Improved Mass Determination of Poly(ethylene glycols) by Electrospray Ion-Mobility Time-of-Flight Mass Spectrometry Coupled with Ion–Molecule Reactions

The authors developed a method to accurately measure the average molecular weight of large poly(ethylene glycols) (PEGs) using ion-mobility time-of-flight mass spectrometry coupled with gas-phase ion–molecule reactions.
Jul 02, 2008

Poly(ethylene glycol) (PEG) is a polymer composed of repeating subunits of ethylene oxide. PEG and its functionalized derivatives can be produced in linear or branched forms with various molecular masses. Because PEG possesses many unique properties, for example, high water solubility and low toxicity, it is widely used in a range of biomedical applications (1). For instance, modification of protein-based therapeutics by chemical attachment of PEG chains to the proteins (i.e., PEGylation) has been demonstrated to be an effective approach for reducing immunogenicity, increasing circulating half-life and improving stability of the biopharmaceuticals (2).

A major challenge faced in PEG applications is to ensure the quality and stability of PEG-based materials before the PEG-ylation process. In contrast to a protein that normally shows a single molecular weight, PEG is a polydisperse material with a molecular weight distribution (MWD). In addition, the polymer chains might have different end groups because of different initiation and termination processes, thereby creating a functionality type distribution (FTD). All of these have created complexity and diversity to the polymer, thus imposing great challenges to analytical characterizations of the materials.

Desired information about PEG characterizations normally includes average molecular weight (MW), MWD or polydispersity, structural, compositional, and end-group identification. Many techniques are available for characterizing PEGs. However, no single technique could completely characterize all the attributes related to the material. Frequently, applications of multiple analytical techniques such as gel-permeation chromatography (GPC), nuclear magnetic resonance (NMR), Raman spectroscopy, and mass spectrometry (MS) are required for thorough structure elucidation, with each technique addressing one aspect of the characterizations (3–6).

Among the analytical techniques currently used in the characterization of synthetic polymers, mass spectrometry is of increasing importance (7). Mass spectrometry can be used to determine the molecular mass of synthetic polymers directly with unparalleled accuracy and precision. In addition, the accurate mass measurement attained by mass spectrometry yields much useful information for the repeat unit and end-group analysis, for providing evidence for the existence of copolymers and impurities, and as a quality control protocol to confirm synthetic pathways and batch-to-batch compositional variation (8–10).

Both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been useful for polymer analysis. However, for synthetic polymers with MW much greater than 5 kDa, MALDI is often a preferred ionization method for MS analysis because of the simplicity of the mass spectra, which show mainly singly charged molecular ions (11). The limited use of ESI in PEG analysis, especially in high molecular weight PEG, is largely restrained by the complex spectra resulted from an ESI process. One of the primary features of ESI is that it tends to generate multiply charged ions, and the propensity for generating multiply charged ions also increases with growing molecular weight for a given polymer (12). The convolution of a charge state distribution with a broad MWD of PEG can result in highly complex spectra from which the MWD is very difficult, if not impossible, to obtain. Multiple charging, therefore, limits the PEG oligomer size range amenable to molecular weight characterization by ESI. The upper limit is essentially defined by the resolving power of the mass analyzer.

A majority of the work thus far using ESI for PEG analysis has focused on relatively low molecular weight PEGs to reduce the number of charges retained on the polymer ions, thus reducing the spectral complexity. For higher molecular mass polymers, a separation method is also helpful in reducing the MS spectra complexity by limiting the range of oligomers being introduced into the electrospray source at a given time (13). To overcome the hindrance in the mass spectrometric analysis of high molecular weight PEGs, a chemical deconvolution approach was also developed which uses ion–ion or ion–molecule reactions to greatly reduce the number of charges PEG ions carried to facilitate mass measurements (14–16). In one method, neutral crown ethers were successfully used to reduce charging of PEG cations in an ion trap mass spectrometer (14). Another interesting method presented a facile study using a base-mediated, gas-phase proton-transfer reaction in the source region of an ESI time-of-flight (TOF) mass spectrometer for the characterization of PEG and PEG-protein conjugates (15). Both methods have demonstrated to be effective at reducing the multiple charging of polymers in ESI analysis and producing accurate MW measurement for a range of PEGs.

The work presented in this article describes an alternative approach to reducing the charge state and thus extending the mass range of PEG amenable for ESI analysis by use of a commercial ion-mobility TOF mass spectrometer. The charge reduction was achieved by conducting an ion–molecule reaction between highly charged PEG ions and a neutral gaseous base inside the mass spectrometer. The charge states of highly charged PEG ions were dramatically stripped down to a few narrowed charge-state distributions, thus greatly reducing the overlapping of PEG peaks from adjacent charge states and resulting in very simplified MS spectra. The chemical deconvolution process allows the molecular weight of PEG to be readily determined and the polydispersity more clearly identified. In contrast to the previous work (15) in which ion reactions were performed in the source region, this approach has the benefits of easy operation and less consumption of superbase materials for a given reaction. More important, the approach presents a simple and flexible method to modify the configurations of a commercial instrument for a tailored application. In addition, the ion-mobility capability of the instrument allowed differentiation of various chain lengths of the polymer, thereby providing enhanced specificity to the characterization of the materials.