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Quantification of polysaccharide in Haemophilus influenzae type b conjugate and polysaccharide vaccines by
1Office of Vaccine Research and Review, Food and Drug Administration, Bethesda, MD 20892.
Insights
A new method accurately quantifies polysaccharide (PS) in Haemophilus influenzae type b (Hib) vaccines. This sensitive technique is ideal for stability studies and vaccines with sugar stabilizers.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Vaccine Development
Background:
- Accurate quantification of polysaccharide (PS) is crucial for vaccine quality control.
- Current methods like the orcinol assay have limitations, especially with sugar stabilizers.
- Haemophilus influenzae type b (Hib) vaccines require precise PS measurement for efficacy.
Purpose of the Study:
- To develop a sensitive and reliable method for quantifying PS in Hib conjugate and PS vaccines.
- To overcome limitations of existing assays, particularly in the presence of interfering substances like lactose.
- To establish a method suitable for vaccine stability studies.
Main Methods:
- Depolymerization of Hib PS in sodium hydroxide to yield a ribitol-ribose-phosphate subunit.
- Analysis of the subunit using high-performance anion-exchange chromatography (HPAEC) with a CarboPak PA-1 column.
- Quantification via pulsed amperometric detection (PAD) and characterization by 31P n.m.r.
Main Results:
- The method successfully quantified PS in various Hib conjugate and PS vaccines from different manufacturers.
- Determined PS values were within expected ranges, demonstrating accuracy.
- The assay demonstrated a sensitivity of 0.1 microgram PS, 30-fold higher than the orcinol assay.
- The method is unaffected by sugar stabilizers like lactose, which interfere with colorimetric assays.
Conclusions:
- A novel, sensitive, and robust method for PS quantification in Hib vaccines has been established.
- This HPAEC-PAD method offers superior sensitivity and specificity compared to the orcinol assay.
- The developed assay is suitable for routine quality control and stability testing of Hib vaccines.
Abstract:
A sensitive method for the quantification of polysaccharide (PS) in Haemophilus influenzae type b (Hib) conjugate and PS vaccines has been developed. It is based on measurement of the Hib PS subunit after depolymerization of the PS in sodium hydroxide to produce the subunit, which is characterized by chemical composition and 31P n.m.r. analyses as ribitol-ribose-phosphate. The Hib vaccines were first treated with 0.1 M sodium hydroxide. The Hib PS subunit in the treated vaccines was then analysed directly by high-performance anion-exchange chromatography using a CarboPak PA-1 column, and quantified by pulsed amperometric detection. The PS contents of three conjugate vaccines and three PS vaccines from different manufacturers were determined. Their values were in the expected ranges. This method is particularly useful for vaccines with a sugar stabilizer such as lactose which would interfere with the colorimetric orcinol assay currently used for determination of the PS. The method can measure 0.1 microgram of PS and its sensitivity is at least 30-fold higher than that of the orcinol assay. It may be used for stability studies of conjugate vaccines since a breakdown as low as 5% of the PS from the PS-protein conjugates would be detected.