Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

Abstract

The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state‐of‐the‐art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid‐chromatographic modes that are discussed include aqueous size‐exclusion chromatography, hydrophobic interaction chromatography, and ion‐exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid‐chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody–drug conjugates is discussed.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass‐spectrometry‐based approach

“…6,10,19 Arguably less wellrecognized is the rapidly developing capability of MS instruments to analyze large, intact biomolecules under native-like conditions. [20][21][22][23][24][25] This, combined with coupled techniques like ion mobility spectrometry (IMS) 26,27 and hydrogen/deuterium exchange (HDX) [28][29][30] allows MS to support higher order structure analysis, in some cases at single residue resolution. 31,32 IMS is a relatively recent addition to the MS-based analysis toolbox, with a number of commercial instruments now including IMS technologies.…”

“…Additional innovations in coupled separation techniques, particularly liquid chromatography (LC–MS) have made MS an exceedingly powerful tool for peptide‐level analyses of mAbs, which allows for sequencing and post‐translational modification (PTM) analysis . Arguably less well‐recognized is the rapidly developing capability of MS instruments to analyze large, intact biomolecules under native‐like conditions . This, combined with coupled techniques like ion mobility spectrometry (IMS) and hydrogen/deuterium exchange (HDX) allows MS to support higher order structure analysis, in some cases at single residue resolution …”

Proteoforms: General Concepts and Methodological Process for Identification

“…The proteins migrate through a porous polymeric column and are separated by their hydrodynamic volume, with more abundant proteins eluting before smaller ones due to their lower accessibility to the interior of the packing materials. The selectivity is provided by the column, defined by the size of the intraparticle pore diameter; thus, the efficiency in the Proteoforms: General Concepts and Methodological Process for Identification DOI: http://dx.doi.org /10.5772/intechopen.89914 SEC separation is mainly governed by the particle diameter [60,61]. SEC has the advantage that can be realized in several types of solutions; however, it is not a highresolution separation method, in addition to promoting the dilution of the sample.…”

“…A common material used is either surface-modified silica or polymeric particles coated with short aliphatic groups n-alkyls (propyl, butyl, hexyl, or octyl chains), phenyl and others [61,85]. HIC separation methods have been evaluated and optimized as complementary selectivity to RPLC, which offer efficient separation for highly orthogonal HIC-RPLC for top-down proteomics [14,27].…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…In a review in 2016, nothing was written on HILIC separation of intact proteins, although it introduced RP, SEC, or IEX separation of them . The situation was not changed in a review published in 2018 . Different from RP, SEC, or IEX modes, HILIC requires the use of mobile phases with high content of organic modifiers, particularly AN, and the conditions will result in denature of proteins.…”