TECHNICAL FIELD
The present disclosure relates to analytical methods. Particularly, the present
disclosure relates to a method for separation and quantification of free
polysaccharide as well as total polysaccharide content in polysaccharide protein
5 conjugate(s) bulk or vaccine composition.
We claim:
1. A method for quantification of free polysaccharide or total polysaccharide
or both, in a sample comprising polysaccharide-protein conjugate, said
method comprising the steps of:
5 a) separating free polysaccharide through a chromatographic resin in
presence of at least one reagent to obtain a supernatant containing free
polysaccharide; and
b) subjecting the supernatant to a spectrophotometric assay to quantify
the free polysaccharide or total polysaccharide or both, in the sample
10 comprising the polysaccharide-protein conjugate.
2. The method as claimed in claim 1, wherein the chromatography resin is a
multi-modal chromatography or mixed-mode chromatography resin.
3. The method as claimed in claim 2, wherein the multi-modal
chromatography or the mixed-mode chromatography resin has multiple
15 functionalities selected from a group comprising anion exchange,
hydrophobic or combinations thereof.
4. The method as claimed in any one of claims 1 to 3, wherein the separation
of free polysaccharide in the sample comprising the polysaccharideprotein conjugate comprises:
20 a) loading the multi-modal or mixed-mode chromatography resin in a
micro-centrifuge tube;
b) adding a first reagent to the micro-centrifuge tube of step a) and
spinning the tube, followed by discarding the supernatant;
c) loading the sample comprising the polysaccharide-protein conjugate
25 on to the multi-modal or mixed-mode chromatography resin in the
micro-centrifuge tube, followed by spinning and discarding the
supernatant;
d) adding a second reagent to the micro-centrifuge tube of step c) and
spinning the tube; and
30 e) collecting the supernatant from step d), cont