Appendix XV K. Carrier Proteins for the Production of Conjugated Polysaccharide Vaccines for Human Use
The use of alternative carrier proteins, production methods and tests are acceptable provided they have been authorised by the competent authority.
Bacterial polysaccharides are not able to induce a T-cell-dependent B-cell immune response, which is needed to obtain an immunological memory response, and are generally poorly immunogenic in children under 2 years of age. The limitations are overcome by conjugating polysaccharides to carrier proteins. Carrier proteins are highly immunogenic and, when conjugated to bacterial polysaccharides, increase the capacity of polysaccharides to induce a protective response in infants.
Carrier proteins currently used in polysaccharide vaccines for human use are toxoids, non-toxic mutated toxins, surface or outer membrane proteins extracted from micro-organisms. Micro-organisms used for the production of the protein may be of genetically modified origin.
The production method used for a carrier protein shall have been shown to yield consistently batches suitable for conjugation of the carrier protein to a polysaccharide antigen.
Appropriate acceptance criteria for low bioburden before conjugation with the polysaccharide may be established. It is a prerequisite that the carrier protein is filtered through a bacteria-retentive filter prior to storage and that adequate measures are in place to avoid contamination and growth of micro-organisms during storage.
The production of carrier proteins is based on a seed-lot system. The seed lots are shown to be free from contamination using suitable methods of appropriate sensitivity. The culture may be inactivated and the carrier protein is purified by a suitable method.
The protein is characterised by one or more suitable method(s) (such as SDS-PAGE, isoelectric focusing, HPLC, size-exclusion chromatography with multiple-angle laser light scattering detection (MALLS), amino-acid analysis, amino-acid sequencing, circular dichroism, fluorescence spectroscopy, peptide mapping and mass spectrometry) and its purity is verified by a suitable method. Suitable tests are carried out, for validation or routinely, to demonstrate that where applicable, the product is free from specific toxins. If purification steps are present, the reduction of selected process-related impurities and residuals is monitored to establish consistency of the purification process. In the case of recombinant carrier proteins, tests for at least the following impurities are also carried out:
Only a carrier protein that complies with the following tests may be used in the preparation of the conjugate.
Identification
The carrier protein is identified using a suitable method.
pH (2.2.3)
Where applicable, the pH of the carrier protein prior to conjugation is monitored and is within the limits approved for the particular product.
Protein content (2.5.16)
The content of the carrier protein is determined using a suitable method and is within the limits approved by the competent authority.
Bacterial endotoxins(2.6.14)
The content is within the limits approved for the particular product.
In addition, the following requirements apply for the carrier proteins listed below.
Diphtheria toxoid
It is produced as described in the monograph Diphtheria vaccine (adsorbed) (0443) and complies with the requirements prescribed therein for bulk purified toxoid, except that the test for sterility (2.6.1) is not required.
Tetanus toxoid
It is produced as described in the monograph 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 and that the test for sterility (2.6.1) is not required.
CRM 197 diphtheria protein
It may be prepared from the growth of genetically modified (C7/β197) or non-genetically modified (mCRM) Corynebacterium diphtheriae or prepared by recombinant DNA technology in organisms such as Escherichia coli. The culture supernatant may be concentrated by ultrafiltration and purified by successive precipitation, filtration, and chromatography steps. When it is produced on the same premises as diphtheria toxin, CRM 197 diphtheria protein should be distinguished from the active toxin by a suitable method. The purity is not less than 90 per cent of diphtheria protein.
OMP (Neisseria meningitidis group B outer membrane protein complex)
Neisseria meningitidis group B outer membrane protein complex is extracted from bacterial cell cultures with a buffer containing a detergent. Cell debris is first removed. The membrane protein complex may be concentrated and purified by successive filtration and additional suitable purification steps. The lipopolysaccharide content does not exceed 8 per cent. The relative quantity of the major OMP is set and approved by the competent authority. The OMP complex complies with the test for pyrogens (2.6.8): inject into each rabbit 0.25 µg of OMP per kilogram of body mass.
Recombinant protein D
Protein D is a surface protein of nontypable Haemophilus influenzae. It is produced using a specific strain of E. coli carrying a plasmid containing the coding sequence for protein D. In order to express protein D, the modified strain is grown in a suitable liquid medium. At the end of cultivation, a purification step is carried out. The purity of the product is not less than 95 per cent of protein D.