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RABIES IMMUNOGLOBULIN

 

RABIES IMMUNOGLOBULIN

          Human Rabies Immune Globulin; Antirabies Immunoglobulin Injection; Human Antirabies Immunoglobulin Injection; HRIG

Category Passive immunizing agent.

          Rabies immunoglobulin is a liquid or freeze-dried preparation containing human immunoglobulins, mainly immunoglobulin G (IgG). The preparation is intended for intramuscular administration. It is obtained from plasma from donors immunized against rabies. It contains specific antibodies neutralizing the rabies virus. Immunoglobulin may be added.

          Rabies Immunoglobulin complies with the requirements stated under Immunoglobulin, p. 209, except for the minimum total protein content, and with those under this monograph.

Description The liquid Rabies Immunoglobulin is clear or slightly opalesscent and colourless or pale yellow to light brown.

          The freeze-dried Rabies Immunoglobulin is a white or slightly yellow powder or solid friable mass.

Stability Liquid Rabies Immunoglobulin should be discarded if it has been frozen. The reconstituted solution of freeze-dried Rabies Immunoglobulin should be used immediately or as stated on the label.

Strength available 150 IU per ml.

Dose Intramuscular, 20 IU per kg of body weight with as much as possible of the volume infiltrated at the site(s) of the bite(s) and the remainder should be administered at a distant site, deltoid preferred.

Contra-indication It is not for intravenous administration.

Warning

          1. Rabies immunoglobulin should be administered cautiously to individuals with a specific IgA deficiency since anaphylaxis may occur.

          2. Rabies immunoglobulin should be used with caution in individuals with thrombocytopenia or bleeding disorders, since bleeding may occur following intramuscular administration of the drug.

          3. Rabies immunoglobulin should not be administered to an individual who has been previously immunized with rabies vaccine and has a known adequate rabies antibody titer.

          4. Local tenderness, pain, soreness or stiffness of the muscles may occur at the injection site.

          5. Low-grade fever, urticaria, or angioedema may occur.

Additional information The antibodies in rabies immunoglobulin preparations may interfere with the immune response to certain live virus vaccines such as measles, mumps and rubella including MMR. These vaccines should be administered at least 14 days before or 4 months after treatment of rabies. However, there appears to be no interference between rabies immunoglobulin and oral poliomyelitis vaccine (OPV), yellow fever vaccine or oral typhoid (strain Ty 21a) vaccine.

Expiration date When stored under the prescribed conditions, the expiration date is not later than 2 years from the date of manufacture, or as indicated on the label.

Packaging and storage Liquid Rabies Immunoglobulin shall be kept in a sealed colourless glass container,protected from light, and stored at a temperature of 2º to 8º; avoid freezing.

          Freeze-dried Rabies Immunoglobulin shall be stored in a sealed and colourless glass containers, protected from light at a temperature not exceeding 25º, unless otherwise specified by manufacturers.

Labelling Complies with the “General Information for Biological Products”, p. 177. In addition the label on the container states the number of IU per container.

          See also under Immunoglobulin, p. 209.

Assay The potency is determined by comparing the dose of immunoglobulin required to neutralize the infectivity of a rabies virus suspension with the dose of a reference preparation, calibrated in International Units, required to produce the same degree of neutralization.

          Carry out the determination as described in the “Immunochemical Method” (Appendix 14.5). The test is performed in sensitive cell cultures and the presence of unneutralized virus is revealed by immunofluorescence. Rapid Fluorescent-Focus Inhibition Test (RFFIT) is described as the reference immunofluorescent technique for rabies immunoglobulin. Carry out the test in suitable sensitive cells. It is usual to use the BHK 21 cell line, grown in the medium described below, between the 18th and 30th passage levels counted from the ATCC seed lot. Harvest the cells after 2 to 4 days of growth, treat with trypsin and prepare a suspension containing 500,000 cells per ml (cell suspension). Ten minutes before using the cell suspension, add 10 μg of diethylamino-ethyldextran per ml, if necessary, to increase the sensitivity of the cells.

          Use a fixed virus strain grown in sensitive cells, such as the CVS strain of rabies virus adapted to growth in the BHK 21 cell line (seed virus suspension). Estimate the titre of the seed virus suspension as follows.

          Prepare a series of dilutions of the viral suspension. In the chambers of cell-culture slides (8 chambers per slide), place 0.1 ml of each dilution and 0.1 ml of medium and add 0.2 ml of the cell suspension. Incubate in an atmosphere of carbon dioxide at 37º for 24 hours.

          Carry out fixation, immunofluorescence staining and evaluation as described below. Determine the endpoint titre of the seed virus suspension and prepare the working virus dilution corresponding to 100 CCID50 per 0.1 ml.

          For each assay, check the amount of virus used by performing a control titration: from the dilution corresponding to 100 CCID50 per 0.1 ml, make three tenfold dilutions. Add 0.1 ml of each dilution to four chambers containing 0.1 ml of medium and add 0.2 ml of the cell suspension. The test is not valid unless the titre lies between 30 CCID50 and 300 CCID50.

          Dilute the reference preparation to a concentration of 2 IU per ml using non-supplemented culture medium (stock reference dilution, stored below –80º). Prepare two suitable predilutions (1:8 and 1:10) of the stock reference dilution so that the dilution of the reference preparation that reduces the number of fluorescent fields by 50 per cent lies within the four dilutions of the cell-culture slide. Add 0.1 ml of the medium to each chamber, except the first in each of two rows, to which add respectively 0.2 ml of the two predilutions of the stock reference dilution transferring successively 0.1 ml to the other chambers. Dilute the preparation to be examined 1 in 100 using non-supplemented medium (stock immunoglobulin dilution) to reduce to a minimum errors due to viscosity of the undiluted preparation and make three suitable predilutions so that the dilution of the preparation to be examined that reduces the number of fluorescent fields by 50 per cent lies within the four dilutions of the cellculture slide. Add 0.1 ml of the medium to all the chambers except the first in each of three rows, to which add respectively 0.2 ml of the three predilutions of the stock immunoglobulin dilution. Prepare a series of twofold dilutions transferring successively 0.1 ml to the other chambers.

          To all the chambers containing the dilutions of the reference preparation and the dilutions of the preparation to be examined, add 0.1 ml of the virus suspension corresponding to 100 CCID50 per 0.1 ml (working virus dilution). Shake manually and allow to stand in an atmosphere of carbon dioxide at 37º for 90 minutes. Add 0.2 ml of the cell suspension, shake manually and allow to stand in an atmosphere of carbon dioxide at 37º for 24 hours.

          After 24 hours, discard the medium and remove the plastic walls. Wash the cell monolayer with phosphate buffered saline pH 7.4 and then with a mixture of 20 volumes of water and 80 volumes of acetone and fix in a mixture of 20 volumes of water and 80 volumes of acetone at –20º for 3 minutes. Spread on the slides fluorescein-conjugated rabies antiserum ready for use. Allow to stand in an atmosphere with a high level of moisture at 37º for 30 minutes. Wash with phosphate buffered saline pH 7.4 and dry. Examine twenty fields in each chamber at a magnification of 250×, using a microscope equipped for fluorescence readings. Note the number of fields with at least one fluorescent cell. Check the test dose used in the virus titration slide and determine the dilution of the reference preparation and the dilution of the preparation to be examined that reduce the number of fluorescent fields by 50 per cent, calculating the two or three dilutions together using probit analysis. The test is not valid unless the statistical analysis shows a significant slope of the dose response curve and no evidence of deviation from linearity or parallelism.

          The stated potency is not less than 150 IU per ml. The estimated potency is not less than the stated potency and is not more than two times the stated potency. The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated potency.

          CULTURE MEDIUM FOR GROWTH OF BHK 21 CELLS

          Commercially available media that have a slightly different composition from that shown below may also be used.

  Sodium chloride 6.4 g
  Potassium chloride 0.40 g
  Calcium chloride, anhydrous 0.20 g
  Magnesium sulfate 0.20 g
  Sodium dihydrogenphosphate, monohydrate 0.124 g
  Dextrose monohydrate  4.5 g
  Iron(III) nitrate nonahydrate 0.10 mg
  L-Arginine hydrochloride 42.0 mg
  L-Cystine 24.0 mg
  L-Histidine 16.0 mg
  L-Isoleucine 52.0 mg
  L-Leucine 52.0 mg
  L-Lysine hydrochloride 74.0 mg
  L-Phenylalanine 33.0 mg
  L-Threonine 48.0 mg
  L-Tryptophan 8.0 mg
  L-Tyrosine 36.0 mg
  L-Valine 47.0 mg
  L-Methionine  15.0 mg
  L-Glutamine  0.292 g
  i-Inositol  3.60 mg
  Choline chloride  2.0 mg
  Folic acid  2.0 mg
  Nicotinamide  2.0 mg
  Calcium pantothenate  2.0 mg
  Pyridoxine hydrochloride  2.0 mg
  Thiamine hydrochloride  2.0 mg
  Riboflavine  0.2 mg
  Phenol red  15.0 mg
  Sodium hydrogencarbonate  2.75 g
  Water to 1000 ml
 

The medium is supplemented with: Fetal calf serum

(heated at 56º for 30 minutes)

 10 per cent
  Tryptose phosphate broth  10 per cent
  Benzylpenicillin sodium 60 mg/l
  Streptomycin 0.1 g/l

 

MONOGRAPHS • RABIES IMMUNOGLOBULIN
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หมายเหตุ / Note : TP II 2011 PAGE 216-219