Category: Publications

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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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Block-Length Distributions Using Fragmentation Data Obtained from Tandem Mass Spectrometry

Researchers Rick van den Hurk, Dr. Tijmen Bos, and Dr. Bob Pirok have, together with scientists Dr. Ynze Mengerink (Brightlands Chemelot Campus) and Prof. Dr. Ron Peters (Covestro, HIMS), developed a new algorithm that can analyze copolymers and determine their block structure, something that was previously out of reach with existing techniques.

Polymers are all around us, from the coatings on your phone and the materials in your running shoes to life-saving drug-delivery systems and medical implants. Many of these advanced materials are copolymers, which are made by combining different types of chemical building blocks.

Interestingly, even when two copolymers have the same overall composition, the way these building blocks are arranged can lead to drastically different properties. For example, one polymer might be rigid while another is flexible, or one transparent while another is opaque. Within a single batch, this arrangement can vary from molecule to molecule. This variability can be described using a concept called the block-length distribution (BLD), which captures how frequently different block arrangements occur. This distribution plays a key role in determining a material’s performance characteristics, including its flexibility, strength, and biodegradability.

Until now, accurately measuring these distributions at the molecular level has been a major challenge. Traditional techniques like nuclear magnetic resonance could only offer averaged information. The team’s newly developed algorithm changes that by combining tandem mass spectrometry (MS/MS) data with a smart computational approach that takes fragmentation behavior into account. The algorithm allows researchers to reconstruct how blocks are distributed within a copolymer sample, giving a much more detailed picture of the material’s internal structure.

This method has already been successfully applied to study polyamides and polyurethanes, important industrial polymers found in everything from textiles to insulation foams. Notably, the findings showed that even polymers with the same chemical makeup can have very different block distributions, depending on how they were synthesized. These subtle differences can explain variations in material performance that would otherwise remain hidden.

 

The ability to determine BLDs with such precision not only improves our understanding of polymer chemistry but also opens the door to the rational design of next-generation materials. By fine-tuning the block arrangement, scientists and engineers can tailor materials more precisely to specific applications. It could also support the development of more sustainable materials, as better control over structure may lead to improved recyclability or allow for the use of bio-based feedstocks.

This work is part of the PARADISE project, a collaboration between academic institutions (VU Amsterdam and the University of Amsterdam) and industrial partners including Covestro, DSM, Shell, and Genentech, aimed at driving forward innovation in polymer research.

Relevant article: https://doi.org/10.1021/acs.macromol.5c00297,

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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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Educational textbook Analytical Separation Science launched by Pirok and Schoenmakers

Bookframe

At the renowned HPLC2025 conference, a milestone in the field of separation science was celebrated with the official launch of Analytical Separation Science, a comprehensive new textbook authored by Dr. Bob Pirok and Prof. Peter Schoenmakers. The launch event, held on June 16 at the Historium Bruges and organized by the Royal Society of Chemistry (RSC), brought together leading scientists and educators in the field.

The first official copy was presented to Prof. Govert Somsen of the Vrije Universiteit Amsterdam, a long-time colleague and co-educator in analytical chemistry.

Structured around Basic, Master, and Advanced modules, the book serves both as a teaching tool and as a professional reference. It introduces fundamental concepts, offers in-depth treatments for graduate-level study, and explores cutting-edge developments in chromatographic and electrophoretic techniques.

“This book reflects our shared commitment to educating the next generation of analytical scientists,” said Prof. Peter Schoenmakers. “By combining foundational theory with real-world case studies and emerging methods, we aim to make separation science engaging and relevant across career stages.”

Figure 1. Cover of the book.

An interactive companion website (https://ass-ets.org) extends the book’s reach. It offers additional resources including a literature repository, academic lectures with interactive figures, and exercises based on decades of teaching experience at the University of Amsterdam and Vrije Universiteit Amsterdam.

The website is a community based effort with universities to be supporting their expertise to complete the analytical portfolio as much as possible.

Figure 2. Prof. Wolfgang Lindner (University of Vienna) and Prof. Peter Schoenmakers (University of Amsterdam) draw the winners of the book competition.

“Our goal was to make learning separation science both accessible and inspiring,” added Dr. Bob Pirok. “This project combines our classroom experience with insights from industry collaborations, bridging education and practice.”

Figure 3. Photograph from the launch event.

Conference participants were able to win a copy of the book by solving a series of puzzles and Prof. Wolfgang Lindner (University of Vienna) and Prof. Schoenmakers (University of Amsterdam) drew the five winners from the entries.

The book is now available at the Royal Society of Chemistry or any other book vendor.

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Introducing an algorithm to accurately determine copolymer block-length distributions

Copolymers are the foundation of many high-performance materials used in advanced applications such as medical devices, implants, electronics, and self-healing coatings for aerospace and space exploration. Their material properties—such as flexibility, toughness, or responsiveness—can be finely tuned by adjusting polymer characteristics like molecular weight, chemical composition, and block-length distribution (BLD).

While molecular weight and composition are routinely analyzed, the BLD—describing how monomer blocks are arranged along the polymer chain—remains difficult to measure, particularly for copolymers composed of more than one type of monomer. Understanding and controlling BLD is crucial because it plays a pivotal role in determining mechanical, thermal, and phase-separation behavior. However, current methods, such as NMR or pyrolysis-GC-MS, have limitations in accurately and comprehensively characterizing BLDs.

 

Our Solution
In this study, we introduce a computational approach that enables the quantitative determination of block-length distributions from copolymer fragmentation data. We developed and validated an algorithm using both simulated copolymer sequences and analytical solutions to generate ground-truth fragment data. This allowed for an objective evaluation of algorithm performance—something not previously achievable.

The algorithm incorporates a trust-region-reflective optimization strategy and was tested under various conditions, including data noise and fragment size limitations. When fragment data containing chains of up to four monomers (tetramers) were included, the algorithm consistently reconstructed BLDs with high accuracy, achieving similarity coefficients (SC) above 0.99 compared to the known distributions.

https://doi.org/10.1016/j.aca.2025.343990

 

Key Innovations

  • High Accuracy: Outperforms existing algorithms in BLD reconstruction from mass spectrometry-based data.

  • Versatile: Capable of handling complex distribution shapes, including non-unimodal and asymmetric distributions.

  • Robust to Noise: Maintains accuracy even when fragment data includes measurement noise.

  • Objective Evaluation: Enables benchmarking of BLD algorithms using simulated data with known parameters.

 

Practical Relevance
The algorithm was also applied to experimental polymer systems such as polyamides and polyurethanes, demonstrating its applicability to real-world materials. This makes it a powerful tool for synthetic chemists seeking to design materials with tailored properties by manipulating block structures.

 

Future Directions
Translating this approach from simulation to experimental data introduces new challenges. Mass spectrometry data may be affected by ionization efficiencies and fragmentation biases, while NMR may suffer from overlapping signals in complex systems. To address this, future research will focus on:

  • Incorporating fragmentation preferences based on bond type or analytical method.

  • Developing preprocessing pipelines tailored to specific instrumentation.

  • Extending the algorithm to support more than two monomer types, while managing the increased computational complexity.

 

Conclusion
This work represents a significant advancement in the field of polymer analytics. For the first time, researchers can objectively and accurately reconstruct the block-length distribution of complex copolymers from fragment data. By making this tool available, we aim to empower chemists and materials scientists in designing next-generation materials with precisely engineered microstructures.

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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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NPLC method to characterize end groups of poly lactic acid co-glycolic acid copolymers

Vertical image laboratory test tubes for research with white plastic polymer or medical supplies lie on a black background. Conceptual image.

The CAST scientist Masashi Serizawa recently published a manuscript in which he investigated a novel method of using gradient elution normal-phase liquid chromatography with basic and acidic additives to separate PLGAs in the different end groups and in the different chemical compositions at the same time.

PLGA is an important material in drug delivery systems. It is used in nanoparticle-containing drugs to prevent a sudden increase in drug concentration in the body when the drug is ingested. The LA/GA ratio and differences in the terminal structure of PLGA have a significant effect on the degradation rate of PLGA in the body.

To distinguish these distinctions, we created a unique ternary gradient liquid chromatography method utilizing base and acid additives. Initially, we used a gradient of hexane, a poor solvent, and ethyl acetate, a good solvent, with a mobile phase containing a base additive to separate non-ester-terminated PLGAs (ester-terminated PLGA and cyclic PLGA) based on their chemical composition. Subsequently, by switching the mobile phase to THF containing an acid additive, we were able to elute acid-terminated PLGA.

This method offers the advantage of quick analysis compared to traditional NMR methods, making it potentially valuable for future industrial research. Furthermore, it can be applied to high molecular weight PLGA of 180 kDa, making it useful for the development of high molecular weight PLGA, which is challenging to analyze using mass spectrometry techniques such as MALDI-TOF-MS.

 

Figure: (left) schematic illustration of the working principle of the NPLC separation. (right): key results obtained in the study
Figure: (left) schematic illustration of the working principle of the NPLC separation. (right): key results obtained in the study

 

The study is supported by the COAST/ TKI-Chemistry POLY-SEQU-ENCHY project between the UvA and Corbion (Gorinchem, The Netherlands) and is funded by Mitsubishi Chemical Corporation.

The link to the publication is reported below.

https://doi.org/10.1016/j.chroma.2024.465137

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Parallel Gradients 2DLC-HRMS of complex protein digest

2D-LC System
2D-LC System

Investigating the proteins in biological samples can help us understand and identify diseases and improve the effectiveness of medication. To study proteins in these samples, they are typically digested into peptides and subsequently analyzed by liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS).

Comprehensive two-dimensional LC (LC×LC) offers increased separation power over traditional LC methods. However, most common gradient designs require re-equilibration of every second-dimension run, resulting in high flow rate operations to limit the empty separation space. This also limits MS sensitivity as flow splitting is required to handle such flow rates.

In this work, we developed an LC×LC method using a so-called parallel-gradient design, which omits the need for column re-equilibration and enables the use of the entire separation space. Moreover, this allows for lower flow rates and maintains the sensitivity for low-abundant analytes. The parallel-gradient design achieved higher surface coverages and sensitivity at lower effective peak capacities. Most importantly, both methods were applied to analyze a Human IMR90 lung fibroblast cell line digest to assess its applicability to real complex samples. The parallel-gradient method was able to identify significantly more proteins than the current state-of-the-art methods while using the same analysis time and at a lower solvent consumption. The applicability of the parallel-gradient design could be improved even further by shortening the modulation times, as it was not limited by column re-equilibration.

The study is a collaborative work done thanks for the contribution of many colleagues and students. The link to the publication is reported below.

https://doi.org/10.1021/acs.analchem.4c02172

 

 

 

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Nanoflow IEC-HRMS to study complex proteoform mixtures

Clinical Materials Molecules
Clinical Materials Molecules

The CAST scientist Ziran Zhai published a manuscript investigating a novel method of using nanoflow strong cation exchange – native mass spectrometry to characterize non-denaturing complex proteoforms mixtures from the intact level. Zhai focuses on three critical aspects: i) extending the MW that can be observed by top-down proteomics, ii) increasing the MS sensitivity to create conditions of detecting low-abundant proteins, and iii) apply mild desolvation conditions to maintain the native structures of proteins and complexes. 

Proteoforms, which are protein products arising from homologous genes due to sequence variations, alternative splicing, and post-translational modifications, play a crucial role in a wide range of critical functions. However, the standard approach to characterize proteins, known as bottom-up proteomics, faces limitations. This approach cannot directly identify proteoforms as the presence of proteins is inferred from peptides. While top-down proteomics and intact protein mass spectrometry offer solutions to these limitations, the most common top-down methods employ denaturing LC-MS approaches, which unfold proteins and lead to the loss of non-covalent protein complexes.

In this work, we directly coupled nanoflow (250 or 500 nL min-1) strong cation exchange chromatography (SCX) to nano-electrospray-ionization (nESI) under native MS (nMS) conditions. Proteins were separated on packed capillary SCX columns and eluted according to their pI values by a salt-mediated pH gradient method. The low flow promoted desolvation/ionization efficiency allowing for sensitive detection of low-abundant proteins and complexes. We successfully applied our method to analyze an E. coli cell lysate and observed hundreds of proteins with masses up to 150 kDa. We believe that the proposed nanoSCX-nMS is a promising approach for characterizing proteoforms and provides a universal strategy to overcome detection limitations in native top-down proteomics.

 

Screenshot

The study is part of the FFF (From Form to Function) project of Zhai, Astefanei,
Corthals, and Gargano and was funded by the Chinese Scholarship Council (CSC) and was recently published in Analytica Chimica Acta and can be accessed freely at the link below.

https://pubs.acs.org/doi/10.1021/acs.analchem.4c01760.