title: Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations.
abstract: This study investigates whether Raman spectroscopy combined with multivariate analysis (MVA) enables a rapid and direct differentiation between two classes of conformational states, i.e., native-like and non-native proteins, in freeze-dried formulations. A data set comprising of 99 spectra, both from native-like and various types of non-native freeze-dried protein formulations, was obtained by freeze-drying lactate dehydrogenase (LDH) as model protein under various conditions. Changes in the secondary structure in the solid freeze-dried proteins were determined through visual interpretation of the blank corrected second derivative amide I band in the ATR-FTIR spectra (further called FTIR spectra) and served as an independent reference to assign class labels. Exploratory analysis and supervised classification, using Principal Components Analysis (PCA) and Partial Least Squares - Linear Discriminant Analysis (PLS-LDA), respectively, revealed that Raman spectroscopy is with 95% accuracy able to correctly discriminate between native-like and non-native states in the tested freeze-dried LDH formulations. Backbone (i.e., amide III) and side chain sensitive spectral regions proved important for making the discrimination between both classes. As discrimination was not influenced by the spectral signals from the tested excipients, there was no need for blank corrections. The Raman model may allow direct and automated analysis of the investigated quality attribute, opening possibilities for a real time and in-line quality indication as a future step. However, the sensitivity of the method should be further investigated and where possible improved.
authors.affiliation: Department of Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit Brussel - VUB, Brussels, Belgium.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Pieters
authors.forename: Yvan
authors.initials: Y
authors.lastname: Vander Heyden
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Roger
authors.forename: Matthias
authors.initials: M
authors.lastname: D'Hondt
authors.forename: Laurent
authors.initials: L
authors.lastname: Hansen
authors.forename: Bernard
authors.initials: B
authors.lastname: Palagos
authors.forename: Bart
authors.initials: B
authors.lastname: De Spiegeleer
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Remon
authors.forename: Chris
authors.initials: C
authors.lastname: Vervaet
authors.forename: Thomas
authors.initials: T
authors.lastname: De Beer
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods; D005079:Excipients / Q000737:chemistry; D005612:Freeze Drying / Q000379:methods; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D016018:Least-Squares Analysis; D015999:Multivariate Analysis; D025341:Principal Component Analysis / Q000379:methods; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D005079:Excipients; D011506:Proteins; D007770:L-Lactate Dehydrogenase
keywords: Conformational state; Freeze-drying; Lactate dehydrogenase; Multivariate analysis; PAT; Raman spectroscopy