Insulin SEC analysis

• Insulin Samples:– Lispro, identical in structure to Insulin Human,

except that it has lysine and proline at positions 28 and 29, respectively, of chain B, whereas this sequence is reversed in Insulin Human.

– Sigma human recombinant insulin, P/N 910776 – Custom insulin sample for column life time study– Degraded insulin sample (40 hours at 60 oC)

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Insulin-Lispro (Humalog)-mobile phase effect on ZenixTM-C SEC-80 ( 7830 )

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: as indicated, Flow rate: 1 mL/minDetector: UV 276 nm, Column temperature: 30 ℃, Injection volume: 10 µLSample: Insulin Lispro 3.47 mg/mL in phosphate buffer containing m-cresol

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

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A. 0.1% TFA, 50% ACNB. 0.1% Arginine:Acetonitrile:Acetic acid

(65:20:15)A. 10 mM Tris, 140 mM NaCl, pH 7.4

1. Insulin monomer2. M-cresol

Organic mobile phase minimizes the interaction between sample and column resin.

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Zoom in-Insulin Lispro (monomer and aggregate)

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Rs = 2.11

Rs = 3.28

Rs = 3.26 0.1% TFA, 50% ACN

0.1% Arginine:Acetonitrile:Acetic acid (65:20:15)

10 mM Tris, 140 mM NaCl, pH 7.4

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Column temperature- insulin Lispro

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: 0.1% Arginine:Acetonitrile:Acetic acid (65:20:15), Flow rate: 0.5 mL/min, Pressure: 38 barDetector: UV 276 nm, Column temperature: 37 ℃ and 30 ℃, Injection volume: 10 µL, Sample: Insulin Lispro 3.47 mg/mL in phosphate containing m-cresol

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Insulin monomer

M-cresol

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Pore size effect-insulin Lispro

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Pressure: 103 bar ZenixTM-C SEC-150 (3 µm, 150 Å, 7.8 x 300 mm), Pressure: 103 bar

Mobile phase: 0.1% Arginine:Acetonitrile:Acetic acid (65:20:15), Flow rate: 1 mL/min,Detector: UV 276 nm, Column temperature: 37 ℃ and 30 ℃, Injection volume: 10 µL, Sample: Insulin Lispro 3.47 mg/mL in phosphate containing m-cresol

ZenixTM-C SEC-80 provides a better resolution for insulin aggregate and monomer serparation.

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ZenixTM-C SEC-80ZenixTM-C SEC-150

Zoom in

Rs = 3.28

Rs = 2.14

M-cresol

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Column size effect – Insulin Lispro

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 4.6 x 300 mm), Flow rate: 0.35 mL/min, Injection volume: 5 µLZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm), Flow rate: 1 mL/min, Injection volume: 10 µL

Mobile phase: 0.1% Arginine:Acetonitrile:Acetic acid (65:20:15)Detector: UV 276 nm, Column temperature: 30 ℃Sample: Insulin Lispro 3.47 mg/mL in phosphate buffer containing m-cresol

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Rs = 2.79

Rs = 3.28

7.8 x 300 mm

4.6 x 300 mm

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Flow rate effect- insulin Lispro

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: 0.1% Arginine:Acetonitrile:Acetic acid (65:20:15), Flow rate: 0.5 and 1 mL/minDetector: UV 276 nm, Column temperature: 30 ℃, Injection volume: 10 µL, Sample: Insulin Lispro 3.47 mg/mL in phosphate containing m-cresol

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0.5 mL/min

1 mL/min

Rs=3.84

Rs = 3.39

Slower flow rate gives slightly better resolution at the expense of analysis speed.

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Duplicate injections of insulin Lispro

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: 0.1% Arginine:Acetonitrile:Acetic acid (65:20:15), Flow rate: 1 mL/min, Pressure: 103 barDetector: UV 276 nm, Column temperature: 30 ℃, Injection volume: 10 µLSample: Insulin Lispro 3.47 mg/mL in phosphate containing m-cresol

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Insulin

M-cresolHigh run to run reproducibility

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Degraded insulin on ZenixTM-C SEC-80

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: 0.1% TFA, 50% acetonitrile, Flow rate: 1 mL/min, Pressure: 75 barDetector: UV 276 nm, Column temperature: 30 ℃, Injection volume: 10 µL, Sample: Degraded insulin 3.75 mg/mL in phosphate buffer containing m-cresol (40 hours at 60 oC)

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6.087

6.802

7.056

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Insulin

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Insulin Analysis on ZenixTM-C SEC-80 ( 4630 )

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Column: ZenixTM-C SEC-80 (3 µm, 80 Å, 4.6 x 300 mm) Mobile phase0.1% Arginine:Acetonitrile:Acetic acid (65:20:15), Flow rate: 0.35 mL/min,Detector: UV 276 nm, Column temperature: 30 ℃, Injection volume: 5 µL, Pressure: 90 bar,Sample: Insulin Lispro 3.47 mg/mL in phosphate buffer containing m-cresol, sigma insulin 3.8 mg/mL in 0.01 N HCl containing phenol and m-cresol

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Zoom in

Lispro

Sigma

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Column: ZenixTM-C SEC-80 ( 3 µm, 80 Å, 7.8 x 300 mm) Mobile phase: 0.1% Arginine : ACN : HAc = 65 : 20 : 15 ( v/v ), Flow rate: 0.5 mL/minDetector: UV 276 nm, Column temperature: 40 ℃, Injection volume: 25 µLSample: Insulin 4 mg/mL in 0.01 N HCl, Pressure: 52 bar

Life time testInsulin Analysis on ZenixTM-C SEC-80 ( 7830 )

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Aggregate

Insulin monomer

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Insulin analysis parameters-life time test

injection RT Plates Tailing Rs Area% dimer

1 16.08 19767 0.79 4.32 3.37

50 16.13 20116 0.79 4.43 3.21

100 16.17 20327 0.8 4.44 3.18

150 16.22 21287 0.82 4.42 3.34

200 16.28 21629 0.83 4.44 3.15

RSD/% 0.48 3.86 2.25 1.16 3.04

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