Insulin SEC analysis. Insulin Samples:

Transcription

1 EE1 Insulin SEC analysis Insulin Samples: Lispro, identical in structure to Insulin Human, except that it has lysine and proline at positions 8 and 9, respectively, of chain B, whereas this sequence is reversed in Insulin Human. Sigma human recombinant insulin, P/N 9177 Custom insulin sample for column life time study Degraded insulin sample ( hours at o C)

2 EE1 Insulin-Lispro (Humalog)-mobile phase effect on Zenix TM -C SEC-8 ( 783 ) Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm) Mobile phase: as indicated, Flow rate: 1 ml/ Detector: UV 7 nm, Column temperature: 3, Injection volume: 1 µl Sample: Insulin Lispro 3.7 mg/ml in phosphate buffer containing m-cresol A..1% TFA, 5% ACN B..1% Arginine:Acetonitrile:Acetic acid (5::15) A. 1 mm Tris, 1 mm NaCl, ph Insulin monomer. M-cresol C B A Organic mobile phase imizes the interaction between sample and column resin.

3 1 1 8 Zoom in-insulin Lispro (monomer and aggregate) EE1 Rs = 3..1% TFA, 5% ACN Rs = 3.8.1% Arginine:Acetonitrile:Acetic acid (5::15) Rs =.11 1 mm Tris, 1 mm NaCl, ph

4 Column temperature- insulin Lispro EE1 Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm) Mobile phase:.1% Arginine:Acetonitrile:Acetic acid (5::15), Flow rate:.5 ml/, Pressure: 38 bar Detector: UV 7 nm, Column temperature: 37 and 3, Injection volume: 1 µl, Sample: Insulin Lispro 3.7 mg/ml in phosphate containing m-cresol M-cresol Insulin monomer

5 Pore size effect-insulin Lispro Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Pressure: 13 bar Zenix TM -C SEC-15 (3 µm, 15 Å, 7.8 x 3 mm), Pressure: 13 bar Mobile phase:.1% Arginine:Acetonitrile:Acetic acid (5::15), Flow rate: 1 ml/, Detector: UV 7 nm, Column temperature: 37 and 3, Injection volume: 1 µl, Sample: Insulin Lispro 3.7 mg/ml in phosphate containing m-cresol Zoom in Rs = 3.8 Rs = M-cresol EE Zenix TM -C SEC-8 Zenix TM -C SEC Zenix TM -C SEC-8 provides a better resolution for insulin aggregate and monomer serparation.

6 1 Column size effect Insulin Lispro Column: Zenix TM -C SEC-8 (3 µm, 8 Å,. x 3 mm), Flow rate:.35 ml/, Injection volume: 5 µl Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Flow rate: 1 ml/, Injection volume: 1 µl Mobile phase:.1% Arginine:Acetonitrile:Acetic acid (5::15) Detector: UV 7 nm, Column temperature: 3 Sample: Insulin Lispro 3.7 mg/ml in phosphate buffer containing m-cresol. x 3 mm EE1 1 8 Rs = x 3 mm Rs =

7 Flow rate effect- insulin Lispro EE1 Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm) Mobile phase:.1% Arginine:Acetonitrile:Acetic acid (5::15), Flow rate:.5 and 1 ml/ Detector: UV 7 nm, Column temperature: 3, Injection volume: 1 µl, Sample: Insulin Lispro 3.7 mg/ml in phosphate containing m-cresol.5 ml/ Rs= ml/ Rs = Slower flow rate gives slightly better resolution at the expense of analysis speed.

8 Duplicate injections of insulin Lispro Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm) Mobile phase:.1% Arginine:Acetonitrile:Acetic acid (5::15), Flow rate: 1 ml/, Pressure: 13 bar Detector: UV 7 nm, Column temperature: 3, Injection volume: 1 µl Sample: Insulin Lispro 3.7 mg/ml in phosphate containing m-cresol High run to run reproducibility M-cresol EE1 5 Insulin

9 Degraded insulin on Zenix TM -C SEC-8 Column: Zenix TM -C SEC-8 (3 µm, 8 Å, 7.8 x 3 mm) Mobile phase:.1% TFA, 5% acetonitrile, Flow rate: 1 ml/, Pressure: 75 bar Detector: UV 7 nm, Column temperature: 3, Injection volume: 1 µl, Sample: Degraded insulin 3.75 mg/ml in phosphate buffer containing m-cresol ( hours at o C) M-cresol EE Insulin

10 Insulin Analysis on Zenix TM -C SEC-8 ( 3 ) EE1 Column: Zenix TM -C SEC-8 (3 µm, 8 Å,. x 3 mm) Mobile phase.1% Arginine:Acetonitrile:Acetic acid (5::15), Flow rate:.35 ml/, Detector: UV 7 nm, Column temperature: 3, Injection volume: 5 µl, Pressure: 9 bar, Sample: Insulin Lispro 3.7 mg/ml in phosphate buffer containing m-cresol, sigma insulin 3.8 mg/ml in.1 N HCl containing phenol and m-cresol Zoom in Lispro m-cresol Sigma phenol

11 Life time test Insulin Analysis on Zenix TM -C SEC-8 ( 783 ) Column: Zenix TM -C SEC-8 ( 3 µm, 8 Å, 7.8 x 3 mm) Mobile phase:.1% Arginine : ACN : HAc = 5 : : 15 ( v/v ), Flow rate:.5 ml/ Detector: UV 7 nm, Column temperature:, Injection volume: 5 µl Sample: Insulin mg/ml in.1 N HCl, Pressure: 5 bar EE1 3 Insulin monomer Injection 1 Injection 5 Injection 1 Injection 15 Injection Aggregate

12 Insulin analysis parameters-life time test EE1 injection RT Plates Tailing Rs Area% dimer RSD/%