Separation of Unconjugated and Conjugated Saccharide by Solid Phase Extraction

Abstract

The invention is based on the use of solid phase extraction for separating conjugated saccharide from unconjugated saccharide in sample, e.g. a vaccine. Solid phase extraction (SPE) provides faster and more reproducible separation of conjugated saccharides from unconjugated saccharides, thereby allowing quantitative separation of these saccharides. The separation of conjugated and unconjugated saccharide using SPE may be advantageously combined with a quantitative conjugate analysis to provide improved quality control for conjugate vaccines. The SPE separation is compatible with existing quantitative conjugate analysis techniques, such as high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Description

[0001]All documents cited herein are incorporated by reference in their entirety.TECHNICAL FIELD[0002]This invention concerns the analysis and quality control of vaccines that include saccharides (e.g. bacterial capsular saccharides), and especially those where the saccharides are conjugated to a carrier.BACKGROUND OF THE INVENTION[0003]Immunogens comprising capsular saccharide antigens conjugated to carrier proteins are well known in the art. Conjugation converts T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop, and the prototype conjugate vaccine was for Haemophilus influenzae type b (Hib) [e.g. see chapter 14 of ref. 1]. Since the Hib vaccine, conjugated saccharide vaccines for protecting against Neisseria meningitidis (meningococcus) and against Streptococcus pneumoniae (pneumococcus) have been developed. Other organisms where conjugate vaccines are of interest are Streptococcus agalactiae (group B s...

AI Technical Summary

Benefits of technology

The patent text describes a method of separating and analyzing saccharides in a sample. This method uses solid phase extraction to separate conjugated saccharides from unconjugated saccharides, allowing for easier analysis of the unconjugated saccharides. The method is faster and more reproducible than other techniques and does not interfere with the analysis of unconjugated saccharides. The invention also provides a solid phase extraction device for use in this method. The technical effects of this invention include improved quality control for conjugate vaccines and more efficient analysis of saccharide content in samples.

Problems solved by technology

Problems when dealing with conjugate vaccines include stability and batch-to-batch consistency.

Instability of conjugates undesirably leads to a reduction in effective dose of immunogenic conjugate over time, variation between batches, and increased levels of uncharacterised breakdown products.

The need for separation raises a number of problems since conjugate vaccines, especially combination vaccines, typically contain a number of components which may interfere with each other during the separation.

Known methods of separation include ultrafiltration, hydrophobic chromatography and selective precipitation, but these techniques are slow and suffer from poor reproducibility.

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