Heterologous protein production using euchromatin-containing expression vectors in mammalian cells.

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Expression of functionally active sialylated human erythropoietin in plants.

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.

Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) ß1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

SNEV overexpression extends the life span of human endothelial cells.

In a recent screening for genes down regulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.