Umana, Pablo (1998) Engineering of protein glycosylation in Chinese hamster ovary cells. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11162005-152226
Improved versions of therapeutic glycoproteins may be produced by manipulation of their glycosylation patterns. Engineering of glycoform biosynthesis in CHO cells is studied here as a route to produce new variants of a recombinant, therapeutic protein and to maximize the proportion of beneficial glycoforms within the glycoform population. An anti-neuroblastoma monoclonal antibody (chCE7) was used as a model therapeutic glycoprotein, and the target glycoforms were those carrying bi-antennary complex Nlinked oligosaccharides modified with a bisecting N-acetylglucosamine (GlcNAc).
A mathematical model of N-linked glycoform biosynthesis was constructed and used to simulate the qualitative effects of overexpression of GlcNAc-transferase III (GnTIII), the enzyme catalyzing the transfer of a bisecting GlcNAc to various oligosaccharide substrates. These simulations indicated a maximum level for bisected complex oligosaccharides, and accumulation of hybrid bisected oligosaccharides.
To investigate the effects of GnTIII overexpression experimentally, a CHO cell line with tetracycline-regulated overexpression of a rat GnTIII cDNA was established. Expressed GnTIII was localized in the Goigi complex of CHO cells by means of immunoelectron microscopy using an antibody against a peptide epitope tag added to the carboxy-terminus of the enzyme. The enzyme concentrated on one side of the Golgi, mainly in cisternal compartments.
Using the experimental system, it was found that overexpression of GnTIII to high levels led to growth inhibition and was toxic to the cells. Another CHO cell line with tetracycline-regulated overexpression of GnTV, a distinct glycosyltransferase, showed the same inhibitory effect, indicating that this may be a general feature of glycosyltransferase overexpression. The growth effect set an upper limit to glycosyltransferase overexpression and may therefore also limit the extent to which poorly accessible glycosylation sites can be modified by engineering of glycosylation pathways.
A set of chCE7 mAb samples differing in their glycoform distributions was produced by controlling GnTIII expression in a range between basal and toxic levels. Measurement of the ADCC activity of these samples showed an optimal range of GnTIII expression for maximal chCE7 in vitro biological activity. The activity correlated with the level of Fc-associated bisected, complex oligosaccharides. Expression of GnTIII within the biotechnologically practical range, i.e., where no significant growth inhibition and toxicity are observed, led to a consumption of more than 90% of non-bisected, non-galactosylated bi-antennary complex oligosaccharides, but, at most, 50% was converted to the target bisected, complex structures for this set of chCE7 samples. The pattern of oligosaccharide peaks in MALDUTOF-mass spectrometric analysis of samples produced at high levels of GnTIII, indicate that a significant proportion of potential GnTIII substrates is diverted to bisected hybrid oligosaccharide by-products. Minimization of these by-products by further engineering of the pathway could therefore be valuable.
The new optimized variants of chCE7 are promising candidate reagents for the treatment of neuroblastoma. The strategy presented here may also be applicable to other therapeutic IgGs.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||6 April 1998|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||17 Nov 2005|
|Last Modified:||26 Dec 2012 03:10|
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