Degree Type

Dissertation

Date of Award

2001

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

First Advisor

Carole Heath

Second Advisor

Marit Nilsen-Hamilton

Abstract

Hybridoma cells are widely used to produce large quantities of monoclonal antibodies for use as medicines and diagnostics. Bcl-2 is an oncogene that has been shown to have an anti-apoptotic effect in many cell lines including hybridomas. This study investigates the effect of various levels of bcl-2 expression on hybridoma cell grown and antibody productivity in both batch and perfusion culture.;H166 hybridomas were transfected with a plasmid containing a bcl-2 insert and stable clones were selected. The cells were grown in spinner flasks in batch mode for twelve days and it was confirmed that expression at high levels allow for significant protection from apoptosis. Protection from apoptosis first becomes evident at 2--5 units/cell of BCL-2 protein content, where one unit of expression is the amount of BCL-2 content per HL-60 cell taken at 48 hr of batch culture. Further protection from apoptosis is obtained as the level of bcl-2 expression increases to 27 units. Although there is a significant increase in the viability during the decline phase of cell culture, there is no improvement in the antibody productivity. The antibody concentration levels off after 7 days of culture. Bcl-2 expression had no effect on nutrient consumption or cell cycle progression.;Growth of the cells in long-term perfusion culture did not result in an increase in viable cell density, viability or antibody productivity. At low cell bleed rates the vector only control grew better than any of the clones tested. At high cell bleed rates, where cell death due to apoptosis was expected to be low, there was an increase in cell density for the highest expressing clone. This was not due to prevention of apoptosis and was likely due to culture variations. The extent of apoptosis remained low in all the cultures studied. These results suggest that induction of bcl-2 expression is not an effective method to increase the antibody productivity of H166 hybridoma cells in batch or perfusion culture.

DOI

https://doi.org/10.31274/rtd-180813-11019

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Mark Christopher Mowry

Language

en

Proquest ID

AAI3034208

File Format

application/pdf

File Size

176 pages

Share

COinS