Native and Denaturing MS Protein Deconvolution for Biopharma: Monoclonal Antibodies and Antibody–Drug Conjugates to Polydisperse Membrane Proteins and Beyond

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH] n+ or negative [M – nH] n− depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; −203.1 Da).

Proteoform-Resolved FcɤRIIIa Binding Assay for Fab Glycosylated Monoclonal Antibodies Achieved by Affinity Chromatography Mass Spectrometry of Fc Moieties

“…For example, Fc glycoforms G0F/G0F and G0F/G1F will be extracted as the same mass, when combined with Fab glycans of composition H7N4F1/H7N4F1 and H7N4F1/H6N4F1, respectively. The MS analysis of mAbs is generally affected by an increased signal heterogeneity derived from additional non-resolved proteoforms and adducts (Campuzano et al, 2019). In particular for native MS of complex proteins, the applied deconvolution (algorithm, settings) can have an influence on the data evaluation with respect to resolving heterogeneous mass spectra (Campuzano et al, 2019).…”

“…The MS analysis of mAbs is generally affected by an increased signal heterogeneity derived from additional non-resolved proteoforms and adducts (Campuzano et al, 2019). In particular for native MS of complex proteins, the applied deconvolution (algorithm, settings) can have an influence on the data evaluation with respect to resolving heterogeneous mass spectra (Campuzano et al, 2019). Cetuximab glycoforms, containing H7N4F1/H6N4F1S1 and H6N4F1/H6N4F1S1 Fab glycoform pairings (Figure 5 and Supplementary Table 3), were not resolved.…”

Rational selection of building blocks for the assembly of bispecific antibodies

“…The resulting spectra were summed, then deconvoluted using either the Agilent Mass Hunter Qualitative Analysis software (Version B.07.00) or Intact Program module from Protein Metrics, followed by relative quantification of each deconvoluted species using the MS signal intensity. 36 An Excel-based tool was used to calculate the intact mass and intensity of various correct and mispaired species. In the case of a 4-chain hetero-IgG, the following species are calculated: IgG species with four unique chains, IgG species with HC heterodimer but 2× of one LC, IgG species with HC homodimer, and half-mAb species.…”

Deconvolving Native and Intact Protein Mass Spectra with UniDec

“…To overcome these challenges, multiple deconvolution software programs have been created to help assign charge states and deconvolve intact MS data. [15][16][17][18][19][20][21][22][23][24][25] A recent review has covered a wide variety of deconvolution software. [26] UniDec (Universal Deconvolution) software uses Bayesian deconvolution to quickly assign the charge states of m/z peaks and thus determine the mass.…”