TY - JOUR
T1 - Holographic imaging of insect cell cultures
T2 - Online non-invasive monitoring of adeno-associated virus production and cell concentration
AU - Pais, Daniel A. M.
AU - Galrão, Paulo R. S.
AU - Kryzhanska, Anastasiya
AU - Barbau, Jérémie
AU - Isidro, Inês A.
AU - Alves, Paula M.
PY - 2020/4/1
Y1 - 2020/4/1
N2 - The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing product exposure to detrimental proteases. With this purpose, new bioprocess monitoring solutions are needed to accurately estimate culture progression. Herein, we used online digital holographic microscopy (DHM) to monitor bioreactor cultures of Sf9 insect cells. Batches of baculovirus-infected Sf9 cells producing recombinant adeno-associated virus (AAV) and non-infected cells were used to evaluate DHM prediction capabilities for viable cell concentration, culture viability and AAV titer. Over 30 cell-related optical attributes were quantified using DHM, followed by a forward stepwise regression to select the most significant (p < 0.05) parameters for each variable. We then applied multiple linear regression to obtain models which were able to predict culture variables with root mean squared errors (RMSE) of 7 x 105 cells/mL, 3% for cell viability and 2 x 103 AAV/cell for 3-fold cross-validation. Overall, this work shows that DHM can be implemented for online monitoring of Sf9 concentration and viability, also permitting to monitor product titer, namely AAV, or culture progression in lytic systems, making it a valuable tool to support the time of harvest decision and for the establishment of controlled feeding strategies.
AB - The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing product exposure to detrimental proteases. With this purpose, new bioprocess monitoring solutions are needed to accurately estimate culture progression. Herein, we used online digital holographic microscopy (DHM) to monitor bioreactor cultures of Sf9 insect cells. Batches of baculovirus-infected Sf9 cells producing recombinant adeno-associated virus (AAV) and non-infected cells were used to evaluate DHM prediction capabilities for viable cell concentration, culture viability and AAV titer. Over 30 cell-related optical attributes were quantified using DHM, followed by a forward stepwise regression to select the most significant (p < 0.05) parameters for each variable. We then applied multiple linear regression to obtain models which were able to predict culture variables with root mean squared errors (RMSE) of 7 x 105 cells/mL, 3% for cell viability and 2 x 103 AAV/cell for 3-fold cross-validation. Overall, this work shows that DHM can be implemented for online monitoring of Sf9 concentration and viability, also permitting to monitor product titer, namely AAV, or culture progression in lytic systems, making it a valuable tool to support the time of harvest decision and for the establishment of controlled feeding strategies.
KW - AAV-adeno-associated virus
KW - Cell culture monitoring
KW - Digital holographic microscopy
KW - Insect cell-baculovirus
KW - Process analytical technology
UR - http://www.scopus.com/inward/record.url?scp=85084639818&partnerID=8YFLogxK
U2 - 10.3390/PR8040487
DO - 10.3390/PR8040487
M3 - Article
AN - SCOPUS:85084639818
SN - 2227-9717
VL - 8
JO - Processes
JF - Processes
IS - 4
M1 - 487
ER -
