Tag: mass spectrometry

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HILIC-MS impurity profiling of therapeutic PS- oligonucleotides

DNA complex spiral structure , medical, science, genetic biotechnology, gene cell concept ,3D rendering .

Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides

Oligonucleotides are short strands of synthetic DNA or RNA that are synthesized via a solid-phase synthesis, in which numerous of closely-related impurities are generated. Ion-pairing reversed-phase liquid chromatography (IP-RPLC), anion exchange chromatography (AEX), and hydrophilic interaction chromatography (HILIC) are often used to profile these impurities, which allows for good separation of impurities comprising different number of nucleotides as the full-length product (FLP). However, impurities comprising the same number of nucleotides as the FLP are often not separated. Therefore, ion-paring HILIC (IP-HILIC) was explored as an alternative separation mode to overcome these challenges.

Key points:

  • Changed selectivity: by adding ion-pairing reagents (IPRs) to the HILIC eluent, the relative contribution of the highly polar phosphate moieties on HILIC retention is reduced and, thereby, increasing the relative contribution of the nucleobase composition and conjugated groups.
  • Suppressed diastereomer separation: Phosphorothioation of the phosphate groups results in the formation of diastereomers, with 2n possible diastereomers (n = phosphorothioate groups). IPRs in the HILIC eluent reduced diastereomer separation, leading to sharper peaks.
  • Separation of same-length impurities: IP-HILIC shows increased separation of impurities comprising of the same number of nucleotides as the FLP, such as deaminated products that differ less than 1 Da from the FLP. This is noteworthy as no other MS-compatible, one-dimensional LC separation can achieve this.

Figure 1: IP-HILIC-MS total and extracted ion chromatograms of GalNAc-conjugated oligonucleotides (top) and non-conjugated oligonucleotides (bottom) and the mass spectra of peaks A-D (A & C: FLPs, B & D: deaminated products)

The developed IP-HILIC method shows great potential as a screening method for quality control. The work is published in Analytical Chemistry and can be found with the following link: https://pubs.acs.org/doi/full/10.1021/acs.analchem.5c01407

 

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New Frontiers in  Intact Protein Characterization by LC-MS at CAST

CAST scientists Annika van der Zon and Ziran Zhai have just published two manuscripts
showcasing significant advances in the low flow analysis of intact antibodies and protein complexes, offering improved sensitivity and performance. The CAST team will utilize these novel nano SEC-MS and HILIC-MS methods in future bioanalysis projects.

Analyzing Minute Amounts of Protein Complexes with Nanoflow Size Exclusion Chromatography–Native Mass Spectrometry

Characterizing intact proteoforms and protein complexes often faces challenges in maintaining native structures and high sample requirements. CAST scientist Ziran Zhai developed a novel nanoflow size exclusion chromatography–native mass spectrometry (nanoSEC-nMS) method to overcome these limitations.

Key Advancements:

  • Optimized Capillary SEC Columns & Reduced Peak Broadening: The method includes techniques for preparing high-performance capillary SEC columns and optimizing injection to reduce peak widths.
  • Direct Coupling under Challenging Conditions: It enables direct coupling of nanoflow SEC with native MS even in salt-rich environments.
  • Milder Desolvation for Native Structures: Nanoflow allows for milder ESI desolvation, preserving the native structures of proteins and complexes.
  • High Sensitivity and Throughput: The method requires limited sample (approx. 100 nL per injection) and significantly enhances native MS throughput, enabling online desalting and oligomer separations within 25 minutes.

Figure 1: Analysis of urine samples and Ovitrelle with the nanoSEC-nMS: (a) EIC of the urine hCG samples; (b) MS spectrum of hCG proteins; (c) deconvoluted results of hCG proteins; (d) EIC of the Ovitrelle sample; (e) MS spectrum of Ovitrelle; (f) deconvoluted results of Ovitrelle.

This nanoSEC-nMS method enables the analysis of proteins and complexes across a broad molecular weight range (10 to 250 kDa) in their native states, preserving noncovalently bound metal ions. This study was published in Analytical Chemistry and can be accessed freely at the link below:

https://pubs.acs.org/doi/10.1021/acs.analchem.5c01019 

Precise Glycoform Profiling of Intact Antibodies with HILIC-MS

Traditional methods struggle with comprehensive intact antibody glycoform profiling. To address this,CAST scientist Annika van der Zon et al. at developed a novel hydrophilic interaction chromatography (HILIC) method based on lab made acrylamide-based monolithic columns directly coupled to mass spectrometry.

Key Innovations:

  • Optimized Monolithic Stationary Phase: The porogen composition was optimized, enhancing separation efficiency
  • Enhanced Glycoform Resolution: The method achieved baseline separations for single and double Fc glycosylation, and partial separations for glycoforms differing by a single glycan unit.
  • Sensitive Detection of Minor Glycoforms: It enabled sensitive measurement of low-abundance glycoforms in the nanogram injection range.

Figure 2. Analysis of intact trastuzumAb at the intact level. Base Peak Chromatogram of the analysis and Extracted Ion Currents of selected glycoforms are shown.  

This HILIC-MS method significantly enhances glycoform selectivity for intact antibodies, providing a more comprehensive characterization essential for bioanalytical applications. This work was published in the Journal of Analytical Chemistry and can be accessed freely at the link below:

https://doi.org/10.1021/acs.analchem.5c02033

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SEC-MS and Enzymes for polyester polymers analysis

Abstract representation of a molecular connection crystallized in the hexagonal system.. 3d illustration.

CAST scientist Masashi Serizawa recently published a manuscript in which he investigated a novel size-exclusion chromatography hyphenated with mass spectrometry and ultraviolet (SEC-MS/UV) method to characterize poly lactide-co-glycolide (PLGA), and also co-authored work on the use of enzymes for polyester polymer degradation.

 

·         SEC-MS characterization of PLGA:

Size-exclusion chromatography (SEC) hyphenated with MS is valuable for microstructure analysis. While SEC-UV/RI determines molecular weight distribution (MWD), SEC-MS is used for chemical composition distribution (CCD) and functionality-type distribution (FTD). However, previous applications of SEC-MS have failed to address the risk of polymer fragmentation during the analysis process. It is crucial to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occurred during the SEC separation or during the ESI-MS process.

 

This study addresses fragmentation in PLGA analysis by optimizing SEC-MS conditions. We demonstrate that cesium iodide (CsI) minimizes fragmentation during electrospray ionization (ESI-MS), simplifying spectra and enabling differentiation of PLGA isomers. This facilitates accurate determination of CCD and FTD, even revealing “blockiness” when coupled with selective degradation.

Figure 1: schematic illustration of in-source fragmentation in SEC-MS, depending on ionization agents

The study is supported by the COAST/TKI-Chemistry POLY-SEQU-ENCHY project between the UvA and Corbion and is funded by Mitsubishi Chemical Corporation. This work was recently published in the Journal of the American Society for Mass Spectrometry and can be accessed freely at the link below:

https://doi.org/10.1021/jasms.4c00447

 

·         Insights in the selectivity of enzymes for polyester co-polymer degradation

Another example of the SEC-MS/UV polymer application conducted by our group is the structural analysis of aromatic/aliphatic polyesters. To understand the polymer chain structure of aromatic/aliphatic polyesters, Eman et al. successfully developed a two novel thermostable cutinase that primarily degrade aliphatic ester bonds. These enzymes maintain activity at elevated temperatures of up to 90 °C thanks to enzyme engineering.

For both enzymes, higher hydrolysis rates were observed for aliphatic compared to aromatic homo-polyesters. SEC-MS analysis revealed that the hydrolysis of aliphatic/aromatic co-polyesters occurred at the aliphatic monomers, significantly reducing the molecular weight and changing the end-group composition. These results underline the importance of co-polymer composition in the biodegradation of co-polymer systems and demonstrate the applicability of enzymes for the analytical characterization of synthetic polymers by selectively reducing their molecular weight.

Figure 2: Results of SEC-MS/UV analysis of a copolymer containing aromatic/aliphatic polyesters,
comparing between before and after enzymatic degradation (The degradations were
performed at 71°C, using thermostable cutinase)

This research was funded by Topconsortium voor Kennis en Innovatie (TKI) Chemie, deployment project PPS-programma toeslag 2019 (CHEMIE.PGT.2020.020). This work was recently published in the Chemistry – A European Journal and can be accessed at the link below:

https://doi.org/10.1002/chem.202403879

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Separation of mAb charge variants by CZE-MS method under near-native pH conditions

CAST scientist Annika van der Zon recently introduced a near-native separation method for characterizing charge variants of intact monoclonal antibodies (mAbs) using capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS). In this study we used a nanoflow sheath liquid interface, known as nanoCEasy applied thanks to the collaboration via the Uniiversity of Aalen.

The CZE-MS method, employs a neutral static capillary coating made of hydroxypropyl methylcellulose, combined with 50 mM acetic acid at pH 5.0, to create MS-compatible conditions for separating mAb charge variants. Currently, the pharmaceutical industry uses the EACA method of He et al. (2011) method to routinely profile charge variants, but this method relies on a non-volatile background electrolyte (BGE), making it incompatible with MS and hindering the identification of separated charge variants.

The MS-compatible CZE method we introduce allows to obtain similar charge variant profiling as the EACA method but allows for MS analysis. The CZE-MS coupling, enabled by nanoCEasy’s low-flow sheath liquid interface, successfully identified and quantified basic and acidic variants, incomplete pyroglutamate variants, and glycoforms of the mAbs tested. This CZE-MS method provides a powerful tool for assessing mAb heterogeneity and achieving charge variant separation.

 

Figure: Schematic representation of the CZE-UV/MS separation of charge variants of mAbs.

 

This study is published in the journal Analytical Chimica Acta, see here:
Thanks to all the co-authors for their contribution to this study.
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Detection Challenges in Polymer Analysis with LC

With their large distributions culminating in wide envelopes of – almost exclusively – co-eluting peaks, polymers certainly present a unique challenge relative to the analysis of small molecules. If anything, this challenge has spurred innovations which have also benefited other fields. A good example has been the work on polymer analysis with 2D-LC in the first years of this millennium, which have certainly contributed to recent developments on the technique [1,2].

In his review, Wouter Knol (Van ‘t Hoff Institute for Molecular Sciences) reviews another research area which offers a lot of room for innovations: detection. Together with the co-authors, Knol provided an exhaustive overview of applications of detection techniques in LC for polymer analysis. For each detection technique, notable recent applications are discussed and the authors distilled the key advantages and disadvantages of each approach.

One particularly useful trait of the review is this table which summarizes all key strengths and weaknesses of each detection technique employed for LC in polymer analysis. Reprinted with permission from [3].

Knol and co-workers noted that promising approaches receive surprising attention in recent literature. He concluded that the opportunities deserve more attention. You can download and read the paper, which was published open-access in the special Reviews 2021 issue of Journal of Separation Sciences, here.

Example of a separation of complex polyether polyols with LC×LC-MS by Groeneveld et al. showing a clear structure based on the number of ethylene oxide/propylene oxide units in the polymer. Reprinted with permission from [4].

References

[1] Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications
B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem., 2019, 91(1), 240-263, DOI: 10.1021/acs.analchem.8b04841

[2] Comprehensive Two-Dimensional Ultrahigh-Pressure Liquid Chromatography for Separations of Polymers E. Uliyanchenko, P.J.C.H. Cools, Sj. van der Wal and P. J. Schoenmakers, Anal. Chem. 2012, 84, 18, 7802–7809, DOI: 10.1021/ac3011582

[3] Detection challenges in quantitative polymer analysis by liquid chromatography, W.C. Knol, B.W.J. Pirok, and R.A.H. Peters, J. Sep. Sci. 2020, DOI: 10.1002/jssc.202000768

[4] Characterization of complex polyether polyols using comprehensive two-dimensional liquid chromatography hyphenated to high-resolution mass spectrometry G. Groeneveld, M.N. Dunkle, M. Rinken, A.F.G. Gargano, A. de Niet, M. Pursch, E.P.C. Mes, and P.J. Schoenmakers, J. Chromatogr. A, 1569, 2018,  128-138, DOI: 10.1016/j.chroma.2018.07.054

Wouter Knol was a PhD candidate in the group of Peter Schoenmakers and Bob Pirok. He worked in the UNMATCHED project in Amsterdam and mainly focuses on techniques to determine the sequence distribution of polymers.

You can read more about him on our Team page.