Status: discontinued several years ago; being relaunched by new owner

Organizations involved:

Nuron Biotech Inc. - R&D; Tech.; World mark.

Pfizer – Manuf.; Parent

Mitsubishi Tanabe Pharma Corp. – Japan mark.

Praxis Biologics, Inc. – Former

Wyeth – Former

University of Rochester – Tech.

Cross ref: See the Haemophilus b Vaccine Products entry (#453). See also the entry (#451) for a DTP+Hib combination vaccine incorporating HibTITER as a component – Diphtheria & Tetanus Toxoids & Pertussis Vaccine Adsorbed & Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate) or Tetramune.

Description: Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate) or HibTITER vaccine is an aqueous formulation of purified PRP oligosaccharides of the capsular polysaccharide antigen of Haemophilus influenzae type b (Hib) Eagan strain chemically conjugated to purified CRM197 protein, a detoxified variant form of diphtheria toxin isolated from cultures of Corynebacterium diphtheriae C7 (beta197). Conjugation of the Haemophilus influenzae type b polysaccharide to the diphtheria toxin carrier substantially increases the immunogenicity of the polysaccharide.

The Hib oligosaccharides are derived from highly purified capsular polysaccharide, polyribosylribitol phosphate (PRP), isolated from H. influenzae type b cultured in a chemically defined medium (a mixture of mineral salts, amino acids, and cofactors). The PRP oligosaccharides are covalently linked directly to highly purified the CRM197 diphtheria toxin by reductive amidation (i.e, with no linker-spacer groups, as are some other Hib conjugate vaccines). CRM197 is derived from Corynebacterium diphtheriae C7 (beta197) cultured in a cas-amino acids and yeast extract-based medium. CRM197 is purified by ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography to obtain highly purified protein. After linkage, the Hib PRP-CRM197 conjugate is purified to remove unreacted protein, oligosaccharides, and reagents. The bulk is sterilized by filtration and filled into vials. Potency of HibTITER is determined by chemical assay for PRP.

HibTITER is packaged in single-dose vials, containing 10 µg of H. influenzae type b oligosaccharide and 25 µg of CRM197 protein per 0.5 mL dose in 0.9% sodium chloride aqueous solution. The vaccine is available in packages of four 0.5 mL single dose vials, packages of one 2.5 mL five dose vials, and packages of one 5 mL ten-dose vials. The single-dose vial contains no preservative.

HibTITER was previously also packaged in 10-dose vials containing 0.1% thimerosal (mercury derivative) as an antimicrobial preservative, with each 0.5 mL dose deliverying 25 µg of mercury. This dosage form was phased out in 2000.

Nomenclature: Haemophilus b Vaccine/Lederle [BIO]; HibTITER [TR]; Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate) [FDA]; 126161-67-9 [CAS RN]; Hib vaccine [SY]; HIB [CDC]; NDC 0005-0104-41; 0005-0201-10 [NDC current]; NDC 53124-0201-05; 53124-0201-10; and 53124-0104-41 [NDC former]

Biological.: “Cross reacting materials” or CRMs are modified, non-toxic toxins antigenically similar to the parent toxic bacterial toxin. CRM197 has a single amino acid change (mutation) from the native diphtheria toxin and is immunologically indistinguishable from it. A culture of Corynebacterium diphtheriae strain C7 (beta-197), which produces CRM197 protein, has been deposited with the American Type Culture Collection as ATCC 53281. Although CRM197 is immunologically identical to native toxin, treatment of CRM197 with formalin (formaldehyde), as is used to detoxify other toxins (form toxoids), greatly enhances immunological responses, although there is no need to detoxify it. This may be due to stabilization against degradation in vivo and/or aggregation by cross-linking (with immunogenicity increasing with particle size).

Companies: Originally developed, manufactured and marketed by Wyeth, which merged into Pfizer. In April 2011, Nuron acquired the product from Wyeth/Pfizer for the U.S. and many other markets, including Japan and Korea. In Jan. 2012, Nuron licensed Japanese single entity and for combination product use rights to Mitsubishi Tanabe Pharma Corp.

Wyeth/Pfizer discontinued marketing of HibTiter in 2010 following a reassessment of its portfolio and priorities and other available vaccine options.

Manufacture: According to FDA documentation, Haemophilus influenzae type B (Hib) is cultured in chemically--defined medium at facilities in Sanford, NC (originally belonging to Praxis Biologics). Hib oligosaccharides are purified and sized by diafiltration through a series of ultrafiltration membranes, and then covalently coupled by reductive amidation to Diphtheria CRM197 protein. The Diphtheria CRM197 protein is a nontoxic variant of diphtheria toxin isolated from cultures of Corynebacterium diphtheria C7 (beta-197) grown in casamino acids and yeast extract-based medium. The conjugate is purified by diafiltration to remove unreacted protein, oligosaccharides and reagents, and sterilized by filtration. [Apparently, the CRM197 is also treated with formaldehyde (formalin)]. The polysaccharide and protein are directly linked, the conjugate purified, and sterilized by filtration.

Hib conjugate potency is determined by measuring total ribitol using a gas chromatographic method, which is also used to assay total polysaccharide. The final product contains 16.0-24.0 µg/mol of saccharide (HbOC ribitol); with a saccharide:protein (HbOC sac-charide:protein) ratio of 0.50 +/- 0.20 (based on Hemophilus B component). The aluminum content of the final product is ≤0.85 mg per 0.5 mL dose. Residual formaldehyde content is ≤ 0.02%.

The following descriptions are derived from U.S. patents (see Tech. transfer section) – H. influenzae b is grown in a culture, centrifuged, and the cell free supernatant collected. The supernatant is precipitated with ethanol, the precipitate is treated with hexadecyltrimethyl ammonium bromide (a surfactant), and then again with ethanol to recover partially purified PRP. The PRP is then treated with hydroxyapatite (calcium phosphate hydroxide), which adsorbs contaminants, with PRP remaining in the supernatant. If further purification is necessary, the PRP may be applied to a Sepharose 4B-CL column (Phar-ma-cia). The PRP obtained is a relatively high molecular size of PRP, with more than 50% having an elution coefficient (Kav) value less than 0.30 on a column of Sepharose 4B-CL. This value corresponds to a molecular weight greater than 1,000,000 Daltons (1,000 kDa).

In its native form, PRP has no chemically reducing groups (for conjugation with a protein). In order to create PRP fragments with terminal reducing groups (in the form of terminal reactive aldehyde groups), the cis-vicinal hydroxyl group of the polysaccharides is oxidized, preferably with sodium peri-odate, to generate aldehyde functions following the procedure described by Parikh et al., Methods Enzymol., 34, 77-102 (1974) and U.S. patent 4,356,170. The purified PRP is treated in the dark with 0.2-50 mM of sodium periodate at pH 4.0-4.5. The oxidized PRP or [O]PRP is dialyzed extensively against pyrogen-free water to remove small molecular size materials, or the [O]PRP may be purified through a gel filtration column, e.g., a beaded agarose acrylamide copolymer (Sephacryl S-200 or S-300, Pharmacia). The purified [0]PRP is recovered by concentrating and drying. The [O]PRP fragments has chain lengths of about 8-120 monomeric units. This chain length may also be referred to as the degree of polymerization (D.P.).

The [O]PRP is coupled with bacterial protein (CRM197). The aldehyde functions in the [O]PRP react in solution with amino groups of the toxoid protein conjugate at pH 5-9. Toxoid is reduced with sodium cyanobo-rohydride. Purified reduced toxin protein (concentration 1-40 mg/mL) is mixed with [O]PRP (concentration 10-80 mg/ml) in 0.2M potassium phosphate buffer, pH 6.0-6.7, and incubated at 25-37˚C with gentle stirring for 1-6 days to form stable, covalently-bonded conjugate [see Methods Enzymol., 34, 77-102 (1974); Arch. Biochem. Biophys., 181, 542-549 (1977)]. The conjugate is purified on a gel filtration column such as Sephacryl S-200, S-300 or Sepharose CL-4B (Pharmacia). Peak fractions containing the conjugate are pooled, diafiltered and/or lyophilized. Alternatively, the conjugate may be purified by dialysis.

FDA class: Biologic PLA

CBER class: Bacterial Antigens and Vaccines

Approvals: Date = 19881221; first approval, PLA; granted to Praxis Biologics (est. no. 0937); Indication = use in toddlers 18 months of age or older

Date = 19901004; PLA supplement, ref. no. 90-0217; Indication = approval for use in younger infants

Date = 19941206; license revoked and granted (reissued) to new owner, Lederle Labs., CBER est. no. 0017

Date = 20061117; BLA supplement (103578/5063); Indication = changes in the product insert/labeling

Indications: [full text of "INDICATIONS AND USAGE” section of product insert/labeling; 12/2006]:

Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate) HibTITER is indicated for the immunization of children 2 months to 71 months of age against invasive diseases caused by H. influenzae type b.

As with any vaccine, HibTITER may not protect 100% of individuals receiving the vaccine.

The American Academy of Pediatrics (AAP), the Advisory Committee on Immunization Practices (ACIP) and the American Academy of Family Physicians (AAFP) encourage the routine administration of Haemophilus influenzae typ b vaccines with other currently recommended vaccines, but at different sites (see DRUG INTERACTIONS).

Status: Problems in manufacture and supply of Hib-TITER resulted in a disruption in its availability. HibTITER became available again in June 2004.

European Union approval was not granted due to concerns about use in manufacture and potential presence of bovine materials. The vaccine has been approved and is marketed in various individual European countries.

Tech. transfer: The H. influenzae b PRP antigen apparently used in this product has been described in U.S. 4,220,717, assigned to American Cyanamid Co. (Lederle Labs., now Wyeth), concerning PRP isolation and purification. Manufacturing methods are also described in U.S. patent 4,902,506, assigned to the University of Rochester. Example 5 of this patent describes this Haemophilus b conjugate vaccine. See also U.S. patent 5,192,540, assigned to American Cyanamid, concerning conjugates and conjugation methods. See the manufacture section above for manufacturing information derived from these patents.

U.S. 5,192,540, Kuo, et al. (Praxis Biologics), “Haemophilus influenzae type b oxidized polysaccharide-outer membrane protein conjugate vaccine,” March 9, 1993 (filed in 1988), assigned to American Cyanamid (then parent of Lederle Labs.), now Wyeth, discloses immunogenic conjugates of 38 or 40 kDa oxidized polyribosyl-ribi-tol-phosphate polysaccharide fragments of H. influenzae type b. Methods for isolating and purifying the 38 kDA and 40,000 kDa outer membrane proteins (P2 proteins) using anion exchange and hydroxylapatite column chromatography and methods for preparing the oxidized polyribosyl-ribitol-phosphate polysaccharide fragments are also disclosed.

Market: The 2007 Average Wholesale Price (AWP) is $141.91 for five single-dose vials, with a Direct Cost (DC; manufacturer’s discount price) of $114.28; and $256.84/10-dose vial, with DC of $206.97 (no changes from 2004) (Red Book, 2007). These prices include the $.75/dose federal excise tax (funding federal vaccine liability insurance) charged by the manufacturer.

HibTITER is not currently listed among the vaccines purchased by the National Immunization Program (NIP), Centers for Disease Control and Prevention (CDC), for distribution to vaccination/public health programs. As reported by NIP, CDC, in April 005), the average private-sector cost per dose was $22.86 per package of 5 single dose vials. The cost negotiated by NIP, CDC, for bulk contract purchase for public-sector state and local immunization programs was $7.86/dose per package of 5 single dose vials. These prices include the $.75/dose ($.75/covered component vaccine) Federal Excise Tax charged by the manufacturer for the federal vaccine injury compensation program. Wyeth’s contract with NIP, CDC, expired on March 31, 2006.