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

Abstract

Linking 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.

Keywords: 2A peptide; CHO; CHO, Chinese hamster ovary; E2A, 2A peptide derived from the equine rhinitis virus; F2A, 2A peptide derived from the foot-and-mouth disease virus; G, glycine; GE2A, E2A with the GSG linker; GF2A, F2A with the GSG linker; GFP, green fluorescence protein; GP2A, P2A with the GSG linker; GSG linker; GT2A, T2A with the GSG linker; HC, heavy chain; HT, hypoxanthine and thymine; IRES, internal ribosome entry site; IgG, immunoglobulin G; K, lysine; LC, light chain; MS, mass spectrometry; MTX, methotrexate; P, proline; P2A, 2A peptide derived from the porcine teschovirus-1; PFM, protein-free medium; PVDF, polyvinylidene difluoride; SEC, size exclusion chromatography; T2A, 2A peptide derived from the Thosea asigna virus; cleavage efficiency; furin; mAb, monoclonal antibody; monoclonal antibody.

Figure 1.

Schematic representation of the vectors containing different 2A peptides for mAb expression (A) and corresponding amino acid sequences of various 2A peptides (B). The conserved regions of 2A peptides are highlighted in dotted boxes. CMV, human cytomegalovirus IE gene promoter; HC, heavy chain cDNA; Furin, DNA encoding furin recognition sequence RRKR; LC, light chain cDNA; IRESatt, mutated encephalomyocarditis virus (EMCV) internal ribosomal entry site; DHFR, dihydrofolate reductase cDNA; SpA, simian virus 40 early polyadenylation signal.

Figure 2.

Comparison of 2A peptides for mAb expression and cleavage efficiency in transient transfections. (A) Expression of trastuzumab in CHO DG44. (B) Expression of adalimumab in CHO DG44. (C) Expression of bevacizumab in CHO DG44. (D) Expression of trastuzumab in CHO K1. CHO DG44 or K1 cells were co-transfected with an appropriate vector containing a specific 2A peptide and an internal control vector expressing GFP. mAb expression was quantified by ELISA at 48 h post-transfection and then normalized to GFP expression. GFP normalized expression was further normalized to the control vector containing F2A. Each point of normalized mAb expression represents the average and standard deviation of duplicate measurements from 2 independent transfections. Cleavage efficiency of each furin-2A peptide was determined by western blotting analysis of supernatant under reducing conditions. Protein A purified biosimilar trastuzumab expressed from a previously described IRES-mediated tricistronic vector was used as positive control, and supernatant from non-transfected cells as blank.

Figure 3.

Comparison of different 2A peptides for biosimilar trastuzumab expression and cleavage in stably transfected pools. (A) Titer of trastuzumab in stably transfected pools generated using vectors containing various 2A peptides. Each point represents the average and standard deviation of duplicate measurements from 2 independent stably transfected pools. (B) Western blotting of supernatants in stable transfected pools generated using vectors containing various 2A peptides. Protein A purified biosimilar trastuzumab expressed from a previously described IRES-mediated tricistronic vector was used as standard, and supernatant from non-transfected cells as blank.

Figure 4.

Quality analysis of biosimilar trastuzumab expressed from various 2A peptides and purified using traditional clarification method and protein A. (A) SDS-PAGE analysis of purified mAbs under non-reducing and reducing conditions. Protein A purified biosimilar trastuzumab expressed from a previously described IRES-mediated tricistronic vector was used as standard, and supernatant from non-transfected cells as blank. (B) Quantitative comparison of aggregates for various 2A peptides. Each point represents the average and standard deviation of the duplicate measurements from 2 independent stably transfected pools.

Figure 5.

Characterization of aggregate in biosimilar trastuzumab expressed from GT2A and purified using traditional clarification method and protein A. (A) UV chromatogram of a purified sample. Peak P1 and P2 correspond to aggregate and P3 corresponds to IgG monomer. (B) SDS-PAGE analysis of the fractions separated by SEC. Protein A purified biosimilar trastuzumab expressed from a previously described IRES-mediated tricistronic vector was used as standard, and supernatant from non-transfected cells as blank.

Figure 6.

Quality analysis of biosimilar trastuzumab purified using improved purification method. (A) SDS-PAGE analysis of reduced product purified using either traditional or improved clarification methods and protein A. 1X, 2X, and 4X represent loading amount of purified mAb of 10, 5, and 2.5 μg. (B) UV chromatogram of a sample purified using improved clarification and protein A.