Illustra sephadex g 25 dna grade

Example 15

A reaction containing 2.0 mg/mL huMOV19 antibody and 7 molar equivalents of compound 80 (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 20 hours at 4° C. utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).

The purified conjugate was found to have an average of 2.5 molecules of compound 80 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,280 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for compound 80, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 99% monomer (by size exclusion chromatography), <0.1% unconjugated compound 80 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 2.4 mg/ml. The conjugated antibody was found to be >90% intact by gel chip analysis.

Example 13

A reaction containing 2.5 mg/mL huMOV19 antibody and 5 molar equivalents of compound 35, (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 8 hours at room temperature utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 10,000 MWCO).

The purified conjugate was found to have an average of 2.9 molecules of compound 35 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,484 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for IGN128, and ε_{280 nm}=201,400 cm⁻¹ M⁻¹ for huMOV19 antibody), 97% monomer (by size exclusion chromatography), <1% unconjugated compound 35 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 1.4 mg/ml. The MS spectrometry data is shown in FIG. 7A.

Example 13

A reaction containing 2.5 mg/mL huMOV19 antibody and 5 molar equivalents of compound 35, (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 8 hours at room temperature utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 10,000 MWCO).

The purified conjugate was found to have an average of 2.9 molecules of compound 35 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,484 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for IGN128, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 97% monomer (by size exclusion chromatography), <1% unconjugated compound 35 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 1.4 mg/ml. The MS spectrometry data is shown in FIG. 7A.

Example 20

A reaction containing 2.5 mg/mL huMOV19 antibody and 4 molar equivalents of compound 23 (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 8 hours at room temperature utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 10,000 MWCO).

The purified conjugate was found to have an average of 2.8 molecules of compound 23 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,484 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for compound 23, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 98% monomer (by size exclusion chromatography), <3% unconjugated compound 23 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 1.3 mg/ml. The MS spectrometry data is shown in FIG. 7D.

Example 18

An in situ mix containing final concentrations of 1.95 mM compound 99 and 1.5 mM sulfo-SPDB Linker in DMA containing 10 mM N,N-Diisopropylethyl amine (DIPEA) was incubated for 20 min before capping with 4 mM maleimidopropionic acid MPA. A 6-fold excess of of the resulting 99-sulfo-SPDB-NHS was added to a reaction containing 2.5 mg/ml huMOV19 antibody in 15 mM HEPES pH 8.5 (82:18 water:DMA). The solution was allowed to conjugate over night at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 20 mM histidine, 50 mM sodium chloride, 8.5% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer over night at 4° C. utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 10,000 MWCO).

The purified conjugate was found to have an average of 1.6 molecules of compound 99 linked per antibody (by UV/Vis using molar extinction coefficients ε_{330 nm}=15,484 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for compound 99, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 99% monomer (by size exclusion chromatography), and a final protein concentration of 0.59 mg/ml. The MS spectrometry data is shown in FIG. 7C.

Example 17

A reaction containing 2.0 mg/mL huMOV19 antibody and 5 molar equivalents of compound 49 (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 10% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 4 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 4 hours at room temperature utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).

The purified conjugate was found to have an average of 2.8 molecules of compound 49 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,280 cm⁻¹M⁻¹ and ε_{280 nm}=30, 115 cm⁻¹M⁻¹ for compound 49, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 94% monomer (by size exclusion chromatography), <0.1% unconjugated compound 49 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 1.5 mg/ml. The conjugated antibody was found to be >95% intact by gel chip analysis. The MS spectrometry data is shown in FIG. 7C.

Example 14

A reaction containing 2.0 mg/mL huMOV19 antibody and 7 molar equivalents of compound 63 (pretreated with 5-fold excess of sodium bisulfite in 90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA (N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite formulation buffer pH 6.2 using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 20 hours at 4° C. utilizing Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).

The purified conjugate was found to have an average of 2.7 molecules of compound 63 linked per antibody (by UV-Vis using molar extinction coefficients ε_{330 nm}=15,280 cm⁻¹M⁻¹ and E_{280 nm}=30, 115 cm⁻¹M⁻¹ for IGN131, and ε_{280 nm}=201,400 cm⁻¹M⁻¹ for huMOV19 antibody), 99% monomer (by size exclusion chromatography), <0.1% unconjugated compound 63 (by acetone precipitation, reverse-phase HPLC analysis) and a final protein concentration of 1.6 mg/ml. The conjugated antibody was found to be >90% intact by gel chip analysis. The MS spectrometry data is shown in FIG. 7B.