Multi-arm PEG-maleimide conjugation intermediate characterization and hydrolysis study by a selective HPLC method

Wang, Jenny; Yang, Sejung; Zhang, Kelly

doi:10.1016/j.jpba.2018.11.009

Cited by 9 publications

(8 citation statements)

References 23 publications

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“…Studying PEG−Maleimide Ring-Opening Hydrolysis by CRMS. Next, we applied the optimized CRMS method to address an unanswered question from previous work by Wang et al, 22 which details a method to separate each degree of the maleimide ring-opened degradant associated with this same multifunctionalized 40 kDa PEG−maleimide. In this work, there is an acknowledged lack of direct identification of the resolved peaks attributed to be the ring-opening degradants.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The single-dimension LC method reported from previous works for the analysis of PEG–maleimide purity and its degradant forms was used . The same XSelect CSH phenyl-hexyl column (3.5 μm, 4.6 × 150 mm) from Waters (Milford, MA) was employed.…”

Section: Methodsmentioning

confidence: 99%

“…The single-dimension LC method reported from previous works for the analysis of PEG−maleimide purity and its degradant forms was used. 22 The same XSelect CSH phenyl-hexyl column (3.5 μm, 4.6 × 150 mm) from Waters (Milford, MA) was employed. Both 4.6 and 3.0 mm inner-diameter (ID) columns were studied, and a 4.6 mm ID was selected due the high viscosity of the polymers, which caused sample buildup on the columns over time that is more prominent for the 3.0 mm ID column.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…From a mass spectrometry (MS) point of view, the inherent PEG heterogeneity causes extensive numbers of polymer charge states in the electrospray process that result in highly convoluted mass spectra, preventing sensible identification and quantitation . From a chromatographic separation point of view, the intrinsic heterogeneity and HMW of complex polymer samples (e.g., branched-chain multifunctionalized PEG–maleimide molecules) often require more than one mode of separation to obtain the full impurity or degradation profile. − While size variants can be separated by size-exclusion chromatography (SEC), chemical modifications to the pendant functional groups with negligible size differences are difficult to be differentiated by SEC and require reversed-phase (RP) chromatography to provide sufficient separation . In addition to this, the multiple maleimides that attached to the PEG molecule further increase the sample multiplicity and complexity, as the readily occurring succinimide ring-opening hydrolysis can further confound the sample heterogeneity.…”

mentioning

confidence: 99%

“…19−21 While size variants can be separated by sizeexclusion chromatography (SEC), chemical modifications to the pendant functional groups with negligible size differences are difficult to be differentiated by SEC and require reversedphase (RP) chromatography to provide sufficient separation. 22 In addition to this, the multiple maleimides that attached to the PEG molecule further increase the sample multiplicity and complexity, as the readily occurring succinimide ring-opening hydrolysis can further confound the sample heterogeneity.…”

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confidence: 99%

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

Yang¹,

Chen²,

Wang³

et al. 2020

Anal. Chem.

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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.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ■ Experimental Sectionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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