Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

glucagon; protein purification; gel filtration; ion-exchange chromatography; insulin

Authorship

A.S.R. Andrade

Universidade Federal de Minas Gerais, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas

L. Vilela

BIOBRÁS S/A, Departamento de Pesquisa e DesenvolvimentoBIOBRÁS S/ABIOBRÁS S/A, Departamento de Pesquisa e Desenvolvimento

H. Tunes

BIOBRÁS S/A, Departamento de Pesquisa e DesenvolvimentoBIOBRÁS S/ABIOBRÁS S/A, Departamento de Pesquisa e Desenvolvimento

SCIMAGO INSTITUTIONS RANKINGS

Universidade Federal de Minas Gerais, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas

BIOBRÁS S/A, Departamento de Pesquisa e DesenvolvimentoBIOBRÁS S/ABIOBRÁS S/A, Departamento de Pesquisa e Desenvolvimento

Figures | Tables

Figures (5)
Tables (1)

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Figure 1
- Ion-exchange chromatography of glucagon on Q-Sepharose FF. The isoelectric precipitate (400 mg/ml eluent) was applied to a 2.0 x 26-cm column and eluted at room temperature with 50 mM Tris-HCl, 7 M urea, pH 9.0, at a flow rate of 5.0 ml/min. Fractions of 5.0 ml were collected and the amount of glucagon was determined by RIA. The arrow indicates the fraction where the buffer NaCl concentration was increased to 1.0 M NaCl. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

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Figure 2
- Gel filtration chromatography of glucagon on Sephadex G-25. The sample, 70 ml of fraction A (Fr A) from Q-Sepharose FF (Figure 1), was applied to a 3.0 x 100-cm column and eluted at room temperature with 0.1 M acetic acid at a flow rate of 7.0 ml/min. Fractions of 10 ml were collected and the amount of glucagon was determined by HPLC as shown in Figure 4. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

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Figure 3
- Ion-exchange chromatography of glucagon on S-Sepharose FF. The sample, 100 mg of fraction B (Fr B) from Sephadex G-25 (Figure 2), was applied to a 1.6 x 10-cm column and eluted at room temperature with 50 mM sodium citrate, 7 M urea, pH 4.0, at a flow rate of 2.5 ml/min. Fractions of 3.0 ml were collected and the amount of glucagon was determined by HPLC. The arrows indicate the fractions where the buffer NaCl concentration was increased by the addition of NaCl. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

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Figure 4
- Analytical characterization of glucagon by HPLC. A, Purified glucagon (Fr C from S-Sepharose FF). B, Standard glucagon. Purified and standard glucagon have the same retention time (10.97 ± 0.08) and approximately the same purity (84.05 and 85.2% for purified and standard glucagon, respectively). Experimental conditions: after sample application, a stepwise acetonitrile gradient consisting of 50, 60, 62, 70 and 80% buffer "B" (5 min per step) was used (buffer "A", 50 mM sodium phosphate, 10 mM perchloric acid, pH 2.5, and buffer "B", 50% acetonitrile in buffer "A") in an Aquapore RP 300 column (4.6 x 250 mm) at room temperature and at a flow rate of 1 ml/min. The elution was monitored by absorbance at 214 nm.

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Figure 5
- Hyperglycemic action of glucagon on mice. Circles, Control (without glucagon); triangles, purified glucagon (Fr C from S-Sepharose FF); squares, standard glucagon. The mice (fasted females weighing 18 to 25 g) were divided into three groups (8 mice per group), which received by subcutaneous injection 2.5 µg of purified glucagon (Fr C from S-Sepharose), standard glucagon or saline solution. Forty-µl blood samples were taken and centrifuged and plasma glucose was determined by the method of Barhan and Trinder (16). Each point represents the means of six determinations.

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Figure 1 - Ion-exchange chromatography of glucagon on Q-Sepharose FF. The isoelectric precipitate (400 mg/ml eluent) was applied to a 2.0 x 26-cm column and eluted at room temperature with 50 mM Tris-HCl, 7 M urea, pH 9.0, at a flow rate of 5.0 ml/min. Fractions of 5.0 ml were collected and the amount of glucagon was determined by RIA. The arrow indicates the fraction where the buffer NaCl concentration was increased to 1.0 M NaCl. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

Figure 2 - Gel filtration chromatography of glucagon on Sephadex G-25. The sample, 70 ml of fraction A (Fr A) from Q-Sepharose FF (Figure 1), was applied to a 3.0 x 100-cm column and eluted at room temperature with 0.1 M acetic acid at a flow rate of 7.0 ml/min. Fractions of 10 ml were collected and the amount of glucagon was determined by HPLC as shown in Figure 4. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

Figure 3 - Ion-exchange chromatography of glucagon on S-Sepharose FF. The sample, 100 mg of fraction B (Fr B) from Sephadex G-25 (Figure 2), was applied to a 1.6 x 10-cm column and eluted at room temperature with 50 mM sodium citrate, 7 M urea, pH 4.0, at a flow rate of 2.5 ml/min. Fractions of 3.0 ml were collected and the amount of glucagon was determined by HPLC. The arrows indicate the fractions where the buffer NaCl concentration was increased by the addition of NaCl. Absorbance at 280 nm (solid line), mg glucagon (dashed line).

Figure 4 - Analytical characterization of glucagon by HPLC. A, Purified glucagon (Fr C from S-Sepharose FF). B, Standard glucagon. Purified and standard glucagon have the same retention time (10.97 ± 0.08) and approximately the same purity (84.05 and 85.2% for purified and standard glucagon, respectively). Experimental conditions: after sample application, a stepwise acetonitrile gradient consisting of 50, 60, 62, 70 and 80% buffer "B" (5 min per step) was used (buffer "A", 50 mM sodium phosphate, 10 mM perchloric acid, pH 2.5, and buffer "B", 50% acetonitrile in buffer "A") in an Aquapore RP 300 column (4.6 x 250 mm) at room temperature and at a flow rate of 1 ml/min. The elution was monitored by absorbance at 214 nm.

Figure 5 - Hyperglycemic action of glucagon on mice. Circles, Control (without glucagon); triangles, purified glucagon (Fr C from S-Sepharose FF); squares, standard glucagon. The mice (fasted females weighing 18 to 25 g) were divided into three groups (8 mice per group), which received by subcutaneous injection 2.5 µg of purified glucagon (Fr C from S-Sepharose), standard glucagon or saline solution. Forty-µl blood samples were taken and centrifuged and plasma glucose was determined by the method of Barhan and Trinder (16). Each point represents the means of six determinations.

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Purification of bovine pancreatic glucagon as a by-product of insulin production

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