Assay for the separation and quantification of hemagglutinin antigens
AssigneeCrucell Holland B.V.
InventorsJohan Christiaan Kapteyn, Fija Maria Lagerwerf
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Abstract
The present invention relates to novel methods for separating hemagglutinin (HA) antigens, comprising the steps of applying a reduced and derivatized antigen preparation comprising solubilized HA antigens and a detergent in a pH controlled solution, on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column; and eluting the HA antigens from the column with an ion pairing agent in an organic mobile phase. The invention further relates to quantifying methods using the methods for separating the antigens with the further step of measuring the peak area of the eluted antigen in a chromatogram resulting from the elution step.
Claims19
A method for separating hemagglutinin (HA) HA1 and HA2 antigens from an influenza A virus or an influenza B virus, said method comprising:
- applying a reduced and derivatized antigen preparation comprising solubilized HA1 and HA2 antigens and a detergent in a pH controlled solution to a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column, wherein a mature form of HA in an antigen preparation has been cleaved by a protease into HA1 and HA2 prior to applying the antigen preparation to the RP-HPLC column;
- and
- eluting the HA1 and HA2 antigens from the column with an ion pairing agent in an organic mobile phase, wherein the HA antigens are derivatized with an alkylating agent,
- wherein the elution results in separation of HA1from HA2, and
- wherein the elution is performed at a temperature of between about 50° C. and about 70° C.
A method for quantifying a hemagglutinin (“HA”) titer of an influenza HA antigen preparation, said method comprising
- applying a reduced and derivatized antigen preparation comprising solubilized influenza HA that is cleaved into subunits HA1 and HA2 and a detergent in a pH controlled solution to a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column in an RP-HPLC system;
- eluting HA antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein the elution is performed at a temperature of between about 60° C. and about 70° C., and further wherein the HA antigens are derivatized with an alkylating agent, and
- measuring a peak area of the HA 1 subunit of influenza HA in a chromatogram resulting from the elution to determine an HA titer of influenza HA antigen preparation based on calibration of the RP-HPLC system with a reference concentration of influenza HA.
A method for quantifying a hemagglutinin (HA) titer of an influenza HA antigen preparation, the method comprising
- applying a reduced and derivatized antigen preparation comprising solubilized influenza HA cleaved into subunit HA1 and subunit HA2 and a detergent in a pH-controlled solution to a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column in an RP-HPLC system;
- eluting HA antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein the elution is performed at a temperature of between about 50° C. and about 70° C., and further wherein the HA antigens are derivatized with an alkylating agent, and
- measuring a peak area of the HA1subunit of influenza HA in a chromatogram resulting from the elution to determine an HA titer of influenza HA antigen preparation based on calibration of the RP-HPLC system with a reference concentration of influenza HA.
Dependencies are inferred from the claim text.