Assay for the separation and quantification of hemagglutinin antigens
AssigneeCrucell Holland B.V.
InventorsJohan C. Kapteyn, Fija M. Lagerwerf
Life of this patent
Estimating the term…
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 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.
Claims11
A method for quantifying the hemagglutinin (HA) titer of an influenza A or influenza B HA antigen preparation, the method comprising:
- separating hemagglutinin (HA) antigens from an influenza A or influenza B virus by a method comprising:
- obtaining a preparation comprising HA antigens from influenza A or influenza B virus;
- solubilizing the antigens by a detergent in a pH controlled solution;
- cleaving the HA antigens into HA1 and HA2 with a protease;
- reducing the HA1 and HA2 antigens with a reducing agent;
- derivatizing the HA1 and HA2 antigens with an alkylating agent;
- applying the antigen preparation on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column;
- eluting HA1 and HA2 antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein elution is performed at a temperature of between approximately 50° C. and approximately 70° C., thereby separating HA1 from HA2;
- and
- measuring the peak area of eluted HA1 antigen in a chromatogram resulting from the elution to quantify the HA titer of the antigen preparation.
A method for quantifying the hemagglutinin (HA) titer of an influenza A or influenza B HA antigen preparation, the method comprising:
- separating hemagglutinin (HA) antigens from an influenza A or influenza B virus by a method comprising:
- obtaining a preparation comprising HA antigens from influenza A or influenza B virus;
- solubilizing the antigens by a detergent in a pH controlled solution;
- cleaving the HA antigens into HA1 and HA2 with a protease;
- reducing the HA1 and HA2 antigens with a reducing agent;
- derivatizing the HA1 and HA2 antigens with an alkylating agent;
- adding reducing agent after alkylation;
- applying the antigen preparation on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column;
- eluting HA1 and HA2 antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein elution is performed at a temperature of between approximately 50° C. and approximately 70° C., thereby separating HA1 from HA2;
- and
- measuring the peak area of eluted HA1 antigen in a chromatogram resulting from the elution to quantify the HA titer of the antigen preparation.
A method for quantifying the hemagglutinin (HA) titer of an influenza A or influenza B HA antigen preparation, the method comprising:
- separating hemagglutinin (HA) antigens from an influenza A or influenza B virus by a method comprising:
- obtaining a preparation comprising HA antigens from influenza A or influenza B virus
- solubilizing the antigens by a detergent in a pH controlled solution;
- cleaving the HA antigens into HA1 and HA2 with a protease;
- reducing the HA1 and HA2 antigens with a reducing agent;
- derivatizing the HA1 and HA2 antigens with an alkylating agent;
- adding reducing agent after alkylation;
- applying the antigen preparation on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column;
- eluting HA1 and HA2 antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein elution is performed at a temperature of between approximately 60° C. and approximately 70° C., thereby separating HA1 from HA2;
- and
- measuring the peak area of eluted HA1 antigen in a chromatogram resulting from the elution to quantify the HA titer of the antigen preparation.
A method for quantifying the hemagglutinin (HA) titer of an influenza A or influenza B HA antigen preparation, the method comprising:
- separating hemagglutinin (HA) antigens from an influenza A or influenza B virus by a method comprising:
- obtaining a preparation comprising HA antigens from influenza A or influenza B virus;
- solubilizing the antigens by a detergent in a pH controlled solution;
- cleaving the HA antigens into HA1 and HA2 with a protease;
- reducing the HA1 and HA2 antigens with a reducing agent;
- derivatizing the HA1 and HA2 antigens with an alkylating agent;
- adding reducing agent after alkylation;
- applying the antigen preparation on a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) column;
- eluting HA1 and HA2 antigens from the RP-HPLC column with an ion pairing agent in an organic mobile phase, wherein elution is performed at a temperature of approximately 60° C., thereby separating HA1 from HA2;
- and
- measuring the peak area of eluted HA1 antigen in a chromatogram resulting from the elution to quantify the HA titer of the antigen preparation.
Dependencies are inferred from the claim text.