Nile Red fluorescence spectrum decomposition enables rapid screening of large protein aggregates in complex biopharmaceutical formulations like influenza vaccines

Vaccine. 2017 May 25;35(23):3026-3032. doi: 10.1016/j.vaccine.2017.04.066. Epub 2017 May 2.

Abstract

The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components.

Keywords: Biopharmaceutical formulation stability; Influenza split-virus vaccine; Nile Red fluorescence; Protein aggregate screening; Triton X-100.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid
  • Immunodiffusion
  • Influenza Vaccines / analysis
  • Influenza Vaccines / chemistry*
  • Octoxynol
  • Oxazines*
  • Protein Aggregates*
  • Spectrometry, Fluorescence / methods*
  • Vaccine Potency
  • Vaccines / analysis
  • Vaccines / chemistry

Substances

  • Influenza Vaccines
  • Oxazines
  • Protein Aggregates
  • Vaccines
  • Octoxynol
  • nile red