Western blot analyses of purified mAb in triplicate stable cell pools transfected with vectors bearing F2A-mediated expression system.

<p>Light chains with various molecular weights (25, 28 & 30 kDa) were detected. Triplicates of each stable pool were shown with 1, 2 and 3. (A) Lane 1; CHEF-F2A (2), lane 2; CHEF-F2A (3), lane 3; protein molecular ladder. (B) Lane 1; protein molecular ladder, lane 2; CMV-F2A (1), lane 3; CMV-F2A (3).</p

Analysis of integrated transgene copy number in stable cell pools transfected by four bicistronic vectors.

<p>Antibody copy number based on light chain copy number was calculated by qRT-PCR. The error bars represent the standard deviation of three independent measurements.</p

Analysis of the four bicistronic vectors for mAb and mRNA expression in established stable cell lines.

<p>Stably transfected pools were generated by transfection of CHO-S cells with various bicistronic vectors containing either IRES or F2A sequences and different promoters (CHEF or CMV). Levels of mAb and mRNA expression were measured by ELISA and qRT-PCR respectively. Black bars represent the mAb titer and gray bars represent mRNA fold induction. The error bars represent the standard deviation of three independent measurements.</p

Analysis of the stability of antibody expression over time by stable cell pools transfected with two vectors containing F2A sequences.

<p>Both stable cells were cultivated for 18 weeks upon removal of puromycin as a selection marker. Every 2 weeks, antibody expression was monitored and measured by ELISA. The other pools exhibited the same expression pattern; the data from one of them was represented.</p

SDS-PAGE and Western blot analysis of purified mAb in stable cell pools transfected with four bicistronic vectors.

<p>Supernatants of three different cell pools were purified using protein-A. Purified samples were analyzed with SDS-PAGE and western blot under reducing and non-reducing condition. Commercial IgG1 was used as a positive control. (A) SDS-PAGE profile of purified samples in reduced state, lane 1; Protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (B) SDS-PAGE profile of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder. (C) Western blot analysis of samples under reducing condition, lane 1; protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (D) Western blot analysis of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder.</p

Schematic illustration showing insertion of furin/2A sequences between light and heavy chain by overlap extension PCR.

<p>Schematic illustration showing insertion of furin/2A sequences between light and heavy chain by overlap extension PCR.</p

Oligonucleotides used for analysis of mRNA levels and relative gene copy numbers by quantitative real-time PCR.

<p>Oligonucleotides used for analysis of mRNA levels and relative gene copy numbers by quantitative real-time PCR.</p

Primers used for construction of the LC-furin-2A-HC gene.

<p>Primers used for construction of the LC-furin-2A-HC gene.</p

Western-blot analysis of single ORF donor vectors containing cell pools.

<p>IRES and 2A elements were used to express light and heavy chains in bicistronic mRNAs. <b>A</b>, Western-blotting of IRES-containing cells in reduced and non-reduced forms. Lanes 1–3 are reduced and lanes 5–7 are non-reduced. Lane 1 and 7, purified IgG as a positive control, lane 2 and 6 purified mAb produced by N-pBLIH cells (due to their low expression profile western-blot had done on their purified product), lane 3and 5, the supernatant of 1/2.5-pBLIH cell pools, lane 4, pre-stained protein marker (10–170 kDa). <b>B</b>, Western-blot analysis of 2A harboring cells, left figure in reduced and right figure in non-reduced forms. In reduced picture, lane 1, positive control, lane 2, N-pBL2AH cells supernatant, lane 3, 1/2.5-pBL2AH cells supernatant, 25, 28 and 30 kDa light chains appeared in N-pBL2AH and 1/2.5-pBL2AH lanes respectively. Furin peptidase didn't work in the latter. In the non-reduced picture, lane 1, pre-stained protein marker (10–170 kDa), lane 2, N-pBL2AH and lane 3, 1/2.5-pBL2AH cells supernatants.</p

mRNA levels in established pools were assessed by quantitative real-time PCR.

<p><b>A</b>, In dual promoter bearing cells both light and heavy chains, are expressed via an independent expression cassette. Transposition effect was assessed at the level of transcription of each chain and as it is demonstrated in the picture both chains expression rate increase in a similar way. HC to LC ratio analysis showed that HC in all dual promoter cells was produced in a greater amount. <b>B</b>, Light chain mRNA levels in single ORF mAb expressing cells were evaluated by real-time PCR using N-pBLPCH-LC mRNA as the control gene. Error bars indicate SD of triplicate measurements. ANOVA was used to detect statistically significant differences between the generated pools (P< 0.05).</p