Characterization of High Molecular Weight Multi-Arm Functionalized PEG–Maleimide for Protein Conjugation by Charge-Reduction Mass Spectrometry Coupled to Two-Dimensional Liquid Chromatography

Abstract

A current trend in drug development involves the use of high molecular weight, branched, and functionalized polymers for protein conjugation and drug delivery. Accurately characterizing these polymers is critical to control the product quality, to monitor the stability, and ultimately to ensure the drug efficacy and patient safety. However, due to the heterogeneity in size, the multiplicity of functional groups, and the highly convoluted charge-distribution profile in mass spectra, the characterization of these polymers is highly challenging from both chromatography and mass spectrometry perspectives. To overcome these challenges, we developed a strategy utilizing charge-reduction mass spectrometry (CRMS) coupled with two-dimensional HPLC (2D-LC). We then applied the workflow to characterize a 40 kDa 8-arm polyethylene glycol (PEG) functionalized with a maleimide terminal group for protein conjugation. The development was carried out in stages, where first we focused on the development of a CRMS method to simplify the charge profile of the polymers and then coupled it to HPLC to obtain discernible mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of the size-variant impurity resolved by reversed-phase size-exclusion 2D-LC. Finally, a separate size-exclusion reversed-phase 2D-LC-CRMS method was developed to capture a wider range of process-related impurities and reaction intermediates from the PEG–maleimide polymers throughout the conjugation process. The combination of these experiments using the 2D-LC-CRMS strategy enables the sensitive characterization of the entire impurity profile of the high molecular weight multifunctionalized PEG–maleimide conjugation intermediate.

Approaches to Heterogeneity in Native Mass Spectrometry

“…To address these problems, Cleary and Prell introduced the use of the Gábor transform (GT), which is a type of “windowed” or “short-time” Fourier transform in which the intensities of “local” frequencies in the mass spectrum are plotted against the mass spectrum itself, into iFAMS . GT and FT analysis with iFAMS of heterogeneous mass spectra of α-hemolysin complexes in detergent micelles enabled separation of the overlapped frequency signals of two oligomeric states, as well as determination of detergent stoichiometries and reconstruction of zero-charge mass spectra, and iFAMS has also been used to characterize functionalized polymer constructs for protein conjugation . In many cases, GT can readily overcome pitfalls of FT analysis due to separation of frequency signal from different types of ions according to their m / z .…”

“…298 GT and FT analysis with iFAMS of heterogeneous mass spectra of α-hemolysin complexes in detergent micelles enabled separation of the overlapped frequency signals of two oligomeric states, as well as determination of detergent stoichiometries and reconstruction of zero-charge mass spectra, 299 and iFAMS has also been used to characterize functionalized polymer constructs for protein conjugation. 300 In many cases, GT can readily overcome pitfalls of FT analysis due to separation of frequency signal from different types of ions according to their m/z. Another advantage is that salt cluster ions, which typically increase in mass as their charge state increases, can be distinguished at a glance from native biomolecular ions, which typically change little in mass over their charge state envelope and give rise to a "negatively chirped" GT signal (see section 2.4.2.3).…”

Recent Advances in Separation-Based Techniques for Synthetic Polymer Characterization

“…In another example, star branched polyethylene glycol, end functionalized with maleimide groups, was characterized by SEC, RPLC and 2DLC including SEC × RPLC and RPLC × SEC. Charge reduction mass spectrometry was coupled to these separations to greatly facilitate data interpretation and identification of impurities . Additionally, PS–PBD diblocks were coupled with multifunctional coupling agents to produce star branched block copolymers comprising both long and short diblock arms, and these were characterized by a host of separation techniques including comprehensive 2DLC .…”

“…Charge reduction mass spectrometry was coupled to these separations to greatly facilitate data interpretation and identification of impurities. 133 Additionally, PS−PBD diblocks were coupled with multifunctional coupling agents to produce star branched block copolymers comprising both long and short diblock arms, and these were characterized by a host of separation techniques including comprehensive 2DLC. 86 Both comprehensive NP TGIC × SEC and NP TGIC × LCCC under critical conditions for PS provided excellent separations of the components.…”

Mapping the Separation Landscape in Two-Dimensional Liquid Chromatography: Blueprints for Efficient Analysis and Purification of Pharmaceuticals Enabled by Computer-Assisted Modeling

“…Heterogeneities and chemical complexity associated with such molecules include a high-order structure, composition, conformation, complex mixtures, closely related components along with post-translational modifications, and other chemical changes that occur during bioprocesses . Chromatography is at the center of all facets of pharmaceutical analysis and purification; robust analytical and preparative chromatographic methods are badly needed to accelerate drug research and development efforts. − …”