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Hightlight:
- IDeS proteolysis of IgG and Fc fusion protein is a convenient tool in characterization of monoclonal antibody (mAb) and antibody drug conjugate (ADC), which greatly simplifies the sample preparation and mass spec data analysis.
- IDeS proteolysis generates two mAb domains Fc/2 and F(ab')2. Two more fragments Fd and light chain (LC) can be generated by further DTT reduction. Sepax SEC, HIC and Reversed Phase chromatography products can successfully separate the large fragments generated from IDeS proteolysis of mAb and ADC.
- Domain modifications and the molecular weight of each mAb fragments and the associated modified fragment in ADC samples can be quickly identified by Sepax SEC and RP products with LC/MS using mass spec compatible volatile mobile phases.
- IDeS proteolysis with LC separation characterization can be applied to product purity, identification and stability assay. Characterization of biosimilar mAbs, ADCs and fc fusion proteins can benefit the simple workflow of IDeS proteolysis with multiple LC separation methods.
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Product information:
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Downloads:
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>>Learn more details in the full version of this application note here: ZM1022
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on any bioseparation analytical columns
Offer Expires: 12/31/2014
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Technical Manager: Haiying Chen
Ooctober 27-29, 2014 San Diego, CA
Sepax Poster:
Antibody drug conjugate analytical characterization
by multiple liquid chromatographic methods following IDeS proteolysis
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MAb Fragments IDeS Digestion and DTT Reduction
Mab and ADC (2 mg/mL each) were incubated with FragITIDeS resin or PromegaIDeS at 37˚C for 1 hour. Digestion buffer: 10 mM phosphate buffer with 150 mM NaCl at pH 6.6.
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SEC Analysis of MAb Fragments from IDeS Digestion
Column: Zenix� SEC-300 ( 3 �m, 300 �, 7.8 x 300 mm)
Mobile phase: 150 mM Phosphate buffer, pH 7.0
Mab digestion: Mab 5 mg/mL was incubated with FragIT IDeS resin at 37˚C for 1 hour; Flow rate: 1 mL/min; Detector: UV 214 nm; Column temperature: Room temperature; Injection: 15 �g IDeS digested mAb and 12 �g undigested mAb
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Herceptin and Fragment Analysis with SEC-LC/MS
Column: Zenix� - C SEC-300 ( 3 �m, 300 �, 4.6 x 300 mm)
Mobile phase: 0.1% TFA, 0.1% formic acid and 20% ACN
Flow rate: 0.35 mL/min;
Detector: UV 280 nm;
Column temperature: 25�C;
Injection volume: 2 �g for each sample
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HIC Separation of Herceptin and its Cysteine-ADC Fragments after IDeS Proteolysis
Column: Proteomix HIC Butyl-NP5 (4.6 x 35 mm)
Flow rate: 0.8 mL/min, Detector: UV 214 nm
Mobile phase: A: 2M ammonium sulfate in 0.025M sodium phosphate, pH 7.0, B: 0.025M sodium phosphate pH 7.0, C : 100%IPA; Flow rate: 0.8 mL/min, Detector: UV 214 nm;
Column temperature: 25�C; Injection 10 mL for herceptin, 20 mL for cysteine-ADC 1 mg/mL in 750 mM ammonium sulfate 25 mM sodium phosphate pH7.0
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RP Separation of MAb/ADC Fragments from IDeS Digested and Reduced-Herceptin vs. Herceptin-Lys, Cys
Column: Proteomix�RP-1000(5 �m, 1000 �, 2.1 x 50 mm)
Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN;
Flow rate: 0.4 mL/min; Detector: UV 214 nm; Column temperature: 80 ˚C; Column pressure: 45 bar;
Injection: 3mL for IDeS digested Herceptin, Lys-ADC and Cys-ADC (1mg/mL each)
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Deconvoluted Mass for Herceptin-Cys ADC Fragment Generated from IDeS Digestion and DTT Redution
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