Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells

Abstract

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.

Keywords: Cysteamine; IgG4; Monoclonal antibody; Protein disulfide isomerase; Sp2.0 cells; Thiol reducing agents.

Figure 1

Analysis of recombinant vectors by restriction digestion. pFUSE-CHIg-hG4-VH and pFUSE-CLIg-hk-VL were cut by NheI/NotI and NotI/BamHI respectively. According to the vector maps (A), predicted product sizes were 1133 and 3677 bp for pFUSE-CHIg-hG4-VH and 960 and 3234 bp for pFUSE-CLIg-hk-VL. Digest reactions were electrophoresed on a 1% agarose gel (B). Lane 1, cut pFUSE-CHIg-hG4-VH; lane 2, cut pFUSE-CLIg-hk-VL; lane 3, 1kb DNA markers.

Figure 2

PCR analysis of genomic DNA from zeocin/blasticidin-resistant Sp2.0 cells. PCR products were electrophoresed on a 1% agarose gel. Lane 1, 1kb DNA markers; lane 2, amplification of heavy chain gene from DNA of resistant Sp2.0 cells; lane 3, amplification of variable region of heavy chain gene from DNA of resistant Sp2.0 cells; lane 4, amplification of light chain gene from DNA of resistant Sp2.0 cells; lane 5, amplification of variable region of light chain gene from DNA of resistant Sp2.0 cells; lane 6, amplification of variable region of heavy chain from recombinant vector pFUSE-CHIg-hG4-VH; lane 7, amplification of variable region of light chain from recombinant vector pFUSE-CLIg-hk-VL.

Figure 3

RT-PCR analysis of total RNA isolated from untransfected and rSp2.0. PCR products were electrophoresed on a 1% agarose gel. Lane 1, 1kb DNA markers; lane 2, amplification of variable region of heavy chain from recombinant vector pFUSE-CHIg-hG4-VH; lane 3, amplification of variable region of heavy chain gene from RNA of rSp2.0; lane 4, amplification of variable region of light chain gene from RNA of rSp2.0; lane 5, amplification of variable region of heavy chain gene from RNA of untransfected Sp2.0; lane 6, amplification of variable region of light chain gene from RNA of untransfected Sp2.0.

Figure 4

SDS–PAGE and Western blot analysis of expressed IgG₄. The concentrated culture supernatant was separated on 8% resolving gel under non-reducing condition. Purified protein was analyzed under reducing condition on 12% resolving gel. The gels were stained with Coomasie Blue. In Western blot analysis, the blots were probed with a HRP-conjugated chicken Anti-Human IgG. Western blots were visualized by ECL. (A) Coomassie stained gel under non-reduced condition. Lane 1, supernatant of rSp2.0 cells; lane 2, supernatant of untransfected Sp2.0 cells; lane 3, positive control; lane 4, marker (B) Western blot analysis under non-reduced condition. Lane 1, marker; lane 2, supernatant of untransfected Sp2.0 cells (30 µg/mL); lane 3, supernatant of untransfected Sp2.0 cells (10 µg/mL); lane 4, supernatant of rSp2.0 cells (30 µg/mL); lane 5, supernatant of rSp2.0 cells (10 µg/mL); lane 6, positive control (C) Coomassie stained gel under reduced condition. Lane 1, marker; lane 2, purified IgG₄, lane 3, positive control (D) Western blot analysis under reduced condition. Lane 1, marker; lane 2, purified IgG₄, lane 3, positive control.

Figure 5

Effect of cysteamine on viability of rSp2.0. Stable rSp2.0 Cells at cell density of 4 ×10⁵ cells/mL were treated by various concentrations of cysteamine (0, 2, 4, 5 and 7 mM). The viability was measured after 24 h using the trypan blue dye exclusion method. Error bars represent the standard deviations calculated from the data obtained in three independent experiments.

Figure 6

Effect of cysteamine on IgG₄ production. Cysteamine in various concentrations (2, 4, 5, 7 mM) was added to medium of rSp2.0. The supernatants were collected after 24 hours and the amounts of secreted IgG₄ were analyzed by ELISA. Error bars represent the standard deviations calculated from the data obtained in three independent experiments. *P<0.05, two-tailed unpaired t-test

Figure 7

Assessment of late effect of cysteamine on rSp2.0. Cells were treated by 2 mM cysteamine. For 5 days cell viability (A) and IgG₄ concentration (B) were determined daily. The amounts of secreted IgG₄ were analyzed by ELISA. The viability was measured using the trypan blue dye exclusion method. *P<0.05, two-tailed unpaired t-test.

Figure 8

RT-PCR analysis of PDI mRNA in cysteamine treated rSp2.0. Lane 1, amplification of PDI gene from RNA of control cells; lane 2, amplification of PDI gene from RNA of treated cells by 2 mM cysteamine; lane 3, amplification of β-actin gene from RNA of control cells; lane 4, amplification of β-actin gene from RNA of treated cells by 2 mM cysteamine; lane 6, amplification of β-actin gene from RNA of control cells; lane 7, amplification of β-actin gene from RNA of treated cells by 4 mM cysteamine; lane 8, amplification of PDI gene from RNA of control cells; lane 9, amplification of PDI gene from RNA of treated cells by 4 mM cysteamine and lanes 5,10, 1kb DNA ladder. Cysteamine was added to culture medium. After 24 h, cells were harvested and total RNA was isolated from rSp2.0 cells. Levels of mRNA were standardized by β-actin mRNA. The expected size of products was 186 bp for PDI and 318 for β-actin. The experiments were repeated three times independently.