Conformationally selective biophysical assay for influenza vaccine potency determination.
Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines.
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Yingxia
Y
Wen
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Liqun
L
Han
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Giuseppe
G
Palladino
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Annette
A
Ferrari
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Yuhong
Y
Xie
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Andrea
A
Carfi
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA.
Philip R
PR
Dormitzer
NVS Influenza Vaccines, Now owned and operated by the CSL Group, 45 Sidney Street, Cambridge, MA 02139, USA. Electronic address: ethan.settembre@novartis.com.
Ethan C
EC
Settembre
D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion / Q000379:methods*; D009980:Influenza A virus / Q000145:classification / Q000276:immunology*; D007252:Influenza Vaccines / Q000276:immunology*; D013696:Temperature; D064166:Vaccine Potency
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines
Hemagglutinin; Influenza; Potency; Reversed-phase high pressure liquid chromatography; Trypsinization; Vaccine