BP 2025 (Ph. Eur. 11.6 update)
Action and use
Hormone; treatment of hyperglycaemia.
DEFINITION
Human Glucagon for Injection is a sterile material consisting of freeze-dried Human Glucagon (produced by recombinant DNA technology) with or without excipients. It is supplied in a sealed container.
The contents of the sealed container comply with the requirements for Powders for Injections stated under Parenteral Preparations and with the following requirements.
Content of glucagon, C153H225N43O49S
75.0 to 104.0% of the stated amount.
IDENTIFICATION
In the Assay, the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of the principal peak in the chromatogram obtained with solution (2).
TESTS
Acidity
pH of a 0.1% w/v solution, 2.5 to 3.5, Appendix V L.
Related proteins and deamidated forms
Carry out the method for liquid chromatography, Appendix III D, using the following solutions and the normalisation procedure.
(1) Dissolve the powder being examined in 0.01M hydrochloric acid to obtain the equivalent of 0.05% w/v of human glucagon. Maintain the solution at 2° to 8°.
(2) Dissolve the contents of a vial of human glucagon EPCRS in 0.01M hydrochloric acid to obtain a concentration of 0.05% w/v. Maintain the solution at 2° to 8°.
(3) Dissolve the powder being examined in 0.01M hydrochloric acid to obtain a concentration of about 0.05% w/v human glucagon. Heat at 50° for 48 hours (generation of all 4 deamidated forms of glucagon at a total concentration of not less than 7%).
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 3.0 mm) packed with octadecylsilyl silica gel for chromatography (3 μm) (ACE 3 C18 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 0.5 mL per minute.
(d) Use a column temperature of 45°.
(e) Use a detection wavelength of 214 nm.
(f) Inject 15 μL of each solution.
MOBILE PHASE
Mobile phase A 200 volumes of acetonitrile for chromatography and 800 volumes of the solution prepared as described below. Dissolve 16.3 g of potassium dihydrogen orthophosphate in 800 mL of water, adjust to pH 2.7 with orthophosphoric acid.
Mobile phase B 400 volumes of acetonitrile for chromatography and 600 volumes of water.
Use the following gradient elution.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-25 | 61 | 39 | isocratic |
| 25-29 | 61→12 | 39→88 | linear gradient |
| 29-30 | 12 | 88 | isocratic |
| 30-31 | 12→61 | 88→39 | re-equilibration |
Note: The relative retention time with respect to glucagon (retention time about 21 minutes) of deamidated glucagon 1 is about 1.1. The end time of the isocratic elution may be adjusted so that the gradient begins after elution of the peak due to deamidated glucagon 4 (relative retention with reference to glucagon about 1.4).
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3):
the resolution factor between the peak due to glucagon and the following peak due to deamidated glucagon 1 is at least 1.5;
the 4 peaks eluting after the principal peak, that correspond to the deamidated forms, are clearly visible;
and, in the chromatogram obtained with solution (2):
the symmetry factor of the peak due to glucagon is a maximum of 1.8;
the repeatability has a maximum relative standard deviation of 2.0% after 5 injections.
LIMITS
In the chromatogram obtained with solution (1):
the total area of all deamidated forms is not greater than 10.0%;
the total area of all secondary peaks is not greater than 15.0%.
Water
Not more than 5.0%, Appendix IX C, Method III. Use the entire freeze dried contents of the sealed container (containing 1 mg of the powder).
Bacterial endotoxins
Carry out the test for bacterial endotoxins, Appendix XIV C. Dissolve the contents of the sealed container in water BET to give a solution containing 1 mg of Human Glucagon per mL (solution A). The endotoxin limit concentration of solution A is 10 IU of endotoxin per mL.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the method described under Related proteins and deamidated forms, injecting solutions (1) and (2).
DETERMINATION OF CONTENT
Calculate the content of human glucagon (C153H225N43O49S) from the chromatograms obtained and using the declared content of C153H225N43O49S in human glucagon EPCRS.
STORAGE
Human Glucagon for Injection should be protected from light and stored strictly in accordance with the manufacturer’s instructions.
