Human albumin solution EUROPEAN PHARMACOPOEIA 9.

Top of the plate Water content Refractive index at 20 °C

_______ _______ (per cent m/m)
18.4 1.4905
Fructose : an intense brown zone An intense brown zone (fructose)
18.6 1.4900
Glucose : an intense greyish-blue An intense greyish-blue zone
zone (glucose) 18.8 1.4895
_______ _______
19.0 1.4890

19.2 1.4885
Sucrose : a brown zone 19.4 1.4880
2 to 3 brownish-grey zones 19.6 1.4875
Reference solution Test solution 19.8 1.4870

TESTS
Refractive index (2.2.6): minimum 1.487 (equivalent to a
maximum water content of 20 per cent).
Homogenise 100 g and transfer into a flask. Close tightly and
place in a water-bath at 50 ± 0.2 °C until all sugar crystals
have dissolved. Cool the solution to 20 °C and rehomogenise.
Immediately after rehomogenisation, cover the surface of the
refractometer prism evenly with the sample. Determine the
refractive index after 2 min if using an Abbe refractometer and
after 4 min if using a digital refractometer. Use the average
value of 2 determinations.
Conductivity (2.2.38) : maximum 800 μS·cm− 1.
Using the value obtained for the refractive index, determine
the water content of the substance to be examined from
Table 2051.-1. Using this information, dissolve an amount of
the substance to be examined equivalent to 20.0 g of honey
dry solids, in water R to produce 100.0 mL.
Optical rotation (2.2.7) : maximum + 0.6°.
Using the value obtained for the refractive index, determine
the water content of the substance to be examined from
Table 2051.-1. Using this information, dissolve an amount of
the substance to be examined, equivalent to 20.0 g of honey
dry solids, in 50 mL of water R. Add 0.2 mL of concentrated
ammonia R and dilute to 100.0 mL with water R. If necessary
decolourise the solution with activated charcoal R.
Table 2051.-1. – Relationship of water content of honey to
refractive index
Water content Refractive index at 20 °C
(per cent m/m)
15.0 1.4992
15.2 1.4987
15.4 1.4982
15.6 1.4976
20.0 1.4865
5-Hydroxymethylfurfural : maximum 80 ppm, calculated
on dry solids.
Using the value obtained for the refractive index, determine
the water content of the substance to be examined from
Table 2051.-1. Using this information, dissolve an amount of
the substance to be examined, equivalent to 5.0 g of honey
dry solids, in 25 mL of water R and transfer to a 50.0 mL
volumetric flask with the same solvent. Add 0.5 mL of a
150 g/L solution of potassium ferrocyanide R and mix. Add
0.5 mL of a 300 g/L solution of zinc acetate R, mix and dilute
to 50.0 mL with water R (a drop of anhydrous ethanol R may
be added to avoid foaming). Filter. Transfer 5.0 mL of the
filtered solution into each of 2 tubes. To one tube add 5.0 mL
of water R (test solution). To the other tube add 5.0 mL of
a 2.0 g/L solution of sodium hydrogensulfite R (reference
solution). Determine the absorbance (2.2.25) of the test
solution against the reference solution at 284 nm and 336 nm
within 60 min. If the absorbance at 284 nm is greater than
0.8, dilute to the same extent the test solution with water R
and the reference solution with a 2.0 g/L solution of sodium
hydrogensulfite R so as to obtain an absorbance of less than 0.8.
Calculate the content of 5-hydroxymethylfurfural from the
expression :
A1 = absorbance at 284 nm,
A2 = absorbance at 336 nm,
D = dilution factor, where applicable.
Chlorides (2.4.4) : maximum 350 ppm, determined on 15 mL
of a 10 g/L solution.
Sulfates (2.4.13) : maximum 250 ppm, determined on 15 mL
of a 40 g/L solution.

15.8 1.4971

16.0 1.4966 01/2013:0255

corrected 8.0
16.2 1.4961

16.4 1.4956

16.6 1.4951

16.8 1.4946
HUMAN ALBUMIN SOLUTION
17.0 1.4940

17.2 1.4935 Albumini humani solutio

17.4 1.4930 DEFINITION
17.6 1.4925 Sterile liquid preparation of a plasma protein fraction
17.8 1.4920
containing human albumin. It is obtained from plasma that
complies with the monograph Human plasma for fractionation
18.0 1.4915 (0853). The preparation may contain excipients such as
18.2 1.4910
sodium caprylate (sodium octanoate) or N-acetyltryptophan
or a combination of the two.

2660 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 9.0
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
General Notices (1) apply to all monographs and other texts
Human albumin solution
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.
Column :
– size : l = 0.6 m, Ø = 7.5 mm, or l = 0.3 m, Ø = 7.8 mm ;
– stationary phase : hydrophilic silica gel for chromatography R,
of a grade suitable for fractionation of globular proteins
with relative molecular masses in the range 10 000 to
500 000.
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.
2661
Human anti-D immunoglobulin EUROPEAN PHARMACOPOEIA 9.0

Atomisation device : furnace ; fire between readings. 01/2015:0557

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.
Table 0255.-1. – Operating conditions found suitable, cited as
an example HUMAN ANTI-D IMMUNOGLOBULIN
Step Final Ramp time Hold time Gas
temperature (s) (s) Immunoglobulinum humanum anti-D
(°C)
1 120 10 80 argon DEFINITION
2 200 5 20 argon Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G. The
3 650 5 10 argon preparation is intended for intramuscular administration. It
4 1300 5 10 argon contains specific antibodies against erythrocyte D-antigen
and may also contain small quantities of other blood-group
5 1300 1 10 no gas antibodies. Human normal immunoglobulin for intramuscular
6 2500 0.7 4 no gas administration (0338) and/or Human albumin solution (0255)
may be added.
7 2600 0.5 3 argon
It complies with the monograph Human normal
8 20 12.9 3 no gas immunoglobulin for intramuscular administration (0338),
except for the minimum number of donors and the minimum
Injection : each of the following solutions 3 times : blank total protein content.
solution, reference solutions, test solution and monitor The assay of human anti-D immunoglobulin (2.7.13) is carried
solution. out, as prescribed below under Potency.
System suitability : For products prepared by a method that eliminates
– the recovery of aluminium added in preparation of the immunoglobulins with specificities other than anti-D, where
monitor solution is within the range 80-120 per cent. authorised, the test for antibodies to hepatitis B surface
Prepare a calibration curve from the mean of the readings antigen is not required.
obtained with the reference solutions and determine the
aluminium content of the preparation to be examined using PRODUCTION
the calibration curve. Human anti-D immunoglobulin is preferably obtained from
Potassium : maximum 0.05 mmol of K per gram of protein. the plasma of donors with a sufficient titre of previously
Atomic emission spectrometry (2.2.22, Method I). acquired anti-D antibodies. Where necessary, in order to
ensure an adequate supply of human anti-D immunoglobulin,
Wavelength : 766.5 nm. it is obtained from plasma derived from donors immunised
Sodium : maximum 160 mmol/L and 95 per cent to 105 per with D-positive erythrocytes that are compatible in relevant
cent of the content of Na stated on the label. blood group systems in order to avoid formation of undesirable
Atomic emission spectrometry (2.2.22, Method I). antibodies.
Wavelength : 589 nm. ERYTHROCYTE DONORS
Sterility (2.6.1). It complies with the test. Erythrocyte donors comply with the requirements for
donors prescribed in the monograph Human plasma for
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14). It
fractionation (0853).
complies with the test for pyrogens or, preferably and where
justified and authorised, with a validated in vitro test such as IMMUNISATION
the bacterial endotoxin test. Immunisation of the plasma donor is carried out under
For the pyrogen test, for a solution with a protein content of proper medical supervision. Recommendations concerning
35-50 g/L, inject 10 mL per kilogram of the rabbit’s mass ; for donor immunisation, including testing of erythrocyte donors,
a solution with a protein content of 150-250 g/L, inject 5 mL have been formulated by the World Health Organization
per kilogram of the rabbit’s mass. (Requirements for the collection, processing and quality control
Where the bacterial endotoxin test is used, the preparation of blood, blood components and plasma derivatives, WHO
to be examined contains less than 0.5 IU of endotoxin per Technical Report Series, No. 840, 1994 or subsequent revision).
millilitre for solutions with a protein content not greater than POOLED PLASMA
50 g/L, less than 1.3 IU of endotoxin per millilitre for solutions To limit the potential B19 virus burden in plasma pools used
with a protein content greater than 50 g/L but not greater for the manufacture of anti-D immunoglobulin, the plasma
than 200 g/L, and less than 1.7 IU of endotoxin per millilitre pool is tested for B19 virus using validated nucleic acid
for solutions with a protein content greater than 200 g/L but amplification techniques (2.6.21).
not greater than 250 g/L. B19 virus DNA : maximum 10.0 IU/μL.
STORAGE A positive control with 10.0 IU of B19 virus DNA per
Protected from light. microlitre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
LABELLING plasma pool are included in the test. The test is invalid if the
The label states : positive control is non-reactive or if the result obtained with
the internal control indicates the presence of inhibitors.
– the name of the preparation ;
B19 virus DNA for NAT testing BRP is suitable for use as a
– the volume of the preparation ;
positive control.
– the content of protein expressed in grams per litre ;
If Human normal immunoglobulin for intramuscular
– the content of sodium expressed in millimoles per litre ; administration (0338) and/or Human albumin solution (0255)
– that the product is not to be used if it is cloudy or if a are added to the preparation, the plasma pool or pools from
deposit has formed ; which they are derived comply with the above requirement for
– the name and quantity of any added substance. B19 virus DNA.

2662 See the information section on general monographs (cover pages)