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Abstract Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno‐associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV‐producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV‐mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state‐of‐the‐art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV. 

Abstract A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high‐producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three‐level factorial experimental design was performed in fed‐batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter‐influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late‐stage ROS activity in a dose‐dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity. 

Abstract Recombinant adeno‐associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost‐effective, well‐characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two‐dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid‐high setpoints, and PEI:DNA ratio and total DNA/cell were at low‐mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.