Development of a Mass Spectrometry Methodology for the Absolute Quantification of Proteins

Abstract

There are several techniques used in the biopharmaceutical industry to quantify proteins, whether vaccine antigens or HCPs, in a sample. The most common method used to determine the absolute amount of antigen or HCPs in a sample is immunoassay, usually in the form of an enzyme-linked immunosorbent assay (ELISA). ELISA is the gold standard for the quantification of HCPs. However, the main disadvantage of ELISA is that it requires a specific assay to quantify a single protein. More recently, an orthogonal method has emerged: mass spectrometry. Mass spectrometry overcomes the limitations of ELISA by providing absolute quantification of all individual HCPs in a single assay. First reported in 2003, absolute quantification methodologies based on mass spectrometry have been increasingly performed in the biopharmaceutical industry (Doerr A, 2009). This work focused on the development of an MS-based methodology for the absolute quantification of both vaccine antigens and contaminating host cell proteins by using synthetic isotope-labeled peptides as internal controls. The methodology was developed in the context of a project on a recombinant protein-based vaccine against respiratory syncytial virus. Absolute quantification of the antigen (RSVPreF3, which will be referred to as PreF3 in this thesis) and HCPs was achieved using multiple reaction monitoring (MRM) and, as internal standards, AQUA peptides synthesized by incorporating heavy isotope labels (13C and 15N) on lysine or arginine. Finally, the results obtained by absolute and relative quantification are compared. In general, the MS-based method for absolute protein quantification provides accurate results. Most of the heavy peptides selected either for the antigen and the HCPs could be used for quantification as they gave a good linear correlation with the amount spiked in the samples with R-squared factor near 1. Moreover, the reproducibility of the signal responses for both the endogenous and heavy peptides was good, with CV ranging from 3 to 12. The quantification of PreF3 resulted to be lower than expected. SDS-PAGE performed on the digested samples proved that the antigen was not 100% digested which could explain the lower quantification obtained. Other conditions of digestion were tested to then be applied for future improvements of the methodology. A comparison of the results obtained for HCPs using relative and absolute methodologies resulted in a very similar quantification in the dirty sample (harvest clarified) and less similar quantification for the purer sample (drug substance) but with values still in the same order of magnitude. In the future, sample preparation will be modified to include optimal digestion conditions and spiking of labeled peptides prior to the clean-up of the peptides in which a loss of material is expected. In summary, mass spectrometry methods for absolute protein quantification have considerable advantages, such as the ability to quantify individual proteins in a single assay with no need for a control sample whilst maintaining high sensitivity.