Document Detail
Functional Characterisation of
the Cellular Proteome
Abstract/OtherAbstract :
Engineered mammalian (CHO, NS0 and SP2/0) cells are commonly used for large-scale
production of recombinant therapeutic antibodies. Increased antibody production in these
commercial manufacturing systems is often achieved through systematic empirical optimisation
of culture environment and improved expression vectors. To date, our a priori
knowledge of the molecular mechanisms of antibody manufacture in these expression systems
is very limited. As a result, genetic engineering of mammalian host cells to improve
product yields have often produced ‘mixed’ results, further indicating the need for a more
exploratory approach to understanding glycoprotein production.
Identification of coordinated changes specifically related to one particular phenotype
(i.e. cell-specific productivity; qP) at a clonal level will improve our understanding of the
rate limiting steps or intracellular processes related to that phenotype. Using quantitative
two-dimensional polyacrylamide gel electrophoresis, differences in the host cell proteome
were assessed between murine myeloma NS0 cells producing a recombinant monoclonal
antibody at varying cell-specific production rates. Furthermore the variation in synthesis
and abundance of these proteins were evaluated during fed-batch culture.
Conserved and coordinated changes in specific proteins known to interact with antibody
folding and assembly were identified, as were changes in the intracellular abundance
of recombinant heavy and light chain polypeptides. Conserved changes in the abundance
of proteins as functional categories (chaperones, metabolic and mitochondrial, cytoskeletal
and cell signaling) also demonstrated correlation with enhanced cell-specific productivity
between clonal cell lines. However, variation in the synthesis and abundance of these proteins (individually and in categories) during a fed-batch process did not demonstrate
the same coordinated changes with qP observed between clonal cell lines.
In conclusion these data suggest that cell performance in production processes is de-
fined primarily by the basal phenotype selected after transfection. Bioprocess manipulations
modulate management of host cellular processes altering metabolism and recombinant
gene transcription or translation, but only within the confines of the host cell
machinery selected post-transfection. Pre-screening of cells prior to selection for coordinated
changes associated with improved qP will reduce the variation in transfected cell
phenotypes, reducing the time to select cells with good performance characteristics.
Authors :
Stansfield, Scott Hunton
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Contributors :
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Publication Detail :
Publisher :  University of Queensland. School of Engineering     Type :  -     Format :  -    
Date Detail :
2006
Subject :
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Coverage :
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Relation :
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Source :
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Copyright Information :
http://www.library.uq.edu.au/disclaimer.html), Copyright Scott Hunton Stansfield
Other Details :
Languages :  en    
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