Albumin Solution
Ph Eur
DEFINITION
Sterile liquid preparation of a plasma protein fraction containing human albumin. It is obtained from plasma that complies with the monograph Human plasma for fractionation (0853). The preparation may contain excipients such as sodium caprylate (sodium octanoate) or N-acetyltryptophan or a combination of the two.
PRODUCTION
Separation of the albumin is carried out under controlled conditions, particularly of pH, ionic strength 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 150-250 g/L of total protein or as an isotonic solution containing 35-50 g/L of total protein. No antimicrobial preservative or antibiotic is added. The solution is passed through a bacteria-retentive filter and distributed aseptically into sterile containers which are then closed so as to prevent contamination. The solution in its final container is heated to 60 ± 1.0 °C and maintained at this temperature for not less than 10 h. The containers are then incubated at 30-32 °C for not less than 14 days or at 20-25 °C for not less than 4 weeks and examined visually for evidence of microbial contamination.
CHARACTERS
Appearance
Clear, slightly viscous liquid, almost colourless, yellow, amber or green.
IDENTIFICATION
Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal human serum, compare normal human serum and the preparation to be examined, both diluted to contain 10 g/L of protein. The main component of the preparation to be examined corresponds to 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)
6.7 to 7.3.
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.
Total protein
If necessary, dilute an accurately measured volume of the preparation to be examined with a 9 g/L solution 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 2 mL of a mixture of 1 volume of nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulfuric acid digestion (2.5.9) and calculate the quantity of protein by multiplying by 6.25. The protein content is not less than 95 per cent and not more than 105 per cent of the stated content.
Protein composition
Zone electrophoresis (2.2.31).
Use strips of suitable cellulose acetate gel or agarose gel as the supporting medium and barbital buffer solution pH 8.6 R1 as the electrolyte solution.
If cellulose acetate is the supporting material, the method described below can be used. If agarose gels are used, and because they are normally part of an automated system, the manufacturer′s instructions are followed instead.
Test solution Dilute the preparation to be examined with a 9 g/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 test solution as a 10 mm band or apply 0.25 µL per millimetre if a narrower strip is used. To another strip, apply in the same manner the same volume of the 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 3 measurements of each strip.
System suitability In the electropherogram obtained with the reference solution on cellulose acetate or on agarose gels, the proportion of protein in the principal band is within the limits stated in the leaflet accompanying the reference preparation.
Results In the electropherogram obtained with the test solution on cellulose acetate or on agarose gels, not more than 5 per cent of the protein has a mobility different from that of the principal band.
Molecular-size distribution
Size exclusion chromatography (2.2.30).
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 of 4-12 g/L and injection of 50-600 µg of protein are usually suitable.
Mobile phase Dissolve 4.873 g of disodium hydrogen 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 in 1 L of water R.
Flow rate 0.5 mL/min.
Detection Spectrophotometer at 280 nm.
The peak due to polymers and aggregates is located in the part of the chromatogram representing the void volume. Disregard the peak due to the stabiliser. The area of the peak due to polymers and aggregates is not greater than 10 per cent of the total area of the chromatogram. This represents not more than 5 per cent when expressed in percentage of protein considering the difference in response factor between the albumin monomer and the polymers and aggregates.
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)
Maximum 35 IU/mL.
Aluminium
Maximum 200 µg/L.
Atomic absorption spectrometry (2.2.23, Method I or II).
Use a furnace as atomic generator.
Use plastic containers for preparation of the solutions and use plastic equipment where possible. Wash glassware (or equipment) in nitric acid (200 g/L HNO3) before use.
Test solution Use the preparation to be examined, diluted if necessary.
Reference solutions Prepare at least 3 reference solutions in a range spanning the expected aluminium concentration of the test solution, for example by diluting aluminium standard solution (10 ppm Al) R with a 1 g/L solution of octoxinol 10 R.
Monitor solution Add aluminium standard solution (10 ppm Al) R or a suitable certified reference material to the test solution in a sufficient amount to increase the aluminium concentration by 20 µg/L.
Blank solution 1 g/L solution of octoxinol 10 R.
Wavelength 309.3 nm or other suitable wavelength.
Slit width 0.5 nm.
Tube Pyrolytically coated, with integrated platform.
Background corrector Off.
Atomisation device Furnace; fire between readings.
The operating conditions in Table 0255.-1 are cited as an example of conditions found suitable for a given apparatus; they may be modified to obtain optimum conditions.
Injection Each of the following solutions 3 times: blank solution, reference solutions, test solution and monitor solution.
Prepare a calibration curve from the mean of the readings obtained with the reference solutions and determine the aluminium content of the preparation to be examined using the calibration curve.
Potassium
Maximum 0.05 mmol of K per gram of protein.
Atomic emission spectrometry (2.2.22, Method I).
Wavelength 766.5 nm.
Sodium
Maximum 160 mmol/L and 95 per cent to 105 per cent of the content of Na stated on the label.
Atomic emission spectrometry (2.2.22, Method I).
Wavelength 589 nm.
Sterility (2.6.1)
It complies with the test.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
It complies with the test for pyrogens or, preferably and where justified and authorised, with a validated in vitro test such as the bacterial endotoxin test.
For the pyrogen test, for a solution with a protein content of 35-50 g/L, inject 10 mL per kilogram of the rabbit’s mass; for a solution with a protein content of 150-250 g/L, inject 5 mL per kilogram of the rabbit’s mass.
Where the bacterial endotoxin test is used, the preparation to be examined contains less than 0.5 IU of endotoxin per millilitre for solutions with a protein content not greater than 50 g/L, less than 1.3 IU of endotoxin per millilitre for solutions with a protein content greater than 50 g/L but not greater than 200 g/L, and less than 1.7 IU of endotoxin per millilitre for solutions with a protein content greater than 200 g/L but not greater than 250 g/L.
STORAGE
Protected from light.
LABELLING
Ph Eur