Combination of the 2A/furin technology with an animal component free cell line development platform process

Abstract

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.

Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma

“…Transfection of CHO K1PD cells and selection/amplification with G418 and methotrexate were performed as previously described (Jostock et al, 2010).…”

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

“…21,28 Productivities of F2A vector derived clones have been shown to be comparable to those generated using an industry reference vector designed to use separate expression units for each gene. 29 Studies had also shown that using F2A gave over 2-fold higher mAb expression levels than IRES in transient transfections and 20% higher expression in stable transfection. 18,21 However, incomplete cleavage of F2A was observed in some studies, resulting in HC-F2A-LC or LC-F2A-HC fusion proteins and LC and HC attached with F2A remnants.…”

“…26 F2A from the foot-andmouth disease virus, which is the most studied 2A, has been used for mAb expression in mammalian cells and for in vivo gene therapy. 18,21,[27][28][29][30][31] 2A peptides have approximately 20 amino acids and "self-cleavage" occurs between the last 2 amino acids, glycine (G) and proline (P). Adding a furin recognition sequence between the first gene and 2A aids in removing 2A residues from the upstream gene.…”

Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells

“…21 Using combination of furin recognition site and 2A-peptide strategy of expression has shown stable co-expression of transgene in different cell lines. 22 Although the 2A system is appropriate for delivering co-expression of particular sets of transgenes, it is not suited in situations where individual transgenes require a different tissue distribution. 23 Our studies of immunogenic properties of obtained adenoviral construction demonstrated low immunogenicity of 2A peptide sequences when utilizing furin cleavage.…”