide and accelerate the development of novel manufacturing strategies [11,23].…”