2.7.2.

Microbiological assay of antibiotics

EUROPEAN PHARMACOPOEIA 5.0

The potency of an antibiotic is estimated by comparing the inhibition of growth of sensitive micro-organisms produced by known concentrations of the antibiotic to be examined and a reference substance. The reference substances used in the assays are substances whose activity has been precisely determined with reference to the corresponding international standard or international reference preparation. The assay must be designed in a way that will permit examination of the validity of the mathematical model on VALIDATION OF THE METHOD which the potency equation is based. If a parallel-line model Validation criteria is chosen, the 2 log dose-response (or transformed response) A quantitative immunochemical method is not valid unless : lines of the preparation to be examined and the reference 1) The antibody or antigen does not significantly discriminate preparation must be parallel ; they must be linear over the range of doses used in the calculation. These conditions between the test and standard. For a labelled reactant, the must be verified by validity tests for a given probability, corresponding reactant does not significantly discriminate usually P = 0.05. Other mathematical models, such as the between the labelled and unlabelled compound, slope ratio model, may be used provided that proof of validity 2) The method is not affected by the assay matrix, that is demonstrated. is, any component of the test sample or its excipients, Unless otherwise stated in the monograph, the confidence which can vary between samples. These may include high limits (P = 0.95) of the assay for potency are not less than concentrations of other proteins, salts, preservatives or 95 per cent and not more than 105 per cent of the estimated contaminating proteolytic activity, potency. 3) The limit of quantitation is below the acceptance criteria Carry out the assay by method A or method B. stated in the individual monograph, 4) The precision of the assay is such that the variance of A. DIFFUSION METHOD the results meets the requirements stated in the individual Liquefy a medium suitable for the conditions of the assay monographs, and inoculate it at a suitable temperature, for example 48 C 5) The order in which the assay is performed does not give to 50 C for vegetative forms, with a known quantity of a rise to systematic errors. suspension of micro-organisms sensitive to the antibiotic to be examined, such that clearly defined zones of inhibition Validation methods of suitable diameter are produced with the concentrations In order to verify these criteria, the validation design of the antibiotic used for the assay. Immediately pour into includes the following elements : Petri dishes or large rectangular dishes a quantity of the 1) The assay is performed at least in triplicate, inoculated medium to form a uniform layer 2 mm to 5 mm 2) The assay includes at least 3 different dilutions of the thick. Alternatively, the medium may consist of 2 layers, only standard preparation and 3 dilutions of sample preparations the upper layer being inoculated. of presumed activity similar to the standard preparation, Store the dishes so that no appreciable growth or death of the micro-organisms occurs before the dishes are used and 3) The assay layout is randomised, 4) If the test sample is presented in serum or formulated with so that the surface of the medium is dry at the time of use. Using the solvent and the buffer solution indicated in other components, the standard is likewise prepared, 5) The test includes the measurement of non-specific binding Table 2.7.2.-1, prepare solutions of the reference substance and of the antibiotic to be examined having known of the labelled reactant, concentrations and presumed to be of equal activity. Apply 6) For displacement immunoassay : the solutions to the surface of the medium, for example, (a) maximum binding (zero displacement) is determined, in sterile cylinders of porcelain, stainless steel or other suitable material, or in cavities prepared in the agar. The (b) dilutions cover the complete response range from values close to non-specific binding to maximum binding, same volume of solution must be added to each cylinder or cavity. Alternatively, use sterile absorbent paper discs of preferably for both standard and test preparations. suitable quality ; impregnate the discs with the solutions of STATISTICAL CALCULATION the reference substance or the solutions of the antibiotic to To analyse the results, response curves for test and standard be examined and place on the surface of the agar. may be analysed by the methods described in 5.3. Statistical In order to assess the validity of the assay, use not fewer Analysis of Results of Biological Assays and Tests. than 3 doses of the reference substance and 3 doses of the antibiotic to be examined having the same presumed Significant non-parallelism indicates that the antibody or activity as the doses of the reference substance. It is antigen discriminates between test and standard, and the preferable to use a series of doses in geometric progression. results are not valid. 188 See the information section on general monographs (cover pages)

standard for calibration and dilutions of the test material are introduced into a row of wells in a gel and a fixed amount of the corresponding reactant is introduced into an opposite row of wells. The titre of the test material may be determined as the highest dilution showing a precipitation line. A number of modifications of crossed immunoelectrophoresis and electroimmunoassay methods exist. Other techniques combine separation of antigens by molecular size and serological properties. Visualisation and characterisation of immunoprecipitation lines These may be performed by selective or non-selective stains, by fluorescence, by enzyme or isotope labelling or other relevant techniques. Selective staining methods are usually performed for characterisation of non-protein substances in the precipitates. In translucent gels such as agar or agarose, the precipitation line becomes clearly visible in the gel, provided that the concentration of each of the reactants is appropriate.

In displacement immunoassays, the values for non-specific binding and maximum displacement at high test or standard concentration must not be significantly different. Differences may indicate effects due to the matrix, either inhibition of binding or degradation of tracer. 01/2005:20702

2.7.2. MICROBIOLOGICAL ASSAY OF ANTIBIOTICS

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2.7.2. Microbiological assay of antibiotics

In routine assays when the linearity of the system has been subject to agreement by the competent authority. However, demonstrated over an adequate number of experiments in all cases of dispute, a three-point assay as described above using a three-point assay, a two-point assay may be sufficient, must be applied. Table 2.7.2.-1. Diffusion assay
Antibiotic Solvent to be used Buffer solution Reference substance in preparing the (pH) stock solution Amphotericin B CRS Dimethyl sulphoxide R pH 10.5 (0.2 M) Micro-organism Saccharomyces cerevisiae ATCC 9763 IP 1432-83 Micrococcus luteus NCTC 7743 CIP 53.160 ATCC 10240 Mycobacterium smegmatis ATCC 607 Bordetella bronchiseptica NCTC 8344 CIP 53.157 ATCC 4617 Escherichia coli NCIB 8879 CIP 54.127 ATCC 10536 Bacillus subtilis NCTC 8236 CIP 1.83 Water R pH 8.0 (0.05 M) Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Bacillus pumilus NCTC 8241 CIP 76.18 pH 8.0 (0.05 M) Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Bacillus pumilus NCTC 8241 CIP 76.18 Bacillus pumilus NCTC 8241 CIP 76.18 Gentamicin sulphate Gentamicin sulphate CRS Water R pH 8.0 (0.05 M) Staphylococcus epidermidis NCIB 8853 CIP 68.21 ATCC 12228 Bacillus subtilis CIP 52.62 ATCC 6633 NCTC 10400 Bacillus subtilis CIP 52.62 ATCC 6633 NCTC 10400 A - pH 7.9 30-37 C A - pH 7.9 30-37 C Medium and final pH ( 0.1 pH unit) Incubation temperature

Amphotericin B

F - pH 6.1

35-37 C

Bacitracin zinc

0.01 M Bacitracin zinc CRS hydrochloric acid

pH 7.0 (0.05 M)

A - pH 7.0

35-39 C

Bleomycin sulphate

Bleomycin sulphate CRS

Water R

pH 6.8 (0.1 M)

G - pH 7.0

35-37 C

Colistimethate sodium

Colistimethate sodium CRS

Water R

pH 6.0 (0.05 M)

B - pH 7.3

35-39 C

Dihydrostreptomycin sulphate

Dihydrostreptomycin sulphate CRS

A - pH 7.9

30-37 C

Erythromycin estolate

Erythromycin CRS

Methanol R (see the monographs)

E - pH 7.9

30-37 C

Framycetin sulphate

Framycetin sulphate CRS

Water R

pH 8.0 (0.05 M)

E - pH 7.9

30-37 C

A - pH 7.9

35-39 C

A - pH 7.9

35-39 C

Josamycin

Josamycin CRS

Methanol R (see the monograph)

pH 5.6

A - pH 6.6

35-37 C

Josamycin propionate

Josamycin propionate CRS

Methanol R (see the monograph)

pH 5.6

A - pH 6.6

35-37 C

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

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2.7.2. Microbiological assay of antibiotics

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Antibiotic

Solvent to be used Buffer solution Reference substance in preparing the (pH) stock solution

Micro-organism Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633

Medium and final pH ( 0.1 pH unit) A - pH 7.9

Incubation temperature 30-37 C

Kanamycin monosulphate Kanamycin Water R monosulphate CRS Kanamycin acid sulphate pH 8.0 (0.05 M)

Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Bacillus pumilus NCTC 8241 CIP 76.18

A - pH 7.9

35-39 C

E - pH 7.9

30-37 C

Neomycin sulphate Neomycin sulphate for microbiological Water R assay CRS

pH 8.0 (0.05 M)

Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Staphylococcus aureus ATCC 6538P CIP 53.156 Candida tropicalis CIP 1433-83 NCYC 1393

E - pH 7.9

30-37 C

Netilmicin Netilmicin sulphate sulphate CRS

Water R

pH 8.0 0.1

A - pH 7.9 F - pH 6.0

32-35 C 30-37 C

Nystatin

Nystatin CRS

Dimethylformamide R

pH 6.0 (0.05 M) containing 5 per Saccharomyces cent V/V of dimeth- cerevisiae ylformamide R NCYC 87 CIP 1432-83 ATCC 9763 pH 7.0 (0.05 M)

F - pH 6.0

30-32 C

Rifamycin sodium

Rifamycin sodium CRS

Methanol R

Micrococcus luteus NCTC 8340 CIP 53.45 ATCC 9341 Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Bacillus subtilis NCTC 8236 CIP 1.83

A - pH 6.6

35-39 C

Spiramycin

Spiramycin CRS

Methanol R

pH 8.0 (0.05 M)

A - pH 7.9 A - pH 7.9

30-32 C 30-37 C

Streptomycin sulphate

Streptomycin sulphate CRS

Water R

pH 8.0 (0.05 M)

Bacillus subtilis NCTC 10400 CIP 52.62 ATCC 6633 Micrococcus luteus NCTC 8340 CIP 53.45 ATCC 9341 Bacillus subtilis NCTC 8236 CIP 52.62 ATCC 6633

A - pH 7.9

30-37 C

Tylosin for veterinary use Tylosin CRS Tylosin tartrate for veterinary use Vancomycin hydrochloride Vancomycin hydrochloride CRS

2.5 per cent V/V solution of methanol R in 0.1 M phosphate buffer solution pH 7.0 R Water R

A mixture of 40 volumes of methanol R and 60 volumes of 0.1 M phosphate buffer solution pH 8.0 R pH 8.0

A - pH 8.0

32-35 C

A - pH 8.0

37-39 C

Arrange the solutions on each Petri dish or on each rectangular dish according to a statistically suitable design, except for small Petri dishes that cannot accommodate more than 6 solutions, arrange the solutions of the antibiotic to be examined and the solutions of the reference substance in an alternate manner to avoid interaction of the more concentrated solutions. Incubate at a suitable temperature for about 18 h. A period of diffusion prior to incubation, usually 1 h to 4 h, at room temperature or at about 4 C, as appropriate, may be used 190

to minimise the effects of the variation in time between the application of the solutions and to improve the regression slope. Measure the diameters with a precision of at least 0.1 mm or the areas of the circular inhibition zones with a corresponding precision and calculate the potency using appropriate statistical methods. Use in each assay the number of replications per dose sufficient to ensure the required precision. The assay may be repeated and the results combined statistically to obtain the

See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0

2.7.2. Microbiological assay of antibiotics

required precision and to ascertain whether the potency of the antibiotic to be examined is not less than the minimum required. B. TURBIDIMETRIC METHOD Inoculate a suitable medium with a suspension of the chosen micro-organism having a sensitivity to the antibiotic to be examined such that a sufficiently large inhibition of microbial growth occurs in the conditions of the test. Use a known quantity of the suspension chosen so as to obtain a readily measurable opacity after an incubation period of about 4 h. Use the inoculated medium immediately after its preparation. Using the solvent and the buffer solution indicated in Table 2.7.2.-2 prepare solutions of the reference substance and of the antibiotic to be examined having known concentrations presumed to be of equal activity. In order that the validity of the assay may be assessed, use not fewer than 3 doses of the reference substance and 3 doses of the antibiotic to be examined having the same presumed activity as the doses of the reference substance. It is preferable to use a series of doses in geometric progression. In order to obtain the required linearity, it may be necessary to select from a large number 3 consecutive doses, using corresponding doses for the reference substance and the antibiotic to be examined. Distribute an equal volume of each of the solutions into identical test-tubes and add to each tube an equal volume of inoculated medium (for example, 1 ml of the solution and 9 ml of the medium). For the assay of tyrothricin add 0.1 ml of the solution to 9.9 ml of inoculated medium. Prepare at the same time 2 control tubes without antibiotic, both containing the inoculated medium and to one of which is added immediately 0.5 ml of formaldehyde R. These tubes are used to set the optical apparatus used to measure the growth.

Place all the tubes, randomly distributed or in a Latin square or randomised block arrangement, in a water-bath or other suitable apparatus fitted with a means of bringing all the tubes rapidly to the appropriate incubation temperature and maintain them at that temperature for 3 h to 4 h, taking precautions to ensure uniformity of temperature and identical incubation time. After incubation, stop the growth of the micro-organisms by adding 0.5 ml of formaldehyde R to each tube or by heat treatment and measure the opacity to 3 significant figures using suitable optical apparatus. Alternatively use a method which allows the opacity of each tube to be measured after exactly the same period of incubation. Calculate the potency using appropriate statistical methods. Linearity of the dose-response relationship, transformed or untransformed, is often obtained only over a very limited range. It is this range which must be used in calculating the activity and it must include at least 3 consecutive doses in order to permit linearity to be verified. In routine assays when the linearity of the system has been demonstrated over an adequate number of experiments using a three-point assay, a two-point assay may be sufficient, subject to agreement by the competent authority. However, in all cases of dispute, a three-point assay must be applied. Use in each assay the number of replications per dose sufficient to ensure the required precision. The assay may be repeated and the results combined statistically to obtain the required precision and to ascertain whether the potency of the antibiotic to be examined is not less than the minimum required.

Table 2.7.2.-2. Turbidimetric assay

Antibiotic Reference substance Colistimethate sodium CRS Solvent to be used Buffer solution in preparing the (pH) stock solution Water R pH 7.0 Micro-organism Escherichia coli NCIB 8666 CIP 2.83 ATCC 9637 Klebsiella pneumoniae NCTC 7427 CIP 53.153 ATCC 10031 Klebsiella pneumoniae NCTC 7427 CIP 53.153 ATCC 10031 Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Medium and final Incubation pH ( 0.1 pH unit) temperature

Colistimethate sodium

C - pH 7.0

35-37 C

Dihydrostreptomycin sulphate

Dihydrostreptomycin sulphate CRS

Water R

pH 8.0

C - pH 7.0

35-37 C

D - pH 7.0

35-37 C

Erythromycin estolate Erythromycin CRS Erythromycin ethylsuccinate Methanol R (see the monographs) pH 8.0

C - pH 7.0

35-37 C

Framycetin sulphate

Framycetin sulphate CRS

Water R

pH 8.0

C - pH 7.0

35-37 C

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

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Antibiotic

Reference substance

Solvent to be used Buffer solution in preparing the (pH) stock solution

Micro-organism Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Enterococcus hirae CIP 58.55 ATCC 10541 Staphylococcus aureus ATCC 6538 P

Medium and final Incubation pH ( 0.1 pH unit) temperature

Gentamicin sulphate

Gentamicin sulphate CRS

Water R

pH 7.0

C - pH 7.0

35-37 C

Gramicidin CRS Gramicidin

*

Methanol R

pH 7.0*

C - pH 7.0

35-37 C

Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions, for example 0.1 mg/ml of polysorbate 80 R Methanol R (see the monograph) Staphylococcus aureus CIP 53.156 ATCC 6538 P NCTC 7447 Staphylococcus aureus CIP 53.156 ATCC 6538 P NCTC 7447 Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Escherichia coli NCIB 8879 CIP 54.127 ATCC 10536 Staphylococcus aureus NCTC 7447 CIP 53.156 ATCC 6538 P Klebsiella pneumoniae NCTC 7427 CIP 53.153 ATCC 10031 Staphylococcus aureus NCTC 6571 ATCC 9144 CIP 53.154 Enterococcus hirae ATCC 10541 Staphylococcus aureus CIP 53.156 ATCC 6538 P

Josamycin

Josamycin CRS

pH 5.6

C - pH 8.0

35-37 C

Josamycin propionate Kanamycin monosulphate Kanamycin acid sulphate

Josamycin propionate CRS

Methanol R (see the monograph)

pH 5.6

C - pH 8.0

35-37 C

Kanamycin Water R monosulphate CRS

pH 8.0

C - pH 7.0

35-37 C

Neomycin sulphate Neomycin sulphate for microbiological Water R assay CRS

pH 8.0

C - pH 7.0

35-37 C

Rifamycin sodium

Rifamycin sodium CRS

Methanol R

pH 7.0

C - pH 7.0

35-37 C

Spiramycin

Spiramycin CRS

Methanol R

pH 7.0

C - pH 7.0

35-37 C

Streptomycin sulphate

Streptomycin sulphate CRS

Water R

pH 8.0

C - pH 7.0

35-37 C

Tylosin for veterinary use Tylosin tartrate for veterinary use Tyrothricin Vancomycin hydrochloride Tylosin CRS

2.5 per cent V/V solution of methanol R in 0.1 M phosphate buffer solution pH 7.0 R Alcohol R

pH 7.0

C - pH 7.0

37 C

Gramicidin CRS Vancomycin hydrochloride CRS

Alcohol R

C - pH 7.0

37 C

Water R

pH 8.0

C - pH 7.0

37-39 C

antibiotic to be examined and are used in appropriate media and appropriate conditions of temperature and pH. The RECOMMENDED MICRO-ORGANISMS concentrations of the solutions used should be chosen so The following text details the recommended micro-organisms as to ensure that a linear relationship exists between the logarithm of the dose and the response in the conditions and the conditions of use. Other micro-organisms may be of the test. used provided that they are shown to be sensitive to the The following section is published for information. 192 See the information section on general monographs (cover pages)

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2.7.2. Microbiological assay of antibiotics

Preparation of inocula. Bacillus cereus var. mycoides ; Bacillus subtilis ; Bacillus pumilus. Spore suspensions of the organisms to be used as inocula are prepared as follows. Grow the organism at 35-37 C for 7 days on the surface of a suitable medium to which has been added 0.001 g/l of manganese sulphate R. Using sterile water R, wash off the growth, which consists mainly of spores. Heat the suspension at 70 C for 30 min and dilute to give an appropriate concentration of spores, usually 10 106 to 100 106 per millilitre. The spore suspensions may be stored for long periods at a temperature not exceeding 4 C. Alternatively, spore suspensions may be prepared by cultivating the organisms in medium C at 26 C for 4-6 days, then adding, aseptically, sufficient manganese sulphate R to give a concentration of 0.001 g/l and incubating for a further 48 h. Examine the suspension microscopically to ensure that adequate spore formation has taken place (about 80 per cent) and centrifuge. Re-suspend the sediment in sterile water R to give a concentration of 10 106 to 100 106 spores per millilitre, and then heat to 70 C for 30 min. Store the suspension at a temperature not exceeding 4 C. Bordetella bronchiseptica. Grow the test organism on medium B at 35-37 C for 16-18 h. Wash off the bacterial growth with sterile water R and dilute to a suitable opacity. Staphylococcus aureus ; Klebsiella pneumoniae ; Escherichia coli ; Micrococcus luteus ; Staphylococcus epidermidis. Prepare as described above for B. bronchiseptica but using medium A and adjusting the opacity to one which has been shown to produce a satisfactory dose-response relationship in the turbidimetric assay, or to produce clearly defined zones of inhibition of convenient diameter in the diffusion assay, as appropriate.

For bleomycin sulphate, prepare the buffer solution pH 6.8 as follows : dissolve 6.4 g of potassium dihydrogen phosphate R and 18.9 g of disodium hydrogen phosphate R in water R and dilute to 1000 ml with water R. For amphotericin B, prepare the 0.2 M phosphate buffer solution pH 10.5 as follows : dissolve 35 g of dipotassium hydrogen phosphate R in 900 ml of water R, add 20 ml of 1 M sodium hydroxide and dilute to 1000.0 ml with water R. Culture media. The following media or equivalent media may be used. Medium A
Peptone Pancreatic digest of casein Beef extract Yeast extract Glucose monohydrate Agar Water to produce 6g 4g 1.5 g 3g 1g 15 g 1000 ml

Medium B
Pancreatic digest of casein Papaic digest of soya bean Sodium chloride Dipotassium hydrogen phosphate Glucose monohydrate Agar Polysorbate 80 17 g 3g 5g 2.5 g 2.5 g 15 g 10 g

Saccharomyces cerevisiae ; Candida tropicalis. Grow the 1000 ml Water to produce test organism on medium F at 30-37 C for 24 h. Wash off the growth with a sterile 9 g/l solution of sodium chloride R. The polysorbate 80 is added to the hot solution of the other Dilute to a suitable opacity with the same solution. ingredients after boiling, and immediately before adjusting Buffer solutions. Buffer solutions having a pH between 5.8 to volume. and 8.0 are prepared by mixing 50.0 ml of 0.2 M potassium Medium C dihydrogen phosphate R with the quantity of 0.2 M sodium hydroxide indicated in Table 2.7.2.-3. Dilute with freshly 6g Peptone prepared distilled water R to produce 200.0 ml. 1.5 g
Beef extract

Table 2.7.2.-3.
pH 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 0.2 M Sodium hydroxide (ml) 3.72 5.70 8.60 12.60 17.80 23.65 29.63 35.00 39.50 42.80 45.20 46.80

Yeast extract Sodium chloride Glucose monohydrate Dipotassium hydrogen phosphate Potassium dihydrogen phosphate Water to produce

3g 3.5 g 1g 3.68 g 1.32 g 1000 ml

Medium D
Heart extract Yeast extract Peptone-casein Glucose monohydrate Sodium chloride Dipotassium hydrogen phosphate Potassium dihydrogen phosphate Potassium nitrate Water to produce 1.5 g 1.5 g 5g 1g 3.5 g 3.68 g 1.32 g 2g 1000 ml

These buffer solutions are used for all microbiological assays shown in Table 2.7.2.-1 with the exception of bleomycin sulphate and amphotericin B. General Notices (1) apply to all monographs and other texts

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2.7.4. Assay of human coagulation factor VIII

EUROPEAN PHARMACOPOEIA 5.0

Medium E
Peptone Meat extract Disodium hydrogen phosphate,12H2O Agar Water to produce 5g 3g 26.9 g 10 g 1000 ml

The disodium hydrogen phosphate is added as a sterile solution after sterilisation of the medium. Medium F
Peptone Yeast extract Beef extract Sodium chloride Glucose monohydrate Agar Water to produce 9.4 g 4.7 g 2.4 g 10.0 g 10.0 g 23.5 g 1000 ml

Both steps employ reagents that may be obtained commercially from a variety of sources. Although the composition of individual reagents may be subject to some variation, their essential features are described in the following specification. Deviations from this description may be permissible provided that it has been shown, using the International Standard for Human Blood Coagulation Factor VIII concentrate as the standard, that the results obtained do not differ significantly. Commercial assay kits are to be used in accordance with the manufacturers instructions ; it is important to ascertain the suitability for the assay of the kit used. REAGENTS

The coagulation factor reagent comprises purified proteins derived from human or bovine sources. These include 10 g Glycerol factor X, factor IXa, and a factor VIII activator, usually Peptone 10 g thrombin. These proteins are partly purified, preferably to at least 50 per cent, and do not contain impurities that Meat extract 10 g interfere with the activation of factor VIII or factor X. 3g Sodium chloride Factor X is present in amounts giving a final concentration Agar 15 g during the first step of the assay of 10-350 nmol/l, preferably 15-30 nmol/l. Factor IXa is prepared by activating purified Water to produce 1000 ml factor IX to factor IXa using factor XIa, and by subsequent purification of factor IXa from the reaction mixture. Its final pH 7.0 0.1 after sterilisation. concentration during factor Xa generation is less than 30 per cent of the factor X concentration, usually 1-100 nmol/l, 01/2005:20704 preferably 1-10 nmol/l. Thrombin may be present in its precursor form prothrombin, provided that its activation in the reagent is sufficiently rapid to give almost instantaneous, 2.7.4. ASSAY OF HUMAN complete activation of factor VIII in the assay. Phospholipids may be obtained from natural sources such as bovine brain COAGULATION FACTOR VIII or spinal cord or soya-bean extract, or synthetically prepared, Human coagulation factor VIII is assayed by its biological and must consist to a substantial extent, usually 15 per cent activity as a cofactor in the activation of factor X by activated to 35 per cent, of the species phosphatidylserine. The final factor IX (factor IXa) in the presence of calcium ions and phospholipid concentration during factor Xa generation is phospholipids. The potency of a factor VIII preparation is 1-50 mol/l, preferably 10-35 mol/l. The reagent contains estimated by comparing the quantity necessary to achieve calcium ions to give a final concentration of 5-15 mmol/l. a certain rate of factor Xa formation in a test mixture The final factor Xa generation is performed in a solution containing the substances that take part in the activation of containing at least 1 mg/ml of human or bovine albumin factor X, and the quantity of the International Standard, or which is appropriately buffered, at a pH of 7.38.0. The of a reference preparation calibrated in International Units, components of the complete reagent are usually divided into required to produce the same rate of factor Xa formation. at least two separate reagents each lacking the ability to generate factor Xa on its own. After reconstitution, these The International Unit is the factor VIII activity of a stated may be combined provided that no substantial amounts of amount of the International Standard which consists of a factor Xa are generated in the absence of factor VIII. In freeze-dried human coagulation factor VIII concentrate. the final incubation mixture, factor VIII must be the only The equivalence in International Units of the International rate-limiting component. Standard is stated by the World Health Organisation. Human coagulation factor VIII BRP is calibrated in The second step comprises the quantification of the formed International Units by comparison with the International factor Xa employing a chromogenic substrate that is specific Standard. for factor Xa. Generally this consists of a derivatised short The chromogenic assay method consists of two consecutive peptide of between three and five amino acids, joined to a chromophore group. On cleavage of this group from the steps : the factor VIII-dependent activation of factor X peptide substrate, its chromophoric properties shift to a in a coagulation-factor reagent composed of purified wavelength allowing its spectrophotometric quantification. components, and the enzymatic cleavage of a chromogenic The substrate is usually dissolved in water and used at a factor Xa substrate to yield a chromophore that can be quantified spectrophotometrically. Under appropriate assay final concentration of 0.2-2 mmol/l. The substrate may further contain appropriate inhibitors to stop further factor conditions, there is a linear relation between the rate of Xa generation and to suppress thrombin activity, thereby factor Xa formation and the factor VIII concentration. The improving selectivity for factor Xa. assay is summarised by the following scheme : Medium G 194 See the information section on general monographs (cover pages)