Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq.

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a "high" and "low" clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33-39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.

Clonal variation in productivity and proteolytic clipping of an Fc-fusion protein in CHO cells: Proteomic analysis suggests a role for defective protein folding and the UPR.

Product degradation, such as clipping, is a common quality issue in the production of Fc-fusion proteins from Chinese hamster ovary (CHO) cells. Degradation of proteins is mainly due to the action of either intracellular or extracellular host cell proteases. This study was carried out to understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of Fc-fusion protein clipping. The cell lines that displayed the highest levels of clipping also produced less product than the cell lines with a lower level of clipping. In this study we applied differential quantitative label-free LC-MS/MS proteomic analysis to group clonally-derived cell lines (CDCLs) based on the level of clipping of the Fc-fusion protein. The analysis was carried out over two times points in culture and clones were designated as either having 'high' or 'low' clipping phenotypes. We have identified 200 differentially expressed proteins using quantitative label-free LC-MS/MS analysis between the two experimental groups. Functional assessment of the resultant proteomic data using Gene Ontology analysis showed a significant enrichment of biological processes and molecular functions related to protein folding, response to unfolded protein and protein translation. The levels of several proteases were also increased. This study identified protein targets that could be modified using cell line engineering approaches to improve the quality of recombinant Fc-fusion protein production in the biopharmaceutical industry.

Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.

The control of helminth diseases of people and livestock continues to rely on the widespread use of anti-helminthic drugs. However, concerns with the appearance of drug resistant parasites and the presence of pesticide residues in food and the environment, has given further incentive to the goal of discovering molecular vaccines against these pathogens. The exponential rate at which gene and protein sequence information is accruing for many helminth parasites requires new methods for the assimilation and analysis of the data and for the identification of molecules capable of inducing immunological protection. Some promising vaccine candidates have been discovered, in particular cathepsin L proteases from Fasciola hepatica, aminopeptidases from Haemonchus contortus, and aspartic proteases from schistosomes and hookworms, all of which are secreted into the host tissues or into the parasite intestine where they play important roles in host-parasite interactions. Since secreted proteins, in general, are exposed to the immune system of the host they represent obvious candidates at which vaccines could be targeted. Therefore, in this article, we consider the potential values and uses of algorithms for characterising cDNAs amongst the collated helminth genomic information that encode secreted proteins, and methods for their selective isolation and cloning. We also review the variety of prokaryotic and eukaryotic cell expression systems that have been employed for the production and downstream purification of recombinant proteins in functionally active form, and provide an overview of the parameters that must be considered if these recombinant proteins are to be commercialised as vaccine therapeutics in humans and/or animals.

High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler.

A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.