BP_05 - 2001

Monographs

Blood Products

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BLOOD PRODUCTS

Anticoagulant and Preservative Solutions for Blood

Anticoagulant and Preservative Solutions for Blood comply with the requirements of the 3rd edition of theEuropean Pharmacopoeia for Anticoagulant and Preservative Solutions for Human Blood [0209]. Theserequirements are reproduced after the heading Definition below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Anticoagulant and preservative solutions for human blood are sterile and pyrogen-free solutionsprepared with water for injections, filtered, distributed in the final containers and sterilised. Thecontent of sodium citrate (C6H5Na3O7,2H2O), glucose monohydrate (C6H12O6,H2O) or anhy-drous glucose (C6H12O6) and sodium dihydrogen phosphate dihydrate (NaH2PO4,2H2O) is notless than 95.0 per cent and not more than 105.0 per cent of that stated in the formulae below. Thecontent of citric acid monohydrate (C6H8O7,H2O) or anhydrous citric acid (C6H8O7) is not lessthan 90.0 per cent and not more than 110.0 per cent of that stated in the formulae below. Subjectto agreement by the competent authority, other substances, such as red-cell preservatives, may beincluded in the formula provided that their name and concentration are stated on the label.

Anticoagulant and preservative solutions for human blood are presented in airtight, tamper-proofcontainers of glass (3.2.1) or plastic (3.2.3).

ANTICOAGULANT ACID-CITRATE-GLUCOSE SOLUTIONS (ACD)

A B

Sodium citrate (412) 22.0 g 13.2 g

Citric acid monohydrate (456) 8.0 g 4.8 gor Anhydrous citric acid (455) 7.3 g 4.4 g

Glucose monohydrate (178)* 24.5 g 14.7 gor Glucose, anhydrous (177)* 22.3 g 13.4 g

Water for injections (169) to 1000.0 ml 1000.0 ml

Volume to be used per 100 ml 15.0 ml 25.0 mlof blood

*The competent authority may require that the substance comply with the test for pyrogens givenin the monographs on Glucose monohydrate (178) and Glucose, anhydrous (177), respectively.

CHARACTERS

A colourless or faintly yellow, clear liquid, practically free from particles.

IDENTIFICATION

A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.

Test solution. Dilute 2 ml of the solution to be examined (for formula A) or 3 ml (for formula B) to100 ml with a mixture of 2 volumes of water R and 3 volumes of methanol R.

Reference solution (a). Dissolve 10 mg of glucose CRS in a mixture of 2 volumes of water R and 3volumes of methanol R and dilute to 20 ml with the same mixture of solvents.

Reference solution (b). Dissolve 10 mg each of glucose CRS, lactose CRS, fructose CRS and sucroseCRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with thesame mixture of solvents.

Apply separately to the plate 2 l of each solution and thoroughly dry the starting points. Developover a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The volumes of solventshave to be measured accurately since a slight excess of water produces cloudiness. Dry the plate ina current of warm air. Repeat the development immediately, after renewing the mobile phase. Drythe plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixtureof 5 ml of sulphuric acid R and 95 ml of alcohol R. Heat at 130C for 10 min. The principal spot inthe chromatogram obtained with the test solution is similar in position, colour and size to theprincipal spot in the chromatogram obtained with reference solution (a). The test is not valid unlessthe chromatogram obtained with reference solution (b) shows four clearly separated spots.

B. To 2 ml add 5 ml of cupri-citric solution R. Heat to boiling. An orange precipitate is formed andthe solution becomes yellow.

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C. To 2 ml (for formula A) add 3 ml of water R or to 4 ml (for formula B) add 1 ml of water R. Thesolution gives the reaction of citrates (2.3.1).

D. 0.5 ml gives reaction (b) of sodium (2.3.1).

TESTS

pH (2.2.3). The pH of the solution to be examined is 4.7 to 5.3.

Hydroxymethylfurfural To 2.0 ml add 5.0 ml of a 100 g/l solution of p-toluidine R in 2-prop-anol R containing 10 per cent V/V of glacial acetic acid R and 1.0 ml of a 5 g/l solution of barbituricacid R. The absorbance (2.2.25), determined at 550 nm after allowing the mixture to stand for 2min to 3 min, is not greater than that of a standard prepared at the same time in the same mannerusing 2.0 ml of a solution containing 5 ppm of hydroxymethylfurfural R for formula A or 3 ppm ofhydroxymethylfurfural R for formula B.

Sterility (2.6.1). They comply with the test for sterility.

Pyrogens (2.6.8). They comply with the test for pyrogens. Dilute with a pyrogen-free, 9 g/lsolution of sodium chloride R to obtain a solution containing approximately 5 g/l of sodium citrate R.Inject 10 ml of the diluted solution per kilogram of the rabbits mass.

ASSAY

Citric acid To 10.0 ml (for formula A) or to 20.0 ml (for formula B) add 0.1 ml of phenolphthaleinsolution R1. Titrate with 0.2M sodium hydroxide until a pink colour is obtained.

1 ml of 0.2M sodium hydroxide is equivalent to 14.01 mg of C6H8O7,H2O or to 12.81 mg ofC6H8O7.

Sodium citrate Prepare a chromatography column 0.1 m long and 10 mm in internal diameterand filled with strongly acidic ion exchange resin R (300 m to 840 m). Maintain a 1 cm layer ofliquid above the resin at all times. Wash the column with 50 ml of de-ionised water R at a flow rateof 12 ml to 14 ml per minute.

Dilute 10.0 ml of the solution to be examined (for formula A) or 15.0 ml (for formula B) toabout 40 ml with de-ionised water R in a beaker and transfer to the column reservoir, washing thebeaker three times with a few millilitres of de-ionised water R. Allow the solution to run through thecolumn at a flow rate of 12 ml to 14 ml per minute and collect the eluate. Wash the column withtwo quantities, each of 30 ml, and with one quantity of 50 ml, of de-ionised water R. The columncan be used for three successive determinations before regeneration with three times its volume ofdilute hydrochloric acid R. Titrate the combined eluate and washings (about 150 ml) with 0.2Msodium hydroxide, using 0.1 ml of phenolphthalein solution R1 as indicator.

Calculate the content of sodium citrate in grams per litre from the following expressions:For formula A: 1.961n 1.40C or 1.961n 1.53CFor formula B: 1.307n 1.40C or 1.307n 1.53C

n=number of millilitres of 0.2M sodium hydroxide used in the titration,C =content of citric acid monohydrate in grams per litre determined as prescribed above,

C =content of anhydrous citric acid in grams per litre determined as prescribed above.

Reducing sugars Dilute 5.0 ml (for formula A) or 10.0 ml (for formula B) to 100.0 ml withwater R. Introduce 25.0 ml of the solution into a 250 ml conical flask with ground-glass neck andadd 25.0 ml of cupri-citric solution R1. Add a few pieces of porous material, attach a refluxcondenser, heat so that boiling begins within 2 min and boil for exactly 10 min. Cool and add 3 gof potassium iodide R dissolved in 3 ml of water R. Add 25 ml of a 25 per cent m/m solution ofsulphuric acid R with caution and in small quantities. Titrate with 0.1M sodium thiosulphate using0.5 ml of starch solution R, added towards the end of the titration, as indicator (n1 ml). Carry out ablank titration using 25.0 ml of water R (n2 ml).

Calculate the content of reducing sugars as anhydrous glucose or as glucose monohydrate, asappropriate, from the Table 2091.

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Table 2091

Volume of 0.1Msodiumthiosulphate(n2 n1 ml)

Anhydrousglucose inmilligrams

Glucose monohydratein milligrams

8 19.8 21.69 22.4 24.510 25.0 27.411 27.6 30.212 30.3 33.113 33.0 36.114 35.7 39.015 38.5 42.116 41.3 45.2

STORAGE

Store in an airtight, tamper-proof container, protected from light.

LABELLING

The label states: the composition and volume of the solution, the maximum amount of blood to be collected in the container.

ANTICOAGULANT CITRATE-PHOSPHATE-GLUCOSE SOLUTION (CPD)

Sodium citrate (412) 26.3g

Citric acid monohydrate (456) 3.27 gor Anhydrous citric acid (455) 2.99 g

Glucose monohydrate (178)* 25.5 gor Glucose, anhydrous (177)* 23.2 g

Sodium dihydrogen phosphate 2.51 gdihydrate (194)

Water for injections (169) to 1000.0 ml

Volume to be used per 100 ml 14.0 mlof blood

*The competent authority may require that the substance comply with the test for pyrogens givenin the monographs on Glucose monohydrate (178) and Glucose, anhydrous (177), respectively.

CHARACTERS

A colourless or faintly yellow, clear liquid, practically free from particles.

IDENTIFICATION

A. Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating substance.

Test solution. Dilute 2 ml of the solution to be examined to 100 ml with a mixture of 2 volumes ofwater R and 3 volumes of methanol R.

Reference solution (a). Dissolve 10 mg of glucose CRS in a mixture of 2 volumes of water R and 3volumes of methanol R and dilute to 20 ml with the same mixture of solvents.

Reference solution (b). Dissolve 10 mg each of glucose CRS, lactose CRS, fructose CRS and sucroseCRS in a mixture of 2 volumes of water R and 3 volumes of methanol R and dilute to 20 ml with thesame mixture of solvents.

Apply separately to the plate 2 l of each solution and thoroughly dry the starting points. Developover a path of 15 cm using a mixture of 10 volumes of water R, 15 volumes of methanol R, 25volumes of anhydrous acetic acid R and 50 volumes of ethylene chloride R. The volumes of solventshave to be measured accurately since a slight excess of water produces cloudiness. Dry the plate ina current of warm air. Repeat the development immediately, after renewing the mobile phase. Drythe plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in a mixtureof 5 ml of sulphuric acid R and 95 ml of alcohol R. Heat at 130C for 10 min. The principal spot inthe chromatogram obtained with the test solution is similar in position, colour and size to theprincipal spot in the chromatogram obtained with reference solution (a). The test is not valid unlessthe chromatogram obtained with reference solution (b) shows four clearly separated spots.

B. To 2 ml add 5 ml of cupri-citric solution R. Heat to boiling. An orange precipitate is formed andthe solution becomes yellow.

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C. To 2 ml add 3 ml of water R. The solution gives the reaction of citrates (2.3.1).

D. 1 ml gives reaction (b) of phosphates (2.3.1).

E. 0.5 ml gives reaction (b) of sodium (2.3.1).

TESTS

pH (2.2.3). The pH of the solution is 5.3 to 5.9.

Hydroxymethylfurfural To 2.0 ml add 5.0 ml of a 100 g/l solution of p-toluidine R in 2-prop-anol R containing 10 per cent V/V of glacial acetic acid R and 1.0 ml of a 5 g/l solution of barbituricacid R. The absorbance (2.2.25), determined at 550 nm after allowing the mixture to stand for 2min to 3 min, is not greater than that of a standard prepared at the same time in the same mannerusing 2.0 ml of a solution containing 5 ppm of hydroxymethylfurfural R.

Sterility (2.6.1). They comply with the test for sterility.

Pyrogens (2.6.8). They comply with the test for pyrogens. Dilute with a pyrogen-free, 9 g/lsolution of sodium chloride R to obtain a solution containing approximately 5 g/l of sodium citrate R.Inject 10 ml of the diluted solution per kilogram of the rabbits mass.

ASSAY

Sodium dihydrogen phosphate Dilute 10.0 ml to 100.0 ml with water R. To 10.0 ml of thissolution add 10.0 ml of nitro-vanado-molybdic reagent R. Mix and allow to stand at 20C to 25C for30 min. At the same time and in the same manner, prepare a reference solution using 10.0 ml of astandard solution containing 0.219 g of potassium dihydrogen phosphate R per litre. Measure theabsorbance (2.2.25) of the two solutions at 450 nm using as the compensation liquid a solutionprepared in the same manner using 10 ml of water R. Calculate the content of sodium dihydrogenphosphate dihydrate (P) in grams per litre from the expression:

1146 12

. C AA

C =concentration of potassium dihydrogen phos-phate R in the standard solution in grams perlitre,

A1 =absorbance of the test solution,A2 =absorbance of the reference solution.

Citric acid To 20.0 ml add 0.1 ml of phenolphthalein solution R1 and titrate with 0.2M sodiumhydroxide.

Calculate the content of citric acid monohydrate (C), or anhydrous citric acid (C), in grams perlitre from the equations:

C = 0.7005n 0.4490P

C = 0.6404n 0.4105Pn=number of millilitres of 0.2M sodium hydroxide used in the titration,P =content of sodium dihydrogen phosphate dihydrate in grams per litre determined as

prescribed above.

Sodium citrate Prepare a chromatography column 100 mm long and 10 mm in internal diameterand filled with strongly acidic ion exchange resin R (300 m to 840 m). Maintain a 1 cm layer ofliquid above the resin at all times. Wash the column with 50 ml of de-ionised water R at a flow rateof 12 ml to 14 ml per minute.

Dilute 10.0 ml of the solution to be examined to about 40 ml with de-ionised water R in a beakerand transfer to the column reservoir, washing the beaker three times with a few millilitres of de-ionised water R. Allow the solution to run through the column at a flow rate of 12 ml to 14 ml perminute and collect the eluate. Wash the column with two quantities, each of 30 ml, and with onequantity of 50 ml, of de-ionised water R. The column can be used for three successive determina-tions before regeneration with three times its volume of dilute hydrochloric acid R. Titrate thecombined eluate and washings (about 150 ml) with 0.2M sodium hydroxide, using 0.1 ml of phenol-phthalein solution R1 as indicator.

Calculate the content of sodium citrate in grams per litre from the following expressions:

1.961n 1.257P 1.40C

1.961n 1.257P 1.53Cn=number of millilitres of 0.2M sodium hydroxide used in the titration,P =content of sodium dihydrogen phosphate dihydrate in grams per litre determined as

prescribed above,C =content of citric acid monohydrate in grams per litre determined as prescribed above,

C =content of anhydrous citric acid in grams per litre determined as prescribed above.

Reducing sugars Dilute 5.0 ml to 100.0 ml with water R. Introduce 25.0 ml of the solution into a250 ml conical flask with ground-glass neck and add 25.0 ml of cupri-citric solution R1. Add a few

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pieces of porous material, attach a reflux condenser, heat so that boiling begins within 2 min andboil for exactly 10 min. Cool and add 3 g of potassium iodide R dissolved in 3 ml of water R. Add25 ml of a 25 per cent m/m solution of sulphuric acid R with caution and in small quantities. Titratewith 0.1M sodium thiosulphate using 0.5 ml of starch solution R, added towards the end of the titra-tion, as indicator (n1 ml). Carry out a blank titration using 25.0 ml of water R (n2 ml).

Calculate the content of reducing sugars as anhydrous glucose or as glucose monohydrate, asappropriate, from the Table 209-1.

STORAGE

Store in an airtight, tamper-proof container, protected from light.

LABELLING

The label states: the composition and volume of the solution, the maximum amount of blood to be collected in the container.

__________________________________________________________________________________________________________ Ph Eur

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Plasma for Fractionation

revised 1/01

Plasma For Fractionation complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Human Plasma for Fractionation [0853]. These requirements are reproduced after the heading Defini-tion below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Human plasma for fractionation is the liquid part of human blood remaining after separation of thecellular elements from blood collected in a receptacle containing an anticoagulant, or separated bycontinuous filtration or centrifugation of anticoagulated blood in an apheresis procedure; it isintended for the manufacture of plasma-derived products.

PRODUCTION

DONORS

Only a carefully selected, healthy donor who, as far as can be ascertained after medical examination,laboratory blood tests and a study of the donors medical history, is free from detectable agents ofinfection transmissible by plasma-derived products may be used in the collection of plasma forfractionation. Recommendations in this field are made by the Council of Europe [RecommendationNo. R (95) 15 on the preparation, use and quality assurance of blood components, or subsequent revision]and the European Union [Council Recommendation of 29 June 1998 on the suitability of blood and plasmadonors and the screening of donated blood in the European Community (98/463/EC)].

Immunisation of donors Deliberate immunisation of donors to obtain immunoglobulins withspecified activities may be carried out when sufficient supplies of material of suitable quality cannotbe obtained from naturally immunised donors. Recommendations for such immunisation areformulated by the World Health Organisation (Requirements for the collection, processing and qualitycontrol of blood, blood components and plasma derivatives, WHO Technical Report Series, No.840, 1994or subsequent revision).

Records Records of donors and donations made are kept in such a way that, while maintaining therequired degree of confidentiality concerning the donors identity, the origin of each donation in aplasma pool and the results of the corresponding acceptance procedures and laboratory tests can betraced.

Laboratory tests Laboratory tests are carried out for each donation to detect the following viralmarkers:

1. antibodies against human immunodeficiency virus 1 (anti-HIV-1),

2. antibodies against human immunodeficiency virus 2 (anti-HIV-2),

3. hepatitis B surface antigen (HBsAg),

4. antibodies against hepatitis C virus (anti-HCV).

Pending complete harmonisation of the laboratory tests to be carried out, the competent authoritymay require that a test for alanine aminotransferase (ALT) also be carried out.

The test methods used are of suitable sensitivity and specificity and are approved by the competentauthority. If a repeat-reactive result is found in any of these tests, the donation is not accepted.

INDIVIDUAL PLASMA UNITS

The plasma is prepared by a method that removes cells and cell debris as completely as possible.Whether prepared from whole blood or by plasmapheresis, the plasma is separated from the cells by amethod designed to prevent the introduction of micro-organisms. No antibacterial or antifungalagent is added to the plasma. The containers comply with the requirements for glass containers(3.2.1) or for plastic containers for blood and blood components (3.2.3). The containers are closedso as to prevent contamination.

If two or more units are pooled prior to freezing, the operations are carried out using sterileconnecting devices or under aseptic conditions and using containers that have not previously beenused.

When obtained by plasmapheresis, plasma intended for the recovery of proteins that are labile inplasma is frozen by cooling rapidly at 30C or below as soon as possible and at the latest within 24 hof collection.

When obtained from whole blood, plasma intended for the recovery of proteins that are labile inplasma is separated from cellular elements and is frozen by cooling rapidly at30C or below as soon as possible and at the latest within 24 h of collection.

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When obtained from whole blood, plasma intended solely for the recovery of proteins that are notlabile in plasma is separated from cellular elements and frozen at 20C or below as soon as possibleand at the latest within 72 h of collection.

It is not intended that the determination of total protein and factor VIII shown below be carried out on eachunit of plasma. They are rather given as guidelines for good manufacturing practice, the test for factor VIIIbeing relevant for plasma intended for use in the preparation of concentrates of labile proteins.

The total protein content of a unit of plasma depends on the serum protein content of the donor and the degreeof dilution inherent in the donation procedure. When plasma is obtained from a suitable donor and using theintended proportion of anticoagulant solution, a total protein content complying with the limit of 50 g/l isobtained. If a volume of blood or plasma smaller than intended is collected into the anticoagulant solution, theresulting plasma is not necessarily unsuitable for pooling for fractionation. The aim of good manufacturingpractice must be to achieve the prescribed limit for all normal donations.

Preservation of factor VIII in the donation depends on the collection procedure and the subsequent handling ofthe blood and plasma. With good practice, 0.7 I.U. per millilitre can usually be achieved, but units of plasmawith a lower activity may still be suitable for use in the production of coagulation factor concentrates. The aimof good manufacturing practice is to conserve labile proteins as much as possible.

Total protein Carry out the test using a pool of not fewer than ten units. Dilute the pool with a 9 g/lsolution of sodium chloride R to obtain a solution containing about 15 mg of protein in 2 ml. To2.0 ml of this solution in a round-bottomed centrifuge tube add 2 ml of a 75 g/l solution of sodiummolybdate R and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid R and 30 volumes ofwater R. Shake, centrifuge for 5 min, decant the supernatant liquid and allow the inverted tube todrain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion(2.5.9) and calculate the protein content by multiplying the quantity of nitrogen by 6.25. The totalprotein content is not less than 50 g/l.

Factor VIII Carry out the test using a pool of not fewer than ten units. Thaw the samples to beexamined, if necessary, at 37C. Carry out the assay of factor VIII (2.7.4), using a reference plasmacalibrated against the International Standard for blood coagulation factor VIII in plasma. The activityis not less than 0.7 I.U. per millilitre.

POOLED PLASMA

During the manufacture of plasma products, the first homogeneous pool of plasma (for example,after removal of cryoprecipitate) is tested for HBsAg, for hepatitis C virus antibodies and for HIVantibodies using test methods of suitable sensitivity and specificity; the pool must give negative resultsin these tests.

The plasma pool is also tested for hepatitis C virus RNA using a validated nucleic acid amplifica-tion technique (2.6.21). A positive control with 100 I.U./ml of hepatitis C virus RNA and, to test forinhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasmapool are included in the test. The test is invalid if the positive control is non-reactive or if the resultobtained with the internal control indicates the presence of inhibitors. The plasma pool complies withthe test if it is found non-reactive for hepatitis C virus RNA.

CHARACTERS

Before freezing, a clear to slightly turbid liquid without visible signs of haemolysis; it may vary incolour from light yellow to green.

STORAGE

Store frozen plasma at or below 20C; the plasma may still be used for fractionation if a temperatureof 20C is exceeded on at most one occasion for not more than 72 h and if the plasma is at all timesmaintained at a temperature of 5C or lower.

LABELLING

The label enables each individual unit to be traced to a specific donor.__________________________________________________________________________________________________________ Ph Eur

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Albumin SolutionAlbumin; Human Albumin

Albumin Solution complies with the requirements of the 3rd edition of the European Pharmacopoeia for HumanAlbumin Solution [0255]. These requirements are reproduced after the heading Definition below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Human albumin solution is an aqueous solution of protein obtained from plasma that complies withthe requirements of the monograph on Plasma for fractionation, human (853).

PRODUCTION

Separation of the albumin is carried out under controlled conditions, particularly of pH, ionicstrength and temperature so that in the final product not less than 95 per cent of the total protein isalbumin. Human albumin solution is prepared as a concentrated solution containing 150 g/l to250 g/l of total protein or as an isotonic solution containing 35 g/l to 50 g/l of total protein. A suitablestabiliser against the effects of heat, such as sodium caprylate (sodium octanoate) or N-acetyltrypto-phan or a combination of these two, at a suitable concentration, may be added but no antimicrobialpreservative is added at any stage during preparation. The solution is passed through a bacteria-retentive filter and distributed aseptically into sterile containers which are then closed so as to preventcontamination. The solution in its final container is heated to 60 0.5C and maintained at thistemperature for not less than 10 h. The containers are then incubated at 30C to 32C for not lessthan 14 days or at 20C to 25C for not less than 4 weeks and examined visually for evidence ofmicrobial contamination.

CHARACTERS

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

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the test be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative results with antisera specific to plasma proteins of other species.

B. Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal humanserum, compare normal human serum and the preparation to be examined, both diluted to contain10 g/l of protein. The main component of the preparation to be examined corresponds to the maincomponent of normal human serum. The preparation may show the presence of small quantities ofother plasma proteins.

TESTS

pH (2.2.3). Dilute the preparation to be examined with a 9 g/l solution of sodium chloride R to obtaina 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 chloride R toobtain 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 of1 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 thenitrogen in the residue by the method of sulphuric acid digestion (2.5.9) and calculate the quantity ofprotein by multiplying by 6.25. The preparation contains not less than 95 per cent and not more than105 per cent of the quantity of protein stated on the label.

Protein composition Examine by zone electrophoresis (2.2.31), using strips of suitable celluloseacetate 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 9 g/l solution of sodium chloride R to aprotein concentration of 20 g/l.

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

To a strip apply 2.5 l of the test solution as a 10 mm band or apply 0.25 l per millimetre if anarrower strip is used. To another strip apply in the same manner the same volume of the referencesolution. Apply a suitable electric field such that the most rapid band migrates at least 30 mm. Treatthe strips with amido black 10B solution R for 5 min. Decolorise with a mixture of 10 volumes ofglacial acetic acid R and 90 volumes of methanol R until the background is just free of colour. Develop

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the transparency of the strips with a mixture of 19 volumes of glacial acetic acid R and 81 volumes ofmethanol R. Measure the absorbance of the bands at 600 nm in an instrument having a linearresponse over the range of measurement. Calculate the result as the mean of three measurements ofeach strip. In the electrophoretogram obtained with the test solution, not more than 5 per cent of theprotein has a mobility different from that of the principal band. The test is not valid unless, in theelectrophoretogram obtained with the reference preparation, the proportion of protein in the princi-pal band is within the limits stated in the leaflet accompanying the reference preparation.

Polymers and aggregates 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 aconcentration suitable for the chromatographic system used. A concentration in the range 4 g/l to12 g/l and injection of 50 g to 600 g of protein are usually suitable.

The chromatographic procedure may be carried out using: a column 0.6 m long and 7.5 mm in internal diameter packed with hydrophilic silica gel for

chromatography R, as mobile phase at a flow rate of 0.5 ml per minute a solution containing per litre: 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,

a detector set at 280 nm.The peak corresponding to polymers and aggregates 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 thetotal area of the chromatogram.

Haem Dilute the preparation to be examined using a 9 g/l solution of sodium chloride R to obtain asolution containing 10 g/l of protein. The absorbance (2.2.25) of the solution measured at 403 nmusing 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 undergoing dialysis or to premature infants, itcomplies with the test for aluminium. Not more than 200 g of Al per litre, determined by atomicabsorption 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) beforeuse.

Test solution. Use the preparation to be examined.

Validation solution. Use human albumin for aluminium validation BRP.

Reference solutions. Prepare a suitable range of reference solutions by adding suitable volumes ofaluminium standard solution (10 ppm Al) R to known volumes of water R.

Dilute the solutions as necessary using nitric acid (10 g/l HNO3) containing 1.7 g/l of magnesiumnitrate R and 0.05 per cent V/V of octoxinol 10 R. Measure the absorbance at 309.3 nm. The test isnot valid unless the aluminium content determined for human albumin for aluminium validation BRPis 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 emissionspectrometry (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 than105 per cent of the content of Na stated on the label, determined by atomic emission spectrometry(Method I, 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/l to 50 g/l ofprotein, inject per kilogram of the rabbits mass 10 ml of the preparation to be examined. For asolution containing 150 g/l to 250 g/l of protein, inject per kilogram of the rabbits mass 3 ml of thepreparation to be examined.

STORAGE

Store protected from light.

LABELLING

The label states: the name of the preparation, the volume of the preparation, the content of protein expressed in grams per litre, the content of sodium expressed in millimoles per litre, the storage conditions, the expiry date,

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that the product is not to be used if it is cloudy or if a deposit has formed, the name and concentration of any added substance (for example stabiliser), where applicable, that the preparation is suitable for administration to patients undergoing

dialysis and to premature infants.__________________________________________________________________________________________________________ Ph Eur

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Antithrombin III Concentrate

Antithrombin III Concentrate complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Freeze-dried Human Antithrombin III Concentrate [0878]. These requirements are reproduced after theheading Definition below.

Action and use Used to correct deficiencies of antithrombin III.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human antithrombin III concentrate is a preparation of a glycoprotein fractionobtained from human plasma that inactivates thrombin in the presence of an excess of heparin. It isobtained from plasma that complies with the requirements of the monograph on Human plasma forfractionation (853).

When reconstituted in the volume of solvent stated on the label or on the leaflet, the potency isnot less than 25 I.U. of antithrombin III per millilitre.

PRODUCTION

The method of preparation includes a step or steps that have been shown to remove or toinactivate known agents of infection; if substances are used for inactivation of viruses duringproduction, the subsequent purification procedure must be validated to demonstrate that theconcentration of these substances is reduced to a suitable level and any residues are such as not tocompromise the safety of the preparation for patients.

The antithrombin III is purified and concentrated and a suitable stabiliser may be added. Thespecific activity is not less than 3 I.U. of antithrombin III per milligram of total protein, excludingalbumin. The antithrombin III concentrate is passed through a bacteria-retentive filter, distributedaseptically into its final, sterile containers and immediately frozen. It is then freeze-dried and thecontainers are closed under vacuum or in an atmosphere of inert gas. No antimicrobial preservative isadded at any stage of production.

VALIDATION TEST

It shall be demonstrated that the manufacturing process yields a product that consistently complieswith the following test:

Heparin-binding fraction. Examine by agarose gel electrophoresis (2.2.31). Prepare a 10 g/l solution ofagarose for electrophoresis R containing 15 I.U. of heparin R per millilitre in barbital buffer solution pH8.4 R. Pour 5 ml of this solution onto a glass plate 5 cm square. Cool at 4C for 30 min. Cut twowells 2 mm in diameter 1 cm and 4 cm from the side of the plate and 1 cm from the cathode.Introduce into one well 5 l of the preparation to be examined, diluted to an activity of about 1 I.U.of antithrombin III per millilitre. Introduce into the other well 5 l of a solution of a marker dye suchas bromophenol blue R. Allow the electrophoresis to proceed at 4C, using a constant electric field of7 V per centimetre, until the dye reaches the anode.

Cut across the agarose gel 1.5 cm from that side of the plate on which the preparation to beexamined was applied and remove the larger portion of the gel leaving a band 1.5 cm wide containingthe material to be examined. Replace the removed portion with an even layer consisting of 3.5 ml of a10 g/l solution of agarose for electrophoresis R in barbital buffer solution pH 8.4 R, containing a rabbitanti-human antithrombin III antiserum at a suitable concentration, previously determined, to giveadequate peak heights of at least 1.5 cm. Place the plate with the original gel at the cathode so that asecond electrophoretic migration can occur at right angles to the first. Allow this second electro-phoresis to proceed using a constant electric field of 2 V per centimetre for 16 h. Cover the plateswith filter paper and several layers of thick lint soaked in a 9 g/l solution of sodium chloride R andcompress for 2 h, renewing the saline several times. Rinse with water R, dry the plates and stain withacid blue 92 solution R.

Calculate the fraction of antithrombin III bound to heparin, which is the peak closest to the anode,with respect to the total amount of antithrombin III, by measuring the area defined by the twoprecipitation peaks.

The fraction of antithrombin III able to bind to heparin is not less than 60 per cent.

CHARACTERS

A white, friable solid or a powder.

Reconstitute the preparation to be examined as stated on the label or on the leaflet immediately before carryingout the identification, the tests (except those for solubility, total protein and water), and the assay.

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparation

66-13

to be examined and stain the gels with acid blue 92 R. It is recommended that the tests be carried outusing antisera specific to the plasma proteins of each species of domestic animal commonly used inthe preparation of materials of biological origin in the country concerned. The preparation is shownto contain proteins of human origin and gives negative results with antisera specific to plasmaproteins of other species.

B. The assay for antithrombin III activity contributes to the identification of the preparation.

TESTS

pH (2.2.3). The pH of the preparation to be examined is 6.0 to 7.5.

Solubility It dissolves completely under gentle swirling within 10 min in the volume of the solventstated on the label or the leaflet, forming a clear or slightly turbid, colourless solution.

Osmolality (2.2.35). Not less than 240 milliosmoles per kilogram.

Total protein If necessary, dilute an accurately measured volume of the preparation to be examinedwith water R to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of thesolution in a round-bottomed centrifuge tube add 2 ml of a 75 g/l solution of sodium molybdate R and2 ml 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 filterpaper. Determine the nitrogen in the residue by the method of sulphuric acid digestion (2.5.9) andcalculate the amount of protein by multiplying the result by 6.25.

Heparin (2.7.5). Not more than 0.1 I.U. of heparin activity per International Unit of antithrombinIII activity. It is necessary to validate the method for assay of heparin for each specific preparation tobe examined to allow for interference by antithrombin III.

Water (2.5.12). Not more than 3.0 per cent, determined on not less than 0.500 g by the semi-microdetermination of water.


Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass avolume of the preparation to be examined equivalent to 50 I.U. of antithrombin III, calculated fromthe activity stated on the label.

ASSAY

The antithrombin III content of the preparation to be examined is determined by comparing itsability to inactivate thrombin in the presence of an excess of heparin with the same ability of areference preparation of human anti-thrombin III concentrate calibrated in International Units.Varying quantities of the preparation to be examined are mixed with a given quantity of thrombinand the remaining thrombin activity is determined using a suitable chromogenic substrate.

The International Unit is the activity of a stated amount of the International Standard for humanantithrombin III concentrate. The equivalence in International Units of the International Standard isstated by the World Health Organisation.

Method. Prepare two independent series of three or four dilutions in the range 1/75 to 1/200 from1 I.U. per millilitre, for both the preparation to be examined and the reference preparation, using tris-EDTA BSA buffer solution pH 8.4 R containing 15 I.U. of heparin per millilitre.

Warm 200 l of each dilution at 37C for 1 min to 2 min. Add to each dilution 200 l of a solutionof bovine thrombin R containing 2 I.U. per millilitre in tris-EDTA BSA buffer solution pH 8.4 R. Mixand maintain at 37C for exactly 1 min. Add 500 l of a suitable chromogenic substrate (for example,D-phenylalanyl-L-pipecolyl-L-arginyl 4-nitroanilide, reconstituted in water R to give a solutioncontaining 4 mmol per litre and further diluted for the assay using tris-EDTA BSA buffer solution pH8.4 R without albumin). Immediately start measurement of the change in absorbance at 405 nm,continuing the measurement for at least 30 s. Calculate the rate of change of absorbance (DA/min).(Alternatively, an end-point assay may be used by stopping the reaction with acetic acid and measur-ing the absorbance at 405 nm.)

The rate of change of absorbance (DA/min) is inversely proportional to antithrombin III activity.Plot the regression of absorbance or DA/min against concentration on a linear scale and determinethe potency by comparing the slopes for the reference preparation and the preparation to be exam-ined.

Check the validity of the assay and calculate the potency of the test preparation by the usualstatistical methods for a slope-ratio assay (for example, 5.3. Statistical Analysis of Results of BiologicalAssays and Tests).

The estimated potency is not less than 90 per cent and not greater than 110 per cent of the potencystated on the label. The confidence interval (P = 0.95) is not greater than 90 per cent to 110 per centof the estimated potency.

STORAGE


66-14

LABELLING

The label states: the content of antithrombin III expressed in International Units per container. the name and volume of solvent to be used to reconstitute the preparation, where applicable, the amount of albumin present as a stabiliser.

__________________________________________________________________________________________________________ Ph Eur

66-15

Dried Factor VII Fraction

Dried Factor VII Fraction complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Freeze-dried Human Coagulation Factor VII [1224]. These requirements are reproduced after theheading Definition below.

Action and use Used to correct deficiencies of coagulation factor VII.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human coagulation factor VII is a plasma protein fraction that contains the single-chainglycoprotein factor VII and may also contain small amounts of the activated form, the two-chainderivative factor VIIa, as well as coagulation factors II, IX and X and protein C and protein S. It isobtained from human plasma that complies with the monograph on Human plasma for fractionation(853).

The potency of the preparation, reconstituted as stated on the label, is not less than 15 I.U. offactor VII per millilitre.

PRODUCTION

The method of preparation is designed to minimise activation of any coagulation factor (to minimisepotential thrombogenicity) and includes a step or steps that have been shown to remove or toinactivate known agents of infection; if substances are used for inactivation of viruses during produc-tion, the subsequent purification procedure must be validated to demonstrate that the concentrationof these substances is reduced to a suitable level and that any residues are such as not tocompromishe safety of the preparation for patients.

The specific activity is not less than 2 I.U. of factor VII per milligram of protein, before the addi-tion of any protein stabiliser.

The factor VII fraction is dissolved in a suitable liquid. Heparin, antithrombin and other auxiliarysubstances such as a stabiliser may be added. No antimicrobial preservative is added. The solution ispassed through a bacteria-retentive filter, distributed aseptically into the final containers and immedi-ately frozen. It is subsequently freeze-dried and the containers are closed under vacuum or under aninert gas.

CONSISTENCY OF THE METHOD OF PRODUCTION

The consistency of the method of production with respect to the activities of factors II, IX and X ofthe preparation, expressed in International Units relative to the activity of factor VII, shall be demon-strated.

The consistency of the method of production with respect to the activity of factor VIIa of thepreparation shall be demonstrated. The activity of factor VIIa may be determined, for example, usinga recombinant soluble tissue factor that does not activate factor VII but possesses a cofactor functionspecific for factor VIIa; after incubation of a mixture of the recombinant soluble tissue factor withphospholipids reagent and the dilution of the test sample in factor VII-deficient plasma, calciumchloride is added and the clotting time determined; the clotting time is inversely related to the factorVIIa activity of the test sample.

CHARACTERS

A powder or friable solid that may be white, pale yellow, green or blue.

Reconstitute the preparation to be examined as stated on the label immediately before carrying out theidentification, tests (except those for solubility and water) and assay

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the tests be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative results with antisera specific to plasma proteins of other species.

B. The assay for factor VII contributes to the identification of the preparation.

TESTS

pH (2.2.3). 6.5 to 7.5.Solubility To a container of the preparation to be examined add the volume of liquid stated on thelabel at the recommended temperature. The preparation dissolves completely with gentle swirlingwithin 10 min, giving a clear or slightly opalescent solution that may be coloured.

Osmolality (2.2.35). Not less than 240 mosmol/kg.

66-16

Total protein If necessary, dilute an accurately measured volume of the reconstituted preparationwith a 9 g/l solution of sodium chloride R to obtain a solution expected to contain about 15 mg ofprotein in 2 ml. To 2.0 ml of the solution in a round-bottomed centrifuge tube add 2 ml of a 75 g/lsolution of sodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid R and30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid and allow theinverted tube to drain on filter paper. Determine the nitrogen in the residue by the method ofsulphuric acid digestion (2.5.9) and calculate the amount of protein by multiplying the result by 6.25.

Water (2.5.12). Not more than 3.0 per cent. Add a suitable volume of anhydrous methanol R to thecontainer of the preparation to be examined, shake, allow to stand and carry out the determinationon a known volume of the supernatant liquid.

Activated coagulation factors If the preparation to be examined contains heparin, determine theamount present as described in the test for heparin and neutralise the heparin by addition ofprotamine sulphate R (10 g of protamine sulphate neutralises 1 I.U. of heparin). Prepare 1 in 10 and1 in 100 dilutions of the reconstituted preparation to be examined using tris(hydroxymethyl)amino-methane buffer solution pH 7.5 R. Place a series of polystyrene tubes in a water-bath at 37C and addto each tube 0.1 ml of platelet-poor plasma R and 0.1 ml of a suitable dilution of cephalin R or plateletsubstitute R. Allow to stand for 60 s. Add to each tube either 0.1 ml of one of the dilutions or 0.1 mlof the buffer solution (control tube). To each tube, add immediately 0.1 ml of a 3.7 g/l solution ofcalcium chloride R, previously heated to 37C, and measure within 30 min of the original dilution thetime that elapses between addition of the calcium chloride solution and formation of a clot. For eachof the dilutions, the coagulation time is not less than 150 s. The test is not valid unless the coagula-tion time measured for the control tube is 200 s to 350 s.

Heparin If heparin has been added during preparation, determine the amount present by the assayof heparin in coagulation factor concentrates (2.7.12). The preparation to be examined contains notmore than the amount of heparin stated on the label and in any case not more than 0.5 I.U. ofheparin per International Unit of factor VII.

Thrombin If the preparation to be examined contains heparin, determine the amount present asdescribed in the test for heparin and neutralise the heparin by addition of protamine sulphate R (10 gof protamine sulphate neutralises 1 I.U. of heparin). In each of two test-tubes, mix equal volumes ofthe reconstituted preparation and a 3 g/l solution of fibrinogen R. Keep one of the tubes at 37C for6 h and the other at room temperature for 24 h. In a third tube, mix a volume of the fibrinogensolution with an equal volume of a solution of human thrombin R (1 I.U./ml) and place the tube in awater-bath at 37C. No coagulation occurs in the tubes containing the preparation to be examined.Coagulation occurs within 30 s in the tube containing thrombin.


Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass avolume equivalent to not less than 30 I.U. of factor VII.

ASSAY

Carry out the assay of human blood coagulation factor VII (2.7.10).The estimated potency is not less than 80 per cent and not more than 120 per cent of the stated

potency. The confidence interval (P = 0.95) of the estimated potency is not greater than 80 per centto 120 per cent.

STORAGE


LABELLING

The label states: the number of International Units of factor VII per container, the amount of protein per container, the name and quantity of any added substances, including where applicable, heparin, the name and volume of the liquid to be used for reconstitution, the storage conditions, the expiry date, that the transmission of infectious agents cannot be totally excluded when medicinal products

prepared from human blood or plasma are administered.__________________________________________________________________________________________________________ Ph Eur

66-17

Dried Factor VIII FractionDried Human Antihaemophilic Fraction

Dried Factor VIII Fraction complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Freeze-dried Human Coagulation Factor VIII [0275]. These requirements are reproduced after theheading Definition below.

Action and use Used to correct deficiencies of coagulation factor VIII.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human coagulation factor VIII is a plasma protein fraction that contains theglycoprotein coagulation factor VIII together with varying amounts of von Willebrand factor, depend-ing on the method of preparation. It is prepared from human plasma that complies with the mono-graph on Human plasma for fractionation (853).

The potency of the preparation, reconstituted as stated on the label, is not less than 20 I.U. offactor VIII:C per millilitre.

PRODUCTION

The method of preparation includes a step or steps that have been shown to remove or to inactivateknown agents of infection; if substances are used for the inactivation of viruses during production, thesubsequent purification procedure must be validated to demonstrate that the concentration of thesesubstances is reduced to a suitable level and that any residues are such as not to compromise thesafety of the preparation for patients.

The specific activity is not less than 1 I.U. of factor VIII:C per milligram of total protein before theaddition of protein stabiliser.

The factor VIII fraction is dissolved in a suitable liquid. Auxiliary substances such as a stabilisermay be added. No anti-microbial preservative is added. The solution is passed through a bacteria-retentive filter, distributed aseptically into the final containers and immediately frozen. It issubsequently freeze-dried and the containers are closed under vacuum or under an inert gas.

Validation test applied to products stated to have von Willebrand factor activity Forproducts intended for treatment of von Willebrands disease it shall be demonstrated that themanufacturing process yields a product with a consistent composition with respect to von Willebrandfactor. This composition may be characterised in a number of ways. For example, the number andthe relative amount of the different multimers may be determined by sodium dodecyl sulphate (SDS)agarose gel electrophoresis (1 per cent agarose) with or without Western blot analysis onnitrocellulose, using a normal human plasma pool as reference; visualisation of the multimericpattern may be performed using an immunoenzymatic technique and quantitative evaluation may becarried out by densitometric analysis or by other suitable methods.

von Willebrand factor activity For products intended for treatment of von Willebrands diseasethe von Willebrand factor activity is determined by a suitable method using a reference preparation ofthe same type as the preparation to be examined, calibrated against the International Standard forvon Willebrand factor in plasma. Suitable methods include determination of ristocetin cofactoractivity and determination of collagen-binding activity. The following method for determination ofristocetin cofactor activity is given as an example of a suitable method.

Ristocetin cofactor activity. Carry out appropriate dilutions of the preparation to be examined and ofthe reference preparation using as diluent a solution containing 9 g/l of sodium chloride R and 50 g/l ofhuman albumin. Add to each dilution suitable amounts of a von Willebrand reagent containingstabilised human platelets and ristocetin A. Mix on a glass plate by moving it gently in circles for1 min. Allow to stand for a further 1 min and read the result against a dark background with sidelighting. The last dilution which shows clearly visible agglutination indicates the ristocetin cofactortitre of the sample. Use diluent as a negative control.

The estimated potency is not less than 60 per cent and not more than 140 per cent of the potencyapproved for the particular product.

CHARACTERS

A white or pale yellow powder or friable solid.

Reconstitute the preparation to be examined as stated on the label immediately before carrying out theidentification, tests (except those for solubility and water) and assay.

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the test be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of human

66-18

origin and gives negative results with antisera specific to plasma proteins of other species.

B. The assays for factor VIII:C and von Willebrand factor activity (where applicable) contribute tothe identification of the preparation.

TESTS

pH (2.2.3). 6.5 to 7.5.

Solubility To a container of the preparation to be examined add the volume of the solvent stated onthe label at the recommended temperature. The preparation dissolves completely with gentle swirlingwithin 10 min, giving a clear or slightly opalescent, colourless or slightly yellow solution.


Total protein If necessary, dilute an accurately measured volume of the preparation to be examinedwith a 9 g/l solution of sodium chloride R to obtain a solution containing about 15 mg of protein in2 ml. To 2.0 ml of the solution in a round-bottomed centrifuge tube add 2 ml of a 75 g/l solution ofsodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid R and 30 volumesof water R. Shake, centrifuge for 5 min, decant the supernatant liquid and allow the inverted tube todrain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion(2.5.9) and calculate the amount of protein by multiplying the result by 6.25.

For some products, especially those without a protein stabiliser such as albumin, this method may not beapplicable and another validated method for protein determination must therefore be performed.

Haemagglutinins anti-A and anti-B Dilute the preparation with a 9 g/l solution of sodiumchloride R to contain 3 I.U. of factor VIII:C per millilitre. Carry out the indirect determination ofhaemagglutinins A and B (2.6.20). The 1 to 64 dilutions do not show agglutination.

Hepatitis B surface antigen Examine the reconstituted preparation by a suitably sensitive methodsuch as enzyme immunoassay (2.7.1). Hepatitis B surface antigen is not detected.



Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass avolume of the preparation to be examined equivalent to not less than 30 I.U. of factor VIII:C.

ASSAY

Carry out the assay of human coagulation factor VIII (2.7.4).The estimated potency is not less than 80 per cent and not more than 120 per cent of the stated


STORAGE


LABELLING

The label states: the number of International Units of factor VIII:C and, where applicable, of von Willebrand

factor in the container, the amount of protein in the container, the name and quantity of any added substance, the name and volume of the liquid to be used for reconstitution, the storage conditions, the expiry date, that the transmission of infectious agents cannot be totally excluded when medicinal products


66-19

Dried Factor IX Fraction

Dried Factor IX Fraction complies with the requirements of the 3rd edition of the European Pharmacopoeia forFreeze-dried Human Coagulation Factor IX [1223]. These requirements are reproduced after the headingDefinition below.

Action and use Used to correct deficiencies of coagulation factor IX (Christmas disease).

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human coagulation factor IX is a plasma protein fraction containing coagulationfactor IX, prepared by a method that effectively separates factor IX from other prothrombincomplex factors (factors II, VII and X). It is obtained from human plasma that complies with themonograph on Human plasma for fractionation (853).

The potency of the preparation, reconstituted as stated on the label, is not less than 20 I.U. offactor IX per millilitre.

PRODUCTION

The method of preparation is designed to maintain functional integrity of factor IX, to minimiseactivation of any coagulation factor (to minimise potential thrombogenicity) and includes a step orsteps that have been shown to remove or to inactivate known agents of infection; if substances areused for inactivation of viruses during production, the subsequent purification procedure must bevalidated to demonstrate that the concentration of these substances is reduced to a suitable level andthat any residues are such as not to compromise the safety of the aration for patients.

The specific activity is not less than 50 I.U. of factor IX per milligram of total protein, before theaddition of any protein stabiliser.

The factor IX fraction is dissolved in a suitable liquid. Heparin, antithrombin and other auxiliarysubstances such as a stabiliser may be included. No antimicrobial preservative is added. The solutionis passed through a bacteria-retentive filter, distributed aseptically into the final containers andimmediately frozen. It is subsequently freeze-dried and the containers are closed under vacuum orunder an inert gas.

CONSISTENCY OF THE METHOD OF PRODUCTION

The consistency of the method of production is evaluated by suitable analytical procedures that aredetermined during process development and which normally include: assay of factor IX, determination of activated coagulation factors, determination of activities of factors II, VII and X which shall be shown to be not more than

5 per cent of the activity of factor IX.

CHARACTERS

A white, or pale yellow powder or friable solid.

Reconstitute the preparation to be examined as stated on the label, immediately before carrying out theidentification, tests (except those for solubility and water) and assay.

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the tests be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative results with antisera specific to plasma proteins of other species.

B. The assay for coagulation factor IX contributes to the identification of the preparation.

TESTS

pH (2.2.3). 6.5 to 7.5.

Solubility To a container of the preparation to be examined add the volume of the liquid stated onthe label at the recommended temperature. The preparation dissolves completely with gentle swirlingwithin 10 min, giving a clear or slightly opalescent, colourless solution.

Osmolality (2.2.35). Not less than 240 mosmol/kg.Total protein If necessary, dilute an accurately measured volume of the preparation to be examinedwith a 9 g/l solution of sodium chloride R, to obtain a solution which may be expected to contain about15 mg of protein in 2 ml. To 2.0 ml of that solution, in a round-bottomed centrifuge tube, add 2 mlof a 75 g/l solution of sodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric

66-20

acid R and 30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid andallow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the methodof sulphuric acid digestion (2.5.9) and calculate the amount of protein by multiplying the result by6.25.

For some products, especially those without a protein stabiliser such as albumin, this method may not beapplicable. Another validated method for protein determination must therefore be performed.

Activated coagulation factors Where applicable, determine the amount of heparin present asdescribed below and neutralise the heparin by addition of protamine sulphate R (10 g of protaminesulphate neutralises 1 I.U. of heparin). If necessary, dilute the preparation to be examined to contain20 I.U. of factor IX per millilitre. Prepare 1 to 10 and 1 to 100 dilutions using tris(hydroxymethyl)-aminomethane buffer solution pH 7.5 R. Place a series of polystyrene tubes in a water-bath at 37C andadd to each tube 0.1 ml of platelet-poor plasma R and 0.1 ml of a suitable dilution of cephalin R orplatelet substitute R. Allow to stand for 60 s. Add to each tube either 0.1 ml of one of the dilutions or0.1 ml of the buffer solution (control tube). To each tube add immediately 0.1 ml of a 3.7 g/l solu-tion of calcium chloride R (previously warmed to 37C) and measure, within 30 min of the originaldilution, the time that elapses between addition of the calcium chloride solution and the formation ofa clot. For each of the dilutions the coagulation time is not less than 150 s. The test is not validunless the coagulation time measured for the control tube is 200 s to 350 s.

Heparin If heparin has been added during preparation, determine the amount by the assay ofheparin in coagulation factor concentrates (2.7.12). The preparation to be examined contains notmore than the amount of heparin stated on the label and in any case not more than 0.5 I.U. ofheparin per International Unit of factor IX.



Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass avolume equivalent to not less than 30 I.U. of factor IX.

ASSAY

Carry out the assay of human blood coagulation factor IX (2.7.11).The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated


STORAGE


LABELLING

The label states: the number of International Units of factor IX per container, the amount of protein per container, the name and quantity of any added substances, including where applicable heparin, the name and volume of the liquid to be used for reconstitution, the storage conditions, the expiry date, that the transmission of infectious agents cannot be totally excluded when medicinal products


66-21

Dried Prothrombin Complex

Dried Prothrombin Complex complies with the requirements of the 3rd edition of the European Pharmacopoeiafor Freeze-dried Human Prothrombin Complex [0554]. These requirements are reproduced after the headingDefinition below.

Action and use Used to correct deficiencies of coagulation factor IX (Christmas disease).Preparations with appropriate activity may be used to correct deficiencies of coagulation factors IIor X.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human prothrombin complex is a plasma protein fraction containing blood coagula-tion factor IX together with variable amounts of coagulation factors II, VII and X; the presenceand proportion of these additional factors depends on the method of fractionation. It is obtainedfrom human plasma that complies with the monograph on Human plasma for fractionation (853).

The potency of the preparation, reconstituted as stated on the label, is not less than 20 I.U. offactor IX per millilitre.

PRODUCTION

The method of preparation is designed to minimise activation of any coagulation factor (tominimise potential thrombogenicity) and includes a step or steps that have been shown to removeor to inactivate known agents of infection; if substances are used for inactivation of viruses duringproduction, the subsequent purification procedure must be validated to demonstrate that theconcentration of these substances is reduced to a suitable level and that any residues are such asnot to compromise the safety of the preparation for patients.

The specific activity is not less than 0.6 I.U. of factor IX per milligram of total protein, beforethe addition of any protein stabiliser.

The prothrombin complex fraction is dissolved in a suitable liquid. Heparin, antithrombin andother auxiliary substances such as a stabiliser may be added. No antimicrobial preservative is added.The solution is passed through a bacteria-retentive filter, distributed aseptically into the finalcontainers and immediately frozen. It is subsequently freeze-dried and the containers are closedunder vacuum or under an inert gas.

CHARACTERS

A white or slightly coloured powder or friable solid, very hygroscopic.

Reconstitute the preparation to be examined as stated on the label immediately before carrying out theidentification, tests (except those for solubility and water) and assay.

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the test be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative results with antisera specific to plasma proteins of other species.

B. The assay for coagulation factor IX activity and, where applicable, those for factors II, VII and Xcontribute to the identification of the preparation.

TESTS

pH (2.2.3). 6.5 to 7.5.Solubility To a container of the preparation to be examined add the volume of the liquid stated onthe label at the recommended temperature. The preparation dissolves completely with gentle swirlingwithin 10 min, giving a clear solution that may be coloured.


Total protein If necessary, dilute an accurately measured volume of the reconstituted preparationwith a 9 g/l solution of sodium chloride R to obtain a solution expected to contain about 15 mg ofprotein in 2 ml. To 2.0 ml of the solution in a round-bottomed centrifuge tube add 2 ml of a 75 g/lsolution of sodium molybdate R and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid R and30 volumes of water R. Shake, centrifuge for 5 min, decant the supernatant liquid and allow theinverted tube to drain on filter paper. Determine the nitrogen in the residue by the method ofsulphuric acid digestion (2.5.9) and calculate the amount of protein by multiplying the result by 6.25.

Activated coagulation factors Where applicable, determine the amount of heparin present asdescribed in the test for heparin and neutralise it by addition of protamine sulphate R (10 g of

66-22

protamine sulphate neutralises 1 I.U. of heparin). Prepare 1 in 10 and 1 in 100 dilutions of thereconstituted preparation to be examined using tris(hydroxymethyl)aminomethane buffer solution pH7.5 R. Place a series of polystyrene tubes in a water-bath at 37C and add to each tube 0.1 ml ofplatelet-poor plasma R and 0.1 ml of a suitable dilution of cephalin R or platelet substitute R. Allow tostand for 60 s. Add to each tube either 0.1 ml of one of the dilutions or 0.1 ml of the buffer solution(control tube). To each tube, add immediately 0.1 ml of a 3.7 g/l solution of calcium chloride R,previously heated to 37C, and measure within 30 min of the original dilution the time that elapsesbetween addition of the calcium chloride solution and formation of a clot. For each of the dilutions,the coagulation time is not less than 150 s. The test is not valid unless the coagulation time measuredfor the control tube is 200 s to 350 s.

Heparin If heparin has been added during preparation, determine the amount present by the assayof heparin in coagulation factor concentrates (2.7.12). The preparation to be examined contains notmore than the amount of heparin stated on the label and in any case not more than 0.5 I.U. ofheparin per International Unit of factor IX.

Thrombin If the preparation to be examined contains heparin, determine the amount present asdescribed in the test for heparin and neutralise it by addition of protamine sulphate R (10 g ofprotamine sulphate neutralises 1 I.U. of heparin). In each of two test-tubes, mix equal volumes of thereconstituted preparation and a 3 g/l solution of fibrinogen R. Keep one of the tubes at 37C for 6 hand the other at room temperature for 24 h. In a third tube, mix a volume of the fibrinogen solutionwith an equal volume of a solution of human thrombin R (1 I.U./ml) and place the tube in a water-bath at 37C. No coagulation occurs in the tubes containing the preparation to be examined.Coagulation occurs within 30 s in the tube containing thrombin.



Pyrogens (2.6.8). It complies with the test for pyrogens. Inject per kilogram of the rabbits mass avolume of the reconstituted preparation equivalent to not less than 30 I.U. of factor IX.

ASSAY

Factor IX Carry out the assay of coagulation factor IX (2.7.11).The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated

potency. The confidence interval of the estimated potency (P = 0.95) is not greater than 80 per centto 125 per cent.

Factor VII If the label states that the preparation contains factor VII, carry out the assay of coagula-tion factor VII (2.7.10).

The estimated potency is not less than 80 per cent and not more than 125 per cent of the statedpotency. The confidence interval of the estimated potency (P = 0.95) is not greater than 80 per centto 125 per cent.

Factors II and X If the label states that the preparation contains factors II and X, carry out validatedassays for these components.

The estimated potency is not less than 80 per cent and not more than 125 per cent of the statedpotency. The confidence interval of the estimated potency (P = 0.95) is not greater than 80 per centto 125 per cent.

STORAGE


LABELLING

The label states: the number of International Units of factor IX and, where applicable, of factors II, VII and X

per container, where applicable, that the preparation contains protein C and/or protein S, the amount of protein per container, the name and quantity of any added substances, including where applicable heparin, the name and quantity of the liquid to be used for reconstitution, the storage conditions, the expiry date, that the transmission of infectious agents cannot be totally excluded when medicinal products


66-23

Dried Fibrinogen

Dried Fibrinogen complies with the requirements of the 3rd edition of the European Pharmacopoeia for Freeze-dried Human Fibrinogen [0024]. These requirements are reproduced after the heading Definition below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Freeze-dried human fibrinogen contains the soluble constituent of human plasma that istransformed to fibrin on the addition of thrombin. It is obtained from Human plasma for fractionation(853). The preparation may contain auxiliary substances such as salts, buffers and stabilisers.

When dissolved in the volume of the solvent stated on the label, the solution contains not lessthan 10 g/l of fibrinogen.

PRODUCTION

The method of preparation includes a step or steps that have been shown to remove or toinactivate known agents of infection; if substances are used for inactivation of viruses duringproduction, the subsequent purification procedure must be validated to demonstrate that theconcentration of these substances is reduced to a suitable level and any residues are such as not tocompromise the safety of the preparation for patients.

No antibiotic is added to the plasma used and no antimicrobial preservative is included in thepreparation.

The method of preparation is such as to obtain fibrinogen with a specific activity (fibrinogencontent with respect to total protein content) not less than 80 per cent. The fibrinogen content isdetermined by a suitable method such as that described under Assay and the total protein content isdetermined by a suitable method such as that described under Total protein in Human AlbuminSolution (255). If a protein stabiliser (for example, human albumin) is added to the preparation, therequirement for specific activity applies to the fibrinogen before addition of the stabiliser. Albuminmay also be obtained with fibrinogen during fractionation and a specific determination of albumin isthen carried out by a suitable immunochemical method (2.7.1) and the quantity of albumindetermined is subtracted from the total protein content for the calculation of the specific activity.

CHARACTERS

A white or pale yellow powder or friable solid.

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined, reconstituted as stated on the label immediately before use. It is recommended thatthe tests be carried out using antisera specific to the plasma proteins of each species of domesticanimal commonly used in the preparation of materials of biological origin in the country concerned.The preparation is shown to contain proteins of human origin and gives negative results with antiseraspecific to plasma proteins of other species.

B. The assay contributes to the identification of the preparation.

TESTS

pH (2.2.3). The pH of the reconstituted preparation is 6.5 to 7.5.

Osmolality (2.2.35). The osmolality of the reconstituted preparation is not less than 240 mosm/kg.

Solubility Add the volume of solvent stated on the label to the contents of a container. The prepara-tion dissolves within 30 min at 20C to 25C, forming an almost colourless, slightly opalescentsolution.

Stability of solution Allow the reconstituted solution to stand at 20C to 25C. No gel formationappears within 60 min of reconstitution.

Hepatitis B surface antigen Examine the reconstituted preparation by an immunochemicalmethod (2.7.1) of suitable sensitivity, such as radio-immunoassay. Hepatitis B surface antigen is notdetected.

Water (2.5.12). Not more than 3.0 per cent, determined by the semi-micro determination of water.

Sterility (2.6.1). The reconstituted preparation complies with the test for sterility.

Pyrogens (2.6.8). The reconstituted preparation complies with the test for pyrogens. Inject perkilogram of the rabbits mass a volume of the reconstituted preparation equivalent to not less than30 mg of fibrinogen, calculated from the quantity stated on the label.

ASSAY

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Mix 0.2 ml of the reconstituted preparation with 2 ml of a suitable buffer solution (pH 6.6 to 6.8)containing sufficient thrombin (approximately 3 I.U./ml) and calcium (0.05 mol/l). Maintain at 37Cfor 20 min, separate the precipitate by centrifugation (5000 g, 20 min), wash thoroughly with a 9 g/lsolution of sodium chloride R. Determine the nitrogen content by sulphuric acid digestion (2.5.9) andcalculate the fibrinogen (clottable protein) content by multiplying the result by 6.0. The content isnot less than 70 per cent and not more than 130 per cent of the amount of fibrinogen stated on thelabel.

STORAGE


LABELLING

The label states: the amount of fibrinogen in the container, the name and volume of the solvent to be used to reconstitute the preparation, where applicable, the name and quantity of protein stabiliser in the preparation.

__________________________________________________________________________________________________________ Ph Eur

66-25

Fibrin Sealant Kit

Fibrin Sealant Kit complies with the requirements of the 3rd edition of the European Pharmacopoeia [0903].These requirements are reproduced after the heading Definition below.

Ph Eur ___________________________________________________________________________________________________________

DEFINITION

Fibrin sealant kit is essentially composed of two components, namely component 1 (fibrinogenconcentrate), a protein fraction containing human fibrinogen and human factor XIII, and component2, a preparation containing human thrombin; the latter component converts the former to fibrin afterreconstitution and combining in the presence of calcium ions. Other ingredients (for example, humanfibronectin and a plasmin inhibitor such as aprotinin) and stabilisers (for example, human albumin)may be added before or during the thrombin-induced generation of fibrin. No antimicrobialpreservative is added.

Human constituents are obtained from plasma that complies with the requirements of the mono-graph on Human plasma for fractionation (853). No antibiotic is added to the plasma used.

When thawed or reconstituted in the volume of solvent stated on the label, component 1 containsnot less than 60 g/litre of clottable protein and not less than 10 units of factor XIII per millilitre; thethrombin activity of component 2 varies over a wide range (approximately 4 to 500 I.U./ml).

PRODUCTION

The method of preparation includes a step or steps that have been shown to remove or to inactivateknown agents of infection; if substances are used for inactivation of viruses during production, thesubsequent purification procedure must be validated to demonstrate that the concentration of thesesubstances is reduced to a suitable level and any residues are such as not to compromise the safety ofthe preparation for patients.

Constituents or mixtures of constituents are passed through a bacteria-retentive filter anddistributed aseptically into sterile containers. Containers of freeze-dried constituents are closed undervacuum or filled with oxygen-free nitrogen or other suitable inert gas before being closed. In eithercase, they are closed so as to exclude micro-organisms.

CHARACTERS

Freeze-dried constituents are white or pale yellow powders or friable solids. Frozen constituents arecolourless or pale yellow, opaque solids. Liquid constituents are colourless or pale yellow.

For the freeze-dried and frozen constituents, reconstitute or thaw as stated on the label immediately beforecarrying out the identification and the tests, except those for solubility and water.

I Component 1 (Fibrinogen concentrate)

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the test be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative reactions with antisera specific to plasma proteins of other species.

B. The assay for fibrinogen contributes to the identification of component 1.

C. The assay for factor XIII contributes to the identification of component 1.

TESTS

pH (2.2.3). The pH is 6.5 to 8.0.

Solubility Freeze-dried concentrates dissolve within 20 min in the volume of solvent for reconstitu-tion and at the temperature stated on the label, forming an almost colourless, clear or slightly turbidsolution.

Stability of solution No gel formation appears within 120 min of reconstitution or thawing.

Water (2.5.12). For a freeze-dried preparation, not more than 3.0 per cent m/m, determined by thesemi-micro determination of water.


ASSAY

Fibrinogen (clottable protein) Use method A or method B.

The estimated content in milligrams of clottable protein is not less than 70 per cent and not morethan 130 per cent of the stated amount.

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A. Clottable protein (determination of nitrogen by sulphuric acid digestion). Mix 0.2 ml of thereconstituted preparation with 2 ml of a suitable buffer solution (pH 6.6 to 6.8) containing sufficienthuman thrombin R (approximately 3 I.U./ml) and calcium (0.05 mol/litre). Maintain at 37C for20 min, separate the precipitate by centrifugation (5000 g, 20 min), wash thoroughly with a 9 g/lsolution of sodium chloride R and determine the protein as nitrogen by sulphuric acid digestion(2.5.9). Calculate the protein content by multiplying the result by 6.0.

B. Clotting assay. Dilute the reconstituted preparation with a 9 g/l solution of sodium chloride R to afibrinogen content in the range 0.1 to 1 mg/ml. Take 0.2 ml of the dilution, maintain at 37C for60 s and add 0.2 ml of a suitable solution of human thrombin R (approximately 20 I.U./ml andcontaining at least 1 mmol/litre of calcium). Determine the clotting time by a suitablemethod. Repeat the procedure with each of at least three different dilutions, in the range statedabove, of a suitable fibrinogen standard (for example, standard human plasma). Standard humanplasma is calibrated against a pool of fresh plasma (>100 donors) by means of the assay. Draw acalibration curve on log-log graph paper using the measured clotting times for the standard dilutionsand the fibrinogen content; use the curve obtained to determine the fibrinogen content of thepreparation to be examined.

Factor XIII For determination of potency (functional factor XIII), make appropriate dilutions of thereconstituted component 1 and of standard human plasma (reference preparation) using as diluent asolution containing 9 g/l of sodium chloride R and 4 g/l of human albumin. Add to each dilutionsuitable amounts of a solution of bovine fibrinogen free from factor XIII activity (AF XIII) and anexcess of calcium and thrombin, to coagulate the fibrinogen. Allow to stand at 37C for 1 h. Add toeach tube a suitable amount of a 10 g/l solution of chloroacetic acid R, shake every 10 min and after 30min note for each sample the highest dilution factor (D) at which the clot has not yet been dissolved.Calculate the factor XIII activity in units per millilitre from the expression:

AD

DAF XIII

comp

refF XIII (ref=

1)

The estimated potency given in units is not less than 60 per cent and not more than 140 per cent ofthe stated potency.

II Component 2 (Thrombin preparation)

IDENTIFICATION

A. Using a suitable range of species-specific antisera, carry out precipitation tests on the preparationto be examined. It is recommended that the test be carried out using antisera specific to the plasmaproteins of each species of domestic animal commonly used in the preparation of materials ofbiological origin in the country concerned. The preparation is shown to contain proteins of humanorigin and gives negative react

BP_05 - 2001

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Transcript of BP_05 - 2001