No abstract

The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high‐density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high‐density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte‐colony inhibition factor. The protein yield on a cell basis from infecting high‐density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high‐density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high‐density perfusion culture for large‐scale protein expression from Sf9 cells are also highlighted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:351–359, 1998.

A large number of microcarriers are commercially available. The capability of cells to successfully proliferate on microcarriers varies with cell lines and media. Choosing the right microcarrier for a particular cell line is more than a choice of a microcarrier. It is part of an integrated process design. A detailed picture of cell growth and product formation will not only be essential in identifying the kind of microcarrier, but also in determining other parts of the process, such as operation mode and media. Our initial screening on thirteen microcarriers showed that cultures on some microcarriers reached a low cell density but high cell-specific productivity, and high density microcarrier cultures have a low specific productivity. The result is a similar product output per unit volume and time for these two types of cultures. An ideal culture system shall have increased volumetric productivity at elevated cell density. This requires the process goal to be incorporated as early as cell line construction and screening. A high output process can then be realized through high density culture.

A recombinant Chinese hamster ovary clone was cultivated in a 2L Cytopilot Mini fluidized bed bioreactor using Cytoline 1 microcarriers and a 10L B. Braun stirred tank bioreactor with Cytodex 1 microcarriers. Cytoline 1 is a macroporous polyethylene microcarrier and Cytodex 1 is a solid DEAE-dextran microcarrier. Cytoline 1 microcarriers in the fluidized bed bioreactor were gently mixed by an uplifting flow. Circulation and sparging in Cytopilot Mini were separated from the fluidized microcarrier bed. Cytopilot Mini bioreactor with Cytoline 1 microcarriers offered 2.3 times more surface area than the stirred tank bioreactor. The 2L fluidized bed bioreactor accommodated approximately half the cells in the 10L stirred tank bioreactor. Moreover, Cytopilot Mini had approximately three times more product output rate and 5.5 times higher specific productivity than the stirred tank bioreactor.