Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 cells: a novel RP-HPLC method.
The major antigenic determinant of influenza A and B virus is haemagglutinin (HA). The HA content is an important specification of influenza vaccines. HA in vaccines has typically been quantified by single-radial-immunodiffusion (SRID). However, SRID is a laborious and low throughput assay. Moreover, sensitivity, accuracy, and precision, especially for non-purified (in-process) influenza virus is relatively low. We present a novel method for quantification of HA in influenza viral cultures as well as for the identification of HA from individual influenza strains in trivalent vaccines. The method is based on the separation of HA(1), the hydrophilic subunit of HA, from the more hydrophobic viral and matrix components by reversed-phase high performance liquid chromatography (RP-HPLC). The HA(1) peak area is demonstrated to be proportional to the level of HA in non-purified, semi-purified and purified vaccine products of various epidemic and pandemic influenza A and B strains propagated in PER.C6((R)) cell cultures. The RP-HPLC assay selectivity allows for the simultaneous identification and quantification of HA(1) from influenza A and B strains in the yearly revised trivalent vaccines for epidemic outbreaks.
Crucell Holland BV, P.O. Box 2048, 2301 CA Leiden, The Netherlands. h.kaptein@crucell.com
Johan C
JC
Kapteyn
Mohammed Drissi
MD
Saidi
René
R
Dijkstra
Cennet
C
Kars
Joan C M S-K
JC
Tjon
G J
GJ
Weverling
Marcel L
ML
de Vocht
Ronald
R
Kompier
Bart A
BA
van Montfort
Jean-Yves
JY
Guichoux
Jaap
J
Goudsmit
Fija M
FM
Lagerwerf
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis* / Q000145:classification*; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry*; D009981:Influenza B virus / Q000737:chemistry*; D007252:Influenza Vaccines / Q000737:chemistry; D021122:Protein Subunits / Q000302:isolation & purification
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D021122:Protein Subunits