Feb 5, 2017 - The vaccine complies with the test for sterility. ... with a defined molecular size. ... Only PRP that complies with the following requirements.
Haemophilus type b conjugate vaccine
EUROPEAN PHARMACOPOEIA 5.0
Sterility (2.6.1). The vaccine complies with the test for sterility. ASSAY Carry out one of the prescribed methods for the assay of diphtheria vaccine (adsorbed) (2.7.6). The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose.
No complex products of animal origin are included in the menstruum used for preservation of strain viability, either for freeze-drying or for frozen storage.
It is recommended that PRP produced by the seed lot be characterised using nuclear magnetic resonance spectrometry (2.2.33). H. INFLUENZAE TYPE b POLYSACCHARIDE (PRP) H. influenzae type b is grown in a liquid medium that does not contain high-molecular-mass polysaccharides ; LABELLING if any ingredient of the medium contains blood-group The label states : substances, the process shall be validated to demonstrate — the minimum number of International Units per single that after the purification step they are no longer detectable. human dose, The bacterial purity of the culture is verified by methods — the name and the amount of the adsorbent, of suitable sensitivity. These may include inoculation into suitable media, examination of colony morphology, — that the vaccine must be shaken before use, microscopic examination of Gram-stained smears and culture — that the vaccine is not to be frozen. agglutination with suitable specific antisera. The culture may be inactivated. PRP is separated from the culture medium and purified by a suitable method. Volatile matter, 01/2005:1219 including water, in the purified polysaccharide is determined by a suitable method such as thermogravimetry (2.2.34) ; the HAEMOPHILUS TYPE b CONJUGATE result is used to calculate the results of certain tests with reference to the dried substance, as prescribed below.
VACCINE
Only PRP that complies with the following requirements Vaccinum haemophili stirpe b coniugatum may be used in the preparation of the conjugate. Identification. PRP is identified by an immunochemical DEFINITION method (2.7.1) or other suitable method, for example 1H nuclear magnetic resonance spectrometry (2.2.33). Haemophilus type b conjugate vaccine is a liquid or freeze-dried preparation of a polysaccharide, derived Molecular-size distribution. The percentage of PRP eluted from a suitable strain of Haemophilus influenzae type b, before a given K0 value or within a range of K0 values is covalently bound to a carrier protein. The polysaccharide, determined by size-exclusion chromatography (2.2.30) ; an polyribosylribitol phosphate, referred to as PRP, is acceptable value is established for the particular product a linear copolymer composed of repeated units of and each batch of PRP must be shown to comply with this 3-β-D-ribofuranosyl-(1→1)-ribitol-5-phosphate [(C10H19O12P)n], limit. Limits for currently approved products, using the with a defined molecular size. The carrier protein, when indicated stationary phases, are shown for information conjugated to PRP, is capable of inducing a T-cell-dependent in Table 1219.-1. Where applicable, the molecular-size B-cell immune response to the polysaccharide. distribution is also determined after chemical modification of the polysaccharide. PRODUCTION Liquid chromatography (2.2.29) with multiple-angle laser GENERAL PROVISIONS light-scattering detection may also be used for determination The production method shall have been shown to yield of molecular-size distribution. consistently haemophilus type b conjugate vaccines of adequate safety and immunogenicity in man. The production A validated determination of the degree of polymerisation or of the weight-average molecular weight and the dispersion of of PRP and of the carrier are based on seed-lot systems. The production method is validated to demonstrate that the molecular masses may be used instead of the determination of molecular size distribution. product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (2.6.9). Ribose (2.5.31). Not less than 32 per cent, calculated with During development studies and wherever revalidation of the reference to the dried substance. manufacturing process is necessary, it shall be demonstrated Phosphorus (2.5.18) : 6.8 per cent to 9.0 per cent, calculated by tests in animals that the vaccine consistently induces a with reference to the dried substance. T-cell-dependent B-cell immune response. Protein (2.5.16). Not more than 1.0 per cent, calculated The stability of the final lot and relevant intermediates is with reference to the dried substance. Use sufficient PRP to evaluated using one or more indicator tests. Such tests may allow detection of proteins at concentrations of 1 per cent include determination of molecular size, determination of or greater. free PRP in the conjugate and the immunogenicity test in Nucleic acid (2.5.17). Not more than 1.0 per cent, calculated mice. Taking account of the results of the stability testing, with reference to the dried substance. release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the Bacterial endotoxins (2.6.14) : less than 25 IU per microgram period of validity. of PRP. BACTERIAL SEED LOTS Residual reagents. Where applicable, tests are carried out to determine residues of reagents used during inactivation The seed lots of H. influenzae type b are shown to be free from contamination by methods of suitable sensitivity. These and purification. An acceptable value for each reagent is established for the particular product and each batch of PRP may include inoculation into suitable media, examination must be shown to comply with this limit. Where validation of colony morphology, microscopic examination of Gram-stained smears and culture agglutination with suitable studies have demonstrated removal of a residual reagent, the test on PRP may be omitted. specific antisera. 662
See the information section on general monographs (cover pages)
Haemophilus type b conjugate vaccine
EUROPEAN PHARMACOPOEIA 5.0
Table 1219.-1. – Product characteristics and specifications for PRP and carrier protein in currently approved products Carrier Type
Conjugation
Haemophilus polysaccharide
Purity
Nominal amount per dose
Type of PRP
Nominal amount per dose
Coupling method
Procedure
Diphtheria toxoid
> 1500 Lf per milligram of nitrogen
18 µg
Size-reduced PRP K0 : 0.6-0.7, using cross-linked agarose for chromatography R
25 µg
cyanogen bromide activation of PRP
activated diphtheria toxoid (D-AH+), cyanogen bromide-activated PRP
Tetanus toxoid
> 1500 Lf per milligram of nitrogen
20 µg
PRP ≥ 50 % ≤ K0 : 0.30, using cross-linked agarose for chromatography R
10 µg
carbodi-imide mediated
ADH-activated PRP (PRP-cov.AH) + tetanus toxoid + EDAC
CRM 197 diphtheria protein
> 90 % of diphtheria protein
25 µg
Size-reduced PRP Dp = 15-35 or 10-35
10 µg
reductive amination (1-step method) or N-hydroxysuccinimide activation
direct coupling of PRP to CRM 197 (cyanoborohydride activated)
Meningococcal group B outer membrane protein (OMP)
outer membrane protein vesicles : ≤ 8 % of lipopolysaccharide
125 µg or 250 µg
Size-reduced PRP K0 < 0.6, using cross-linked agarose for chromatography R or Mw > 50 × 103
7.5 µg or 15 µg
thioether bond
PRP activation by CDI PRPIM + BuA2 + BrAc = PRPBuA2-BrAc + thioactivated OMP
ADH = adipic acid dihydrazide BrAc = bromoacetyl chloride BuA2 = butane-1,4-diamide CDI = carbonyldiimidazole
Dp = degree of polymerisation EDAC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide IM = imidazolium Mw = weight-average molecular weight
CARRIER PROTEIN The carrier protein is chosen so that when the PRP is conjugated it is able to induce a T-cell-dependent B-cell immune response. Currently approved carrier proteins and coupling methods are listed for information in Table 1219.-1. The carrier proteins are produced by culture of suitable micro-organisms ; the bacterial purity of the culture is verified ; the culture may be inactivated ; the carrier protein is purified by a suitable method. Only a carrier protein that complies with the following requirements may be used in the preparation of the conjugate. Identification. The carrier protein is identified by a suitable immunochemical method (2.7.1). Sterility (2.6.1). Carry out the test using for each medium 10 ml or the equivalent of one-hundred doses, whichever is less. Diphtheria toxoid. Diphtheria toxoid is produced as described in Diphtheria vaccine (adsorbed) (0443) and complies with the requirements prescribed therein for bulk purified toxoid. Tetanus toxoid. Tetanus toxoid is produced as described in Tetanus vaccine (adsorbed) (0452) and complies with the requirements prescribed therein for bulk purified toxoid, except that the antigenic purity is not less than 1500 Lf per milligram of protein nitrogen. Diphtheria protein CRM 197. It contains not less than 90 per cent of diphtheria CRM 197 protein, determined by a suitable method. Suitable tests are carried out, for validation or routinely, to demonstrate that the product is nontoxic. OMP (meningococcal group B Outer Membrane Protein complex). OMP complies with the following requirements for lipopolysaccharide and pyrogens. Lipopolysaccharide. Not more than 8 per cent of lipopolysaccharide, determined by a suitable method. Pyrogens (2.6.8). Inject into each rabbit 0.25 µg of OMP per kilogram of body mass.
BULK CONJUGATE PRP is chemically modified to enable conjugation ; it is usually partly depolymerised either before or during this procedure. Reactive functional groups or spacers may be introduced into the carrier protein or PRP prior to conjugation. As a measure of consistency, the extent of derivatisation is monitored. The conjugate is obtained by the covalent binding of PRP and carrier protein. Where applicable, unreacted but potentially reactogenic functional groups are made unreactive by means of capping agents ; the conjugate is purified to remove reagents. Only a bulk conjugate that complies with the following requirements may be used in the preparation of the final bulk vaccine. For each test and for each particular product, limits of acceptance are established and each batch of conjugate must be shown to comply with these limits. Limits applied to currently approved products for some of these tests are listed for information in Table 1219.-2. For a freeze-dried vaccine, some of the tests may be carried out on the final lot rather than on the bulk conjugate where the freeze-drying process may affect the component being tested. PRP. The PRP content is determined by assay of phosphorus (2.5.18) or by assay of ribose (2.5.31) or by an immunochemical method (2.7.1). Protein. The protein content is determined by a suitable chemical method (for example, 2.5.16). PRP to protein ratio. Determine the ratio by calculation. Molecular-size distribution. Molecular-size distribution is determined by size-exclusion chromatography (2.2.30). Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography, ultrafiltration or other validated methods. Free carrier protein. Determine the content by a suitable method, either directly or by deriving the content by calculation from the results of other tests. The amount is within the limits approved for the particular product.
General Notices (1) apply to all monographs and other texts
663
Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine
EUROPEAN PHARMACOPOEIA 5.0
Table 1219.-2. – Bulk conjugate requirements for currently approved products Test
Protein carrier Diphtheria toxoid
Tetanus toxoid
CRM 197
OMP
Free PRP
< 37 %
< 20 %
< 25 %
< 15 %
Free protein
