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

[0008]The inventors have discovered that solid phase extraction (SPE) provides faster and more reproducible separation of conjugated saccharides from unconjugated saccharides, thereby allowing quantitative separation of these saccharides. Furthermore, it has been discovered that, compared with known techniques, solid phase extraction does not depend on the nature of the saccharide, which is particularly advantageous for combination vaccines. In addition, any adjuvant present does not interfere with unconjugated saccharide analysis by SPE. Consequently, 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).

[0018]Thus unconjugated saccharide can be separated from conjugated saccharide and can be separately analysed, thereby allowing a determination of the amount of unconjugated material in a composition. However, comparing the unconjugated or conjugated amount to the total amount is easier than separately analysing the unconjugated and conjugated amount after separation, since one of the unconjugated and the conjugated saccharide will be retained on the solid phase extraction device.

[0024]However, the measurement of the unconjugated saccharide content of the individual types of saccharide in a combination vaccine is nevertheless possible. First, certain methods of quantitative glycoconjugate analysis discussed in more detail below allow the measurement of individual types of saccharide within combined glycoconjugate vaccines comprising more than one type of glycoconjugate immunogen, even where different saccharides share monosaccharide units. Consequently, the methods of the invention allow the analysis of the unconjugated saccharide content of a single or combined vaccine and, in respect of a combined vaccine comprising more than one type of glycoconjugate immunogen, for each individual type of saccharide or for all types of saccharide. The invention is particularly useful for analysing the unconjugated content of N. meningitidis serogroup C saccharide in a combination vaccine comprising more than one type of glycoconjugate immunogen, e.g. combination vaccines comprising:

[0131]To enhance the retention of the appropriate saccharide, the pH, salt concentration and / or organic solvent content of the sample solution may be adjusted. The sample may also be pre-filtered to avoid clogging of the SPE device, e.g. to remove dust.

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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