Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno‐associated virus production by fluorescence spectroscopy

Abstract

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real‐time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell‐baculovirus expression vector system (IC‐BEVS), a well‐established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real‐time monitoring tool for recombinant adeno‐associated virus (rAAV) production in the IC‐BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm‐based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave‐one‐batch‐out cross‐validation. Our approach shows fluorescence spectroscopy allows real‐time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC‐BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.

Holographic Imaging of Insect Cell Cultures: Online Non-Invasive Monitoring of Adeno-Associated Virus Production and Cell Concentration

“…Recombinant baculovirus was produced using the Bac-to-Bac ® Baculovirus Expression System (Invitrogen), according to the manufacturer's instructions. Baculovirus amplification was performed as described elsewhere [9].…”

“…Most cell culture monitoring methods employing label-free methodologies are based on spectroscopic techniques, which have been widely used for cell culture process monitoring. Examples include the use of dielectric spectroscopy and turbidimetry/light scattering probes for the determination of cell concentration [4,5], as well as the use of Raman [6,7], infrared [8] and fluorescence [9] spectroscopy, which allow the quantification of metabolites based on direct spectra quantification, but also the indirect determination of cell concentration and product formation based on chemometric analysis.…”

“…Our group and others have addressed ways to monitor this system using fluorescence [9] or dielectric [3,20,25,26] spectroscopies, as well as using image-based technologies, in particular for measuring the progress of baculovirus infection [27][28][29]. DHM can go one step further, by monitoring not only the cell diameter increase after baculovirus infection, but also the evolution profile of several cell characteristics, allowing to explore the possibility to observe baculovirus or AAV-induced changes in suspension insect cells in real-time.…”

History and Evolution of Modeling in Biotechnology: Modeling & Simulation, Application and Hardware Performance

“…They computed biomass and substrate concentrations for a continuous stirred tank reactor and described the neural network as “accurate with a certain degree of noise immunity” with the advantage that no a priori knowledge on the interrelations of important variables is required like in other modeling approaches [46] . ANNs have been used in the recent years for monitoring bioprocesses in combination with UV/Vis or fluorescence spectroscopy [56] , [57] . Furthermore, ANNs have been employed for various monitoring purposes in combination with an electronic nose as extensively reviewed by Hu et al [58] .…”

Fiberoptic-Coupled Spectrofluorometer with Array Detection as a Process Analytical Chemistry Tool for Continuous Flow Monitoring of Fluoroquinolone Antibiotics

“…PAT has many applications in the pharmaceutical and antibiotics manufacturing [12][13][14][15][16][17][18][19][20][21][22][23][24][25], chemical [26,27], petrochemical [28], and food industries [29][30][31][32][33]. In addition, there are many recent progress in realtime monitoring of cultivations in bioreactors and cell culture process [34][35][36][37][38][39][40][41], fermentation [42,43] and biological process [44], and electrochemical [45,46] and protein purification [47]. Using PAT enables us to get a deeper understanding of the process.…”