PM1016 Intact and reduced mab/adc fragment separation on reversed phase chromatography with mass spec detection Proteomix RP-1000 1
Possible Different cysteine ADC components under acidic/denaturing condition 2 Intact 2, L1 and H1HL 4 L1, H2H1L, H1H1, LH2 6 L1, H2H2, H3, LH2 8 L1, H3 Non reduced: Intact 2, L1, H1HL, H2H1L, H1H1, LH2, H2H2, H3 Fully reduced: L0, H0, L1, H1, H2, H3 Herceptin: L0 = 23,439 Da, H0 = 50,620 Da 2
MAb Herceptin and its ADCs Separation on Proteomix RP-1000 Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Flow rate: 1.0 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Sample: Herceptin and ADCs 1 mg/ml diluted in water; Injection volume: 10 ml 400 375 350 325 Gradient: 2-22 min, 30-48%B 300 275 250 225 200 175 Cysteine ADC 2 non-cleavable linker Cysteine ADC 1 cleavable linker Herceptin 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 3
Herceptin Cysteine ADC2 Separation on Proteomix RP-1000 Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Flow rate: 1.0 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Sample: Cysteine ADC 2 1 mg/ml diluted in water; Injection volume: 10 ml for gradient 1, 8 ml for gradient 2 140 Each in full scale 130 120 110 100 90 80 70 60 G1: 2-22 min, 25-55%B G2: 2-22 min, 30-48%B min 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 4
Herceptin Cysteine ADC2/Blank Separation on Proteomix RP-1000 Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Flow rate: 1.0 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Sample: Cysteine ADC 2 1 mg/ml diluted in water; Injection volume: 8 ml 350 325 2-22 min, 30-48%B Minimum sample carry over in the blank injection after sample run. 300 275 250 225 200 ADC Blank 175 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 5
Herceptin and its lysine ADC separation Proteomix RP-1000 Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Flow rate: 1.0 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Sample: Herceptin and lysine ADC 1 mg/ml diluted in 0.1% TFA; Injection volume: 10 ml 100 2-22 min, 25-55% B Each in full scale 80 60 Unconjugated mab 40 Lysine-ADC 20 Herceptin 0 2.5 5 7.5 10 12.5 15 17.5 20 min 6
Herceptin Cysteine ADC2 Separation-2.1 x 50 mm mass spec analysis Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Flow rate: 0.4 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Column pressure: 45 bar; Sample: ADC 2 diluted in water; Injection volume: 3 ml cysteine ADC 2 180 160 140 120 1. L1 1 2. Unconjugated mab 4 3, 4 mixture of different fragments 5. H2L0 3 5 6. H3 6 0-25min, 25-50% B 100 80 60 2 * 40 6 8 10 12 14 16 18 20 22 24 min 7
Herceptin Cysteine ADC2 Separation-2.1 x 50 mm Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Flow rate: 0.4 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Column pressure: 70 bar; Sample: ADC 2 diluted in water; Injection volume: 3 ml cysteine ADC 2 Peak 1 L1 Peak 5 H2L0 8
Herceptin/ADC1/ADC2 Separation- small size 2.1 x 50 mm 0.6 ml/min fast analysis Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Flow rate: 0.6 ml/min; Detector: UV 210 nm; Column temperature: 80 C; Column pressure: 70 bar; Sample: Herceptin, ADC 1 and ADC 2 diluted in water; Injection volume: 0.5 ml for Herceptin, 1 ml for ADC 1 and ADC 2 300 0-5 min, 34-51% B 250 Unconjugated mab 200 150 Herceptin-ADC2 Herceptin-ADC1 Herceptin 100 1 2 3 4 5 6 min 9
MAb/ADC Fragment Separation on Reversed Phase Proteomix RP-1000 Reduced-Herceptin vs. Herceptin-Lys, Cys ADCs Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Flow rate: 0.4 ml/min; Detector: UV 214 nm; Column temperature: 80 C; Column pressure: 45 bar; Injection: 1 ml for DTT reduced Herceptin (3 mg/ml), 3 ml Lys-ADC and Cys-ADC (1mg/mL each) 250 L1 0-25min, 25-50% B 225 200 L0 H0 H1 175 H2 H3 150 Cys-ADC2 125 100 75 50 Herceptin Lys-ADC 5 7.5 10 12.5 15 17.5 20 22.5 25 min 10
Cysteine ADC DAR analysis on Proteomix HIC Column: Proteomix HICBu-NP5 (5 mm, 4.6 x 35 mm) Mobile phase: A: 2 M ammonium sulfate in 0.025 M sodium phosphate, ph 7.0, B: 0.025 M sodium phosphate ph 7.0, C : 100% IPA Flow rate: 0.8 ml/min, Detector: UV 214 nm, Column temperature: 25 Sample: Herceptin ADC2, 1 mg/ml in 25 mm sodium phosphate, Injection: 10 ml, 1200 1000 800 D2 DAR= (2*AreaD2+4*AreaD4+6*AreaD6+8*AreaD8) Total peak area * D4a 600 D0 D4b D6 D8 Cysteine ADC 2 400 200 0 Herceptin 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 11 11
ADC HIC fraction 4a-DTT reduced fragment separation Reversed phase Proteomix RP-1000 Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Flow rate: 0.4 ml/min; Detector: UV 214 nm; Column temperature: 80 C; Column pressure: 45 bar; Injection: 30 ml for cysteine ADC separated on HIC, fraction 4a concentrated to 45 ml, reduced with 20 mm DTT 200 180 160 0-25min, 25-50% B LC1 Fully reduced ADC: L0, H0, L1, H1, H2, H3 140 H1 120 100 80 60 40 * * Reduced Cys-ADC 2 Reduced fraction 4a from HIC 5 7.5 10 12.5 15 17.5 20 22.5 25 min 12
TFA effect on the ADC fragments separation Column: Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Mobile phase: A: X % TFA in water; B: X % TFA in 100% ACN; Flow rate: 0.4 ml/min; Detector: UV 214 nm; Column temperature: 80 C; Column pressure: 45 bar; Injection: 3 ml for DTT reduced Herceptin cysteine ADC 0.1 and 0.05% TFA runs, 2 ml for 0.02% TFA) 200 DTT reduced cysteine ADC 150 100 0.1% TFA 50 0.05% TFA 0.02% TFA 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 13
Column Temperature effect Proteomix RP-1000 14
BSA and mab separation with 25 C /40 C / 80 C Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN Flow rate: 1.0 ml/min; Detector: UV 210 nm; Sample: BSA and mab 1mg/mL; Injection volume: 20 ul 2000 1750 BSA 2-22 min, 20-70% 1500 1250 1000 750 500 250 80 C 40 C 25 C 0 0 5 10 15 20 25 30 2000 1750 1500 1250 1000 750 500 250 mab 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 15 80 C 40 C 25 C min
Herceptin Cysteine ADC 2 Separation Column: Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Flow rate: 1.0 ml/min; Detector: UV 210 nm; Column temperature: 25, 80 C; Sample: Herceptin Cysteine ADC 2 1 mg/ml diluted in 0.1% TFA; Injection volume: 20 ml 700 2-22 min, 20-70% 600 500 400 300 200 80 C 25 C 100 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 16
PM1016 Product Information Column Part number Proteomix RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm) 465950-4610 Proteomix RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) 465950-2105 Proteomix HIC Bu-NP5 (5 µm, 4.6 x 35 mm) 413NP5-4603 17