2.7.11. Assay of human coagulation factor IX
Prepare a control solution that includes all components
except factor VII.
Prepare all dilutions in plastic tubes and use within 1 h.
Step 1. Mix dilutions of the factor VII reference preparation
and the preparation to be examined with an appropriate
volume of the prewarmed coagulation factor reagent or
a combination of its separate constituents, and incubate
the mixture in plastic tubes or microplate wells at 37 °C.
The concentrations of the various components during the
factor Xa generation must be as specified above under the
description of the reagents.
Allow the activation of factor X to proceed for a suitable
time, usually terminating the reaction before the factor Xa
concentration has reached its maximal level in order to
obtain a satisfactory linear dose-response relationship. The
activation time is also chosen to achieve linear production of
factor Xa in time. Appropriate activation times are usually
between 2 min and 5 min, but deviations are permissible if
acceptable linearity of the dose-response relationship is thus
obtained.
Step 2. Terminate the activation by the addition of a
prewarmed reagent containing a chromogenic substrate.
Quantify the rate of substrate cleavage, which must be linear
with the concentration of factor Xa formed, by measuring
the absorbance change at an appropriate wavelength using
a spectrophotometer, either monitoring the absorbance
continuously, thus allowing the initial rate of substrate
cleavage to be calculated, or terminating the hydrolysis
reaction after a suitable interval by lowering the pH by
the addition of a suitable reagent, such as acetic acid
(500 g/l C2H4O2) or a citrate solution (1 mol/l) at pH 3.
Adjust the hydrolysis time to achieve a linear development
of chromophore with time. Appropriate hydrolysis times
are usually between 3 min and 15 min, but deviations
are permissible if better linearity of the dose-response
relationship is thus obtained.
Check the validity of the assay and calculate the potency
of the test preparation by the usual statistical methods (for
example, 5.3. Statistical analysis of results of biological
assays and tests).
01/2005:20711
corrected
2.7.11. ASSAY OF HUMAN
COAGULATION FACTOR IX
The potency is determined by comparing the quantity of
the preparation to be examined necessary to reduce the
coagulation time of a test mixture containing the substances,
other than factor IX, that take part in the coagulation of
blood and the quantity of a reference preparation, calibrated
in International Units, required to produce the same effect.
The International Unit is the activity of a stated amount of
the International Standard, which consists of a freeze-dried
concentrate of human coagulation factor IX. The equivalence
in International Units of the International Standard is stated
by the World Health Organisation.
Human coagulation factor IX concentrate BRP is calibrated
in International Units by comparison with the International
Standard.
Reconstitute separately the preparation to be examined
and the reference preparation as stated on the label and
use immediately. Where applicable, determine the amount
of heparin present (2.7.12) and neutralise the heparin by
addition of protamine sulphate R (10 µg of protamine
sulphate neutralises 1 IU of heparin). Dilute the preparation
204 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.
to be examined and the reference preparation with a
sufficient quantity of imidazole buffer solution pH 7.3 R to
produce solutions containing 0.5 IU to 2.0 IU per millilitre.
Prepare twofold dilutions in the range 1 to 10 to 1 to 80 using
a mixture of 1 volume of a 38 g/l solution of sodium citrate R
and 5 volumes of imidazole buffer solution pH 7.3 R. Make
these dilutions accurately and use immediately.
Use, for example, incubation tubes maintained in a
water-bath at 37 °C. Place in each tube 0.1 ml of plasma
substrate R2 and 0.1 ml of one of the dilutions of the
reference preparation or of the preparation to be examined.
Add to each tube 0.1 ml of a suitable dilution of cephalin R
or platelet substitute R and 0.1 ml of a suspension of 0.5 g
of light kaolin R in 100 ml of a 9 g/l solution of sodium
chloride R and allow to stand for about 10 min, tilting
the tubes regularly. To each tube, add 0.1 ml of a 7.4 g/l
solution of calcium chloride R. Using a timer, measure the
coagulation time, i.e. the interval between the moment of the
addition of the calcium chloride and the first indication of
the formation of fibrin, which may be observed visually or by
the use of a suitable apparatus. Calculate the potency using
the usual statistical methods (for example, 5.3. Statistical
analysis of results of biological assays and tests).
To ensure that there is no appreciable contamination
of plasma substrate R2 by factor IX, carry out a blank
test using, instead of the preparation to be examined, a
corresponding volume of a mixture of 1 volume of a 38 g/l
solution of sodium citrate R and 5 volumes of imidazole
buffer solution pH 7.3 R. The test is not valid unless the
coagulation time measured in the blank test is 100 s to 200 s.
01/2005:20712
2.7.12. ASSAY OF HEPARIN IN
COAGULATION FACTORS
Heparin is assayed as a complex with antithrombin III (AT)
via its inhibition of coagulation factor Xa (anti-Xa activity).
An excess of AT is maintained in the reaction mixture to
ensure a constant concentration of the heparin-AT complex.
Factor Xa is neutralised by the heparin-AT complex and
the residual factor Xa hydrolyses a specific chromogenic
peptide substrate to release a chromophore. The quantity
of chromophore is inversely proportional to the activity of
the heparin.
Factor Xa chromogenic substrate. Specific chromogenic
substrate for factor Xa such as : N-benzoyl-L-isoleucyl-L-
glutamyl-glycyl-L-arginine-4-nitroanilide hydrochloride.
Reconstitute according to the manufacturer’s instructions.
Dilution buffer. 6.05 g/l solution of tris(hydroxy-
methyl)aminomethane R. Adjust to pH 8.4 if necessary
using hydrochloric acid R.
Test solution. Dilute the preparation to be examined with
dilution buffer to obtain a solution expected to contain
0.1 IU of heparin per millilitre.
Reference solution. Dilute the heparin reference preparation
with dilution buffer to obtain a solution containing 0.1 IU of
heparin per millilitre.
The following working conditions apply to microtitre plates.
If the assay is carried out in tubes, the volumes are adjusted
while maintaining the proportions in the mixture.
Warm all solutions to 37 °C in a water-bath shortly before
the test.
Distribute in a series of wells, 20 µl of normal human plasma
and 20 µl of antithrombin III solution R1. Add to the wells a
series of volumes (20 µl, 60 µl, 100 µl and 140 µl) of the test
EUROPEAN PHARMACOPOEIA 5.0 2.7.13. Assay of human anti-D immunoglobulin

solution or the reference solution and make up the volume
in each well to 200 µl using dilution buffer (0.02-0.08 IU of
heparin per millilitre in the final reaction mixture).
End-point method. Transfer 40 µl from each well to a second
series of wells, add 20 µl of bovine factor Xa solution R and
incubate at 37 °C for 30 s. Add 40 µl of a 1 mmol/l solution
of factor Xa chromogenic substrate and incubate at 37 °C
for 3 min. Terminate the reaction by lowering the pH by the
addition of a suitable reagent, such as a 20 per cent V/V
solution of glacial acetic acid R and measure the absorbance
at 405 nm (2.2.25). Appropriate reaction times are usually
between 3 min and 15 min, but deviations are permissible
if better linearity of the dose-response relationship is thus
obtained.
Kinetic method. Transfer 40 µl from each well to a second
series of wells, add 20 µl of bovine factor Xa solution R and
incubate at 37 °C for 30 s. Add 40 µl of a 2 mmol/l solution
of factor Xa chromogenic substrate, incubate at 37 °C
and measure the rate of substrate cleavage by continuous
measurement of the absorbance change at 405 nm (2.2.25),
thus allowing the initial rate of substrate cleavage to be
calculated. This rate must be linear with the concentration
of residual factor Xa.
Check the validity of the assay and calculate the heparin
activity of the test preparation by the usual statistical
methods for a slope-ratio assay (for example, 5.3. Statistical
analysis of results of biological assays and tests).
Calculate the potency of the preparation to be examined
01/2005:20713 using the usual statistical methods (5.3).
at 15.0 °C. Pump into the manifold of the apparatus the red
blood cell suspension at a rate of 0.1 ml/min and a 3 g/l
solution of methylcellulose 450 R at a rate of 0.05 ml/min.
Introduce the dilutions of the preparation to be examined
and the reference preparation at a rate of 0.1 ml/min for
2 min, followed by the diluent solution at a rate of 0.1 ml/min
for 4 min before the next dilution is introduced.
Introduce air at a rate of 0.6 ml/min. Incubate at 37 °C
for 18 min and then disperse the rouleaux by introducing
at a rate of 1.6 ml/min a 9 g/l solution of sodium
chloride R containing a suitable wetting agent (for example,
polysorbate 20 R at a final concentration of 0.2 g/l)
to prevent disruption of the bubble pattern. Allow the
agglutinates to settle and decant twice, first at 0.4 ml/min
and then at 0.6 ml/min. Lyse the unagglutinated red blood
cells with a solution containing 5 g/l of octoxinol 10 R,
0.2 g/l of potassium ferricyanide R, 1 g/l of sodium
hydrogen carbonate R and 0.05 g/l of potassium cyanide R
at a rate of 2.5 ml/min. A ten-minute delay coil is introduced
to allow for conversion of the haemoglobin. Continuously
record the absorbance (2.2.25) of the haemolysate at a
wavelength between 540 nm and 550 nm. Determine the
range of antibody concentrations over which there is a linear
relationship between concentration and the resultant change
in absorbance (∆A). From the results, prepare a standard
curve and use the linear portion of the curve to determine
the activity of the preparation to be examined.

2.7.13. ASSAY OF HUMAN ANTI-D METHOD B

IMMUNOGLOBULIN The potency of human anti-D immunoglobulin is
determined by competitive enzyme-linked immunoassay on
METHOD A erythrocyte-coated microtitre plates. The method is based
The potency of human anti-D immunoglobulin is on the competitive binding between a polyclonal anti-D
determined by comparing the quantity necessary to produce immunoglobulin preparation and a biotinylated monoclonal
agglutination of D-positive red blood cells with the quantity anti-D antibody directed against a D-antigen specific epitope.
of a reference preparation, calibrated in International Units, The activity of the preparation to be examined is compared
required to produce the same effect. with a reference preparation calibrated in International Units.
The International Unit is the activity contained in a stated The International Unit is the activity of a stated amount
amount of the International Reference Preparation. The of International Reference Preparation. The equivalence
equivalence in International Units of the International in International Units of the International Reference
Reference Preparation is stated by the World Health Preparation is stated by the World Health Organisation.
Organisation.
Human anti-D immunoglobulin BRP is calibrated in Human anti-D immunoglobulin BRP is calibrated in
International Units by comparison with the International International Units by comparison with the International
Standard and intended for use in the assay of human anti-D Standard and intended for use in the assay of human anti-D
immunoglobulin. immunoglobulin.
Use pooled D-positive red blood cells, collected not more MATERIALS
than 7 days earlier and suitably stored, obtained from not Reagents not specified are of analytical grade.
fewer than 4 group O R1R1 donors. To a suitable volume of
PBS (Phosphate-buffered saline). Dissolve 8.0 g of sodium
the cells, previously washed 3 times with a 9 g/l solution
chloride R, 0.76 g of anhydrous disodium hydrogen
of sodium chloride R, add an equal volume of bromelains
phosphate R, 0.2 g of potassium chloride R, 0.2 g of
solution R, allow to stand at 37 °C for 10 min, centrifuge,
potassium dihydrogen phosphate R and 0.2 g of sodium
remove the supernatant liquid and wash 3 times with a 9 g/l
azide R in water R and dilute to 1000 ml with the same
solution of sodium chloride R. Suspend 20 volumes of the
solvent.
red blood cells in a mixture of 15 volumes of inert serum,
20 volumes of a 300 g/l solution of bovine albumin R and TBS (Tris-buffered saline). Dissolve 8.0 g of sodium
45 volumes of a 9 g/l solution of sodium chloride R. Stand chloride R and 0.6 g of tris(hydroxymethyl) aminomethane R
the resulting suspension in iced water, stirring continuously. in water R. Adjust to pH 7.2 (2.2.3) with 1 M hydrochloric
Using a calibrated automated dilutor, prepare suitable acid and dilute to 1000 ml with the same solvent.
dilutions of the preparation to be examined and of the Papain solution. Prepare a solution by stirring 1 g of
reference preparation using as diluent a solution containing papain R at 37 °C for 30 min in 10 ml of 0.067 M phosphate
5 g/l of bovine albumin R and 9 g/l of sodium chloride R. buffer solution pH 5.4 R, centrifuge at 10 000 g for 5 min
Use a suitable apparatus for automatic continuous analysis. and filter through a membrane with a pore size of 0.22 µm.
The following protocol is usually suitable : maintain the To activate, combine 1 ml of the filtrate with 1 ml of a
temperature in the manifold, except for the incubation coils, 48.44 g/l solution of L-cysteine R and 1 ml of a 3.72 g/l

General Notices (1) apply to all monographs and other texts 205