Abstract
BackgroundThe most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations.FindingsIn this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter.ConclusionThis study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.
Highlights
The most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase
This study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells
The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene that results from modification of the integrated vector or its vicinity, such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation [1,2,3]
Summary
The 1.5 Kb human RNP CpG fragment has previously been shown to confer higher expression and stability of the transgene in production cells when placed ahead of the hCMV promoter. We observed that in combination with the different promoters, the corresponding 1.5 Kb UCOE is better than the 3.2 Kb UCOE at expressing FVIII-BDD in both attached and serum free suspension adapted BHK21 cells. In the case of adherent cells, there was a substantial loss of expression from the mCMV and the SRa promoters when compared to the gCMV promoter and the Ubc promoter based expression seemed to increase relative to the gCMV promoter These changes were not due to large differences in copy numbers. Suspension adapted cells grown for a month showed differences in gene expression levels, but these may be a result of differences in gene copy numbers. Optimizing conditions for transfection in suspension adapted serum free BHK21 cells can be achieved and is beneficial in saving time and resources, and in the purification of the biologic with fewer protein contaminants resulting in reduced immunogenicity
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