Monography

Albumin solution, Human
Albumini humani solutio

Definition
Human albumin solution is an aqueous solution of protein obtained from plasma that complies with the requirements of the monograph on Plasma for fractionation, human.

Production
Separation of the albumin is carried out under controlled conditions, particullary of pH, ionic strenght and temperature so that in the final product not less than 95 per cent of the total protein is albumin. Human albumin solution is prepared as a concentrated solution containing 150 g/l to 250 g/l of total protein or as an isotonic solution containing 35 g /l to 50 g/l of total protein. A suitable stabiliser against the effects of heat, such as sodium caprylate (sodium octanoate) or N-acetyltryptophan or a combination of these two, at a suitable concentration, may be added but no antimicrobial preservative is added at any stage during preparation. The solution is passed through a bacteria-retentive filter and distribute aseptically into sterile containers which are then closed so as to prevent contamination: The solution in its final container is heated to ± 60 0.5 ºC and maintained at this temperature for not less than 10 h. The containers are then incubated at 30 ºC to 32ºC for not less than 14 days or at 20 ºC to 25ºC for not less than 4 weeks and examined visually for evidance of microbal contamination.

Characters
A clear, slightly viscous liquid; it is almoust colourless, yellow or green.

Identification
a. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparation to be examined. It is recommended that the test be carried out using antisera specific to the plasma proteins of each species of domestic animal commonly used in the preparation of materials of biological origin in the country concerned. The preparation is shown to contains proteins of human origin and gives negative results with antisera specific to plasma proteins of other species.
b. Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal serum, compare normal human serum and the preparation to be examined, both diluted to contain 10 g/l of protein. The main component of normal human serum. The preparation may show the presence of small quantities of other plasma proteins.

TESTS
PH (2.2.3). Dilute the preparation to be examined with a 9 g/l solution of sodium chloride R to obtain a solution containing 10 g/l of protein. The pH of the solution is 6.7 to 7.3.

Total protein.
Dilute the preparation to be examined with a 9 g/l solution of sodium of sodium chloride R to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of this solution in a round bottomed centrifuge tube add 2 ml of a 75 g/l solution of sodium molybdate R and 2ml of a mixture of 1 volume of nitrogen-free sulphuric acid R and 30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion and calculate the quantity of protein by multiplying by 6.25. The preparation contains not less than 95 per cent and not more than 105 per cent of the quantity of protein stated on the label.

Protein composition.
Examine by zone electrophoresis, using strips of suitable cellulose acetate gel as the supporting medium and barbital buffer solution pH 8.6 R1 as the electrolyte solution.

Test solution.
Dilute the preparation to be examined with a 9g /l solution of sodium chloride R to a protein concentration of 20 g/l.

Reference solution.
Dilute human albumin for electrophoresis BRP with a 9 g/l solution of sodium chloride R to a protein concentration of 20 g/l.

To a strip apply 2.5 µl of the solution as a 10mm band or apply 0.25 µl per milimetre if a narrower strip is used. To another strip apply in the same manner the same volume of reference solution. Apply a suitable electric field such that the most rapid band migrates at least 30 mm. Treat the strips with amido black 10B solution R for 5 min. Decolorise with a mixture of 10 volumes of glacial acetic acid R and 90 volumes of methanol R until the background is just free of colour. Develop the transparency of the strips with a mixture of 19 volumes of glacial acetic acid R and 81 volumes of methanol R. Measure the absorbance of the bands at 600 nm in an instrument having a linear response over the range of measurement. Calculate the result as the mean of three measurements of each strip. In the electrophoretogram obtained with the test solution, not more than 5 per cent of the protein has a mobility different from that of the principal band. The test is not valid unless, in the electrophoretogram obtained with the reference preparation, the proportion of protein in the principal band is within the limits stated in the leaflet accompanying the reference preparation.

Polymers and aggregrates.
Examine by liquid chromatography (2.2.29).

Test solution.
Dilute the preparation to be examined with a 9 g/l solution of sodium chloride R to a concentration suitable for the chromatographic system used. A concentration in the range 4 g/l to 12 g/l and injection of 50 µg to 600 µg of protein are usually suitable.

The chromatographic procedure may be carried out using:
• a column 0.6m long and 7.5 mm in internal diameter packed with hydrophilic silica gel for chromatography R,
• as a mobile phase at a flow rate of 0.5ml per minute a solsution containing per litre: 4.873g of disodium hydrogend phosphate dihydrate R, 1.741 g of sodium dihydrogen phosphate monohydrate R, 11.688 g of sodium chloride R and 50 mg of sodium azide R,
• a detector set at 280nm

The peak corresponding to polymers and aggregrates is located in the part of the chromatogram representing the void volume. The area of this peak divided by 2 is not greater than 5 per cent of the total area of the chromatogram.

Haem.
Dilute the preparation to be examined using a 9 g/l solution of sodium chloride R to obtain a solution containing 10 g/l of protein. The absorbance (2.2.25) of the solution measured at 403 nm using water R as the compensation liquid is not greater than 0.15.

Prekallikrein activator. (2.6.15). Not more than 35 I.U. per millilitre.

Aluminium.
If intended for administration to patients under going dialysis or to premature infants, it complies with the test for aluminium. Not more than 200 µg of Al per litre, determined by atomic absorption spectrometry (Method I, 2.2.23), using a furnace as atomic generator.

Use plastic containers for preparation of the solutions. Wash equipment in nitric acid (200 g/l HNO3) before use.

Test solution.
Use the preparation to be examined.

Validation solution.
Use human albumin for aluminium validation BRP.

Reference solutions.
Prepare a suiteble range of reference solutions by adding suiteble volumes of aluminium standard solution (10ppm Al) R to knowm volumes of water R.

Dilute the solutions as necessary using nitric acid (10 g/l HNO3) containing 1.7 g/l of magnesium nitrate R and 0.05 per cent V/V of octoxinol 10 R. Measure the absorbance at 309.3 nm. The test is not valid unless the aluminium content determined for human albumin for aluminium validation BRP is within 20 per cent of the value stated in the leaflet accompanying the reference preparation.

Potassium.
Not more than 0.05 mmol of K per gram of protein, determined by atomic emission spectrometry (Method I, 2.2.22). Measure the emission intensity at 766 nm.

Sodium.
Not more than 160 mmol of Na per litre and not less than 95 per cent and not more than 105 per cent of the content of Na stated on the label, determined by atomic emission spectrometry (method 1,2.2.22). Measure the emission intensity at 589 nm.

Sterility (2.6.1).
It complies with the test for sterility.

Pyrogens (2.6.8).
It complies with the test for pyrogens. For a solution containing 35 /g/ to 50 g/l of protein, inject per kilogram of the rabbit's mass 10 ml of the preparation to be examined. For a solution containing 150 g/l to 250 g/l of protein, inject per kilogram of the rabbit's mass 3 ml of the preparation to be examined.

STORAGE

Store protected from light

LABELLING