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

Abstract

(2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Abbreviations: CHO, Chinese hamster ovary; HC, heavy chain; LC, light chain; mAb, monoclonal antibody; F2A, 2A peptide derived from the foot-and-mouth disease virus; E2A, 2A peptide derived from the equine rhinitis virus; P2A, 2A peptide derived from the porcine teschovirus-1; T2A, 2A peptide derived from the Thosea asigna virus; GF2A, F2A with the GSG linker; GE2A, E2A with the GSG linker; GP2A, P2A with the GSG linker; GT2A, T2A with the GSG linker; IgG, immunoglobulin G; IRES, internal ribosome entry site; G, glycine; P, proline; K, lysine; MTX, methotrexate; SEC, size exclusion chromatography; MS, mass spectrometry; PFM, protein-free medium; HT, hypoxanthine and thymine; GFP, green fluorescence protein; PVDF, polyvinylidene difluorideLinking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

“…However, when we placed the LC gene upsreatm of the furin recognition site, we did not observe complete cleavage of furin. In agreement with our studies, several other investigations have also indicated that furin could not cleave efficiently when either LC or HC were arranged as a first cisteron [31, 44, 45]. Furthermore, different furin cleavage patterns were detected in the cell pools receiving either CHEF-F2A or CMV-F2A vectors.…”

“…Considering these finding, it appears that factors other than the arrangement of the light and heavy chains affect the hydrolysis activity of furin. To give an illustration, Fang et al observed complete furin cleavage upon expression of mAb in vivo (Rat) [19] while furin could not cleave completely when mAbs were expressed in CHO and HEK 293T cells [31, 44, 45]. Also, some reports have demonstrated that secretory pathway of CHO cells is not able to provide sufficient amount of proteolytic enzymes to support the complete processing of the recombinant proteins.…”

Sustained secretion of anti‐tumor necrosis factor α monoclonal antibody from ex vivo genetically engineered dermal tissue demonstrates therapeutic activity in mouse model of rheumatoid arthritis

“…This is supported by mass spectromtery analysis, which detects 2A peptide at the 25‐kDa band and not the 50‐kDa band (data not shown). Partial cleavage of the FMDV 2A 24 amino acids residues by the furin protease following ribosome skipping was also reported by Chng et al By contrast, western blot analysis of serum derived from SCID mice that were implanted with the same TARGT‐adalimumab demonstrated two bands at 25 and 50 kDa, corresponding to the native antibody L and H chains at a size similar to adalimumab standard spiked into control SCID sera. This implies a serum derived furin enzyme cleavage, and confirms the correct processing of adalimumab in vivo .…”

“…One of approach is the introduction of the FMDV 2A ribosome skipping signal, between the light (L) and heavy (H) DNA coding sequences that enables equimolar expression of the two antibody separated chains. The addition of a furin cleavage site facilitated the separation of the two chains and the removal of the 2A–encoded additional amino acids …”

Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin

“…These plasmids encode multiple proteins in tandem, each separated by high efficiency cleavage sites of a 2A peptide derived from porcine teschovirus-1 ( Chng et al, 2015 ; Daniels et al, 2014 ; Kim et al, 2011 ). Studies using these engineered plasmids in human cell Lines, drosophila, zebrafish and mice have validated their use for the reliable co-expression of multiple constructs in diverse settings ( Chng et al, 2015 ; Daniels et al, 2014 ; Goedhart et al, 2011 ; Kim et al, 2011 ; Martin et al, 2006 ) . …”