Reduction in C-terminal amidated species of recombinant monoclonal antibodies by genetic modification of CHO cells.

BACKGROUND: During development of recombinant monoclonal antibodies in Chinese hamster ovary (CHO) cells, C-terminal amidated species are observed. C-terminal amidation is catalysed by peptidylglycine α-amidating monooxygenase (PAM), an enzyme known to be expressed in CHO cells. The significant variations between clones during clone selection, and the relatively high content of amidated species (up to 15%) in comparison to reference material (4%), led us to develop a cell line with reduced production of C-terminal amidated monoclonal antibodies using genetic manipulation. RESULTS: Initial target validation was performed using the RNA interference approach against PAM, which resulted in a CHO cell line with C-terminal amidation decreased to 3%. Due to the transient effects of small-interfering RNAs, and possible stability problems using short-hairpin RNAs, we knocked-down the PAM gene using zinc finger nucleases. Plasmid DNA and mRNA for zinc finger nucleases were used to generate a PAM knock-out, which resulted in two CHO cell lines with C-terminal amidation decreased to 6%, in CHO Der2 and CHO Der3 cells. CONCLUSION: Two genetically modified cell lines were generated using a zinc finger nuclease approach to decrease C-terminal amidation on recombinant monoclonal antibodies. These two cell lines now represent a pool from which the candidate clone with the highest comparability to the reference molecule can be selected, for production of high-quality and safe therapeutics.

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

BACKGROUND: Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. RESULTS: To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95 degrees C for 10 minutes prior to storage at -20 degrees C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25 degrees C and the correlation between PCN and protein accumulation was established. CONCLUSION: Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.