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Pusfitasari, E. D., Ruiz-Jimenez, J., Samuelsson, J., Besel, V., Fornstedt, T., Hartonen, K. & Riekkola, M.-L. (2023). Assessment of physicochemical properties of sorbent materials in passive and active sampling systems towards gaseous nitrogen-containing compounds. Journal of Chromatography A, 1703, Article ID 464119.
Open this publication in new window or tab >>Assessment of physicochemical properties of sorbent materials in passive and active sampling systems towards gaseous nitrogen-containing compounds
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2023 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1703, article id 464119Article in journal (Refereed) Published
Abstract [en]

The adsorption and desorption behavior of volatile nitrogen-containing compounds in vapor phase by solid-phase microextraction Arrow (SPME-Arrow) and in-tube extraction (ITEX) sampling systems, were investigated experimentally using gas chromatography-mass spectrometry. Three different SPME-Arrow coating materials, DVB/PDMS, MCM-41, and MCM-41-TP and two ITEX adsorbents, TENAX-GR and MCM-41-TP were compared to clarify the selectivity of the sorbents towards nitrogen-containing compounds. In addition, saturated vapor pressures for these compounds were estimated, both experimentally and theoretically. In this study, the adsorption of nitrogen-containing compounds on various adsorbents fol-lowed the Elovich model well, while a pseudo-first-order kinetics model best described the desorption kinetics. Pore volume and pore sizes of the coating sorbents were essential parameters for the deter-mination of the adsorption performance for the SPME-Arrow sampling system. MCM-41-TP coating with the smallest pore size gave the slowest adsorption rate compared to that of DVB/PDMS and MCM-41 in the SPME-Arrow sampling system. Both adsorbent and adsorbate properties, such as hydrophobicity and basicity, affected the adsorption and desorption kinetics in SPME-Arrow system. The adsorption and desorption rates of studied C6H15N isomers in the MCM-41 and MCM-41-TP sorbent materials of SPME-Arrow system were higher for dipropylamine and triethylamine (branched amines) than for hexylamine (linear chain amines). DVB/PDMS-SPME-Arrow gave fast adsorption rates for the aromatic-ringed pyridine and o-toluidine. All studied nitrogen-containing compounds demonstrated high desorption rates with DVB/PDMS-SPME-Arrow. Chemisorption and physisorption were the sorption mechanisms in MCM-41-and MCM-41-TP-SPME-Arrow, but additional experiments are needed to confirm this. An active sampling technique ITEX gave comparable adsorption and desorption rates on the selective MCM-41-TP and univer-sal TENAX-GR sorbent materials for all the compounds studied. Vapor pressures of nitrogen-containing compounds were experimentally estimated by using retention index approach and these values were compared with the theoretical ones, calculated using the COnductor-like Screening MOdel for Real Sol-vent (COSMO-RS) model. Both values agreed well with those found in the literature proving that these methods can be successfully used in predicting VOC's vapor pressures, e.g. for the formation of secondary organic aerosols.

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Nitrogen-containing compounds, Solid-phase microextraction arrow, In -tube extraction, Kinetics, Vapor pressure
National Category
Analytical Chemistry Physical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-96106 (URN)10.1016/j.chroma.2023.464119 (DOI)001015147000001 ()37271082 (PubMedID)2-s2.0-85163254242 (Scopus ID)
Funder
Swedish Research Council, 2018-04969Vinnova, 2019-02496Swedish Research Council Formas
Available from: 2023-07-13 Created: 2023-07-13 Last updated: 2023-07-13Bibliographically approved
Ravald, H., Jaikishan, S., Samuelsson, J., Sukhova, A., Šolínová, V., Fornstedt, T., . . . Wiedmer, S. K. (2023). Capillary electrokinetic chromatography for studying interactions between β-blockers and Intralipid emulsion. Journal of Pharmaceutical and Biomedical Analysis, 234, Article ID 115554.
Open this publication in new window or tab >>Capillary electrokinetic chromatography for studying interactions between β-blockers and Intralipid emulsion
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2023 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 234, article id 115554Article in journal (Refereed) Published
Abstract [en]

Toxicity of β-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of β-blockers of different hydrophobicity’s (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed β-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the β-blockers. The binding and adsorption constants also show that less hydrophobic β-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of β-blockers is worth further investigation. 

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Adsorption isotherm, β-blockers, Capillary electrokinetic chromatography, Distribution constant, Drug toxicities, Intralipid emulsion
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-96274 (URN)10.1016/j.jpba.2023.115554 (DOI)001033439600001 ()37399701 (PubMedID)2-s2.0-85163825276 (Scopus ID)
Funder
Knowledge Foundation, 20210021
Available from: 2023-08-08 Created: 2023-08-08 Last updated: 2023-08-24Bibliographically approved
Fornstedt, T., Forssén, P. & Samuelsson, J. (2023). Chapter 23 - Modeling of preparative liquid chromatography (3ed.). In: Salvatore Fanali; Bezhan Chankvetadze; Paul R. Haddad; Colin F. Poole; Marja-Liisa Riekkola (Ed.), Liquid Chromatography: Fundamentals and Instrumentation (pp. 603-624). Elsevier, 1
Open this publication in new window or tab >>Chapter 23 - Modeling of preparative liquid chromatography
2023 (English)In: Liquid Chromatography: Fundamentals and Instrumentation / [ed] Salvatore Fanali; Bezhan Chankvetadze; Paul R. Haddad; Colin F. Poole; Marja-Liisa Riekkola, Elsevier, 2023, 3, Vol. 1, p. 603-624Chapter in book (Other academic)
Abstract [en]

Preparative chromatography is the best generic method currently available for purifying small drugs and valuable chemical components at the 10-kg level. Progress in computer technology, the development of new non-chiral/chiral stationary phases, and numerous improvements in reliability and economic performance have considerably increased the interest in modeling in academia and industry. This chapter introduces the modeling of preparative liquid chromatography in order to improve the purification process for valuable chemical components such as drugs and chiral components. We review the most important column and adsorption models and the methods for determining the essential thermodynamic adsorption data for both column characterization and process improvement. We also cover important operational modes (e.g., separation in gradient mode), cases involving additives or ion-pair reagents, and operational conditions sometimes neglected in the modeling process, for example, involving the impact of injection profiles. 

Place, publisher, year, edition, pages
Elsevier, 2023 Edition: 3
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-95429 (URN)10.1016/B978-0-323-99968-7.00003-5 (DOI)2-s2.0-85161168550 (Scopus ID)978-0-323-99968-7 (ISBN)
Available from: 2023-06-19 Created: 2023-06-19 Last updated: 2023-06-19Bibliographically approved
Enmark, M., Samuelsson, J. & Fornstedt, T. (2023). Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case. Journal of Chromatography A, 1691, Article ID 463823.
Open this publication in new window or tab >>Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case
2023 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1691, article id 463823Article in journal (Refereed) Published
Abstract [en]

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Adsorption, Adsorption isotherms, Electrostatics, Inverse problems, Oligonucleotides, Solvents, Cosolvents, Elution profiles, Gradient elution, Ion-pair chromatography, Ion-pairing reagent, Overloaded elution profile, Preparative chromatography, Quantitative models, Retention factors, Solvent gradients, Ions, dyes, reagents, indicators, markers and buffers, oligonucleotide, solvent, adsorption, procedures, reversed phase liquid chromatography, Adsorption, Chromatography, Reverse-Phase, Indicators and Reagents, Oligonucleotides, Solvents
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-93758 (URN)10.1016/j.chroma.2023.463823 (DOI)000925422200001 ()36716595 (PubMedID)2-s2.0-85147190760 (Scopus ID)
Funder
Knowledge Foundation, 20210021
Available from: 2023-02-23 Created: 2023-02-23 Last updated: 2023-03-06Bibliographically approved
Fornstedt, T. & Enmark, M. (2023). Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science. Journal of Chromatography Open, 3, Article ID 100079.
Open this publication in new window or tab >>Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science
2023 (English)In: Journal of Chromatography Open, ISSN 2772-3917, Vol. 3, article id 100079Article in journal (Refereed) Published
Abstract [en]

In recent decades, there has been a trend toward using larger biological molecules as new active pharmaceutical ingredients (APIs) instead of the classical small organic API molecules. More recently, this trend has shifted from very large biomolecules toward intermediate-sized APIs, such as oligonucleotide therapeutics. Because of their fundamental role in gene regulation, therapeutic oligonucleotides can be directed against their specific ribonucleic acid (RNA) targets, representing a promising customized approach for the treatment of hitherto incurable diseases. There are several FDA-approved oligonucleotide-based therapeutics and many more are awaiting approval. The complicated synthesis and degradation pathways of oligonucleotides, involving sophisticated new chemical modifications, generate hundreds of impurities, in contrast to classical small APIs, which typically contain only around three to five well-defined impurities (Fig. 1). Therefore, this new class of putative drugs entails challenging separation tasks: for example, a small mass change such as 1 Da must be distinguished in a 10,000 Da parent molecule for purposes of both quantification and purification and at extremely high resolution. All therapeutic oligonucleotides must be chemically modified before entering the body. One such modification is the phosphorothioate (PS) modification, which generates diastereomers: for a 20-nucleotide-long PS oligonucleotide, this exceeds half a million diastereomers. In this review, we will examine recently published ion-pair liquid chromatographic separation strategies to meet current challenges in oligonucleotide separations. Ion-exchange chromatography will be briefly discussed based on its merits for large-scale purification. The review focuses on studies combining theory and practice and aiming at the analysis and preparative separation necessary for performing reliable quality control as well as purification. All relevant aspects of the separation systems will be discussed, including the stationary phase, pore size, mobile phase, and ion-pairing reagents. We will also discuss how the properties of the oligonucleotide and its impurities can be exploited to increase separation selectivity. A particular focus will be on the adsorption of ion-pairing reagent and the electrostatic surface potential it generates, allowing for interaction with the highly charged oligonucleotides. Furthermore, the effects of various gradient modes to decrease the electrostatic potential and thereby elute oligonucleotides will be covered. 

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Analytical chromatography, Ion-exchange chromatography, Ion-pair chromatography, Oligonucleotides, Preparative chromatography, Separation theory
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-94693 (URN)10.1016/j.jcoa.2023.100079 (DOI)2-s2.0-85150510091 (Scopus ID)
Funder
Knowledge Foundation, 20210021
Available from: 2023-05-15 Created: 2023-05-15 Last updated: 2023-05-16Bibliographically approved
Lesko, M., Kaczmarski, K., Jora, M., Stavenhagen, K., Leek, T., Czechtizky, W., . . . Samuelsson, J. (2023). Strategies for predictive modeling of overloaded oligonucleotide elution profiles in ion-pair chromatography. Journal of Chromatography A, 1711, Article ID 464446.
Open this publication in new window or tab >>Strategies for predictive modeling of overloaded oligonucleotide elution profiles in ion-pair chromatography
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2023 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1711, article id 464446Article in journal (Refereed) Published
Abstract [en]

Due to their potential for gene regulation, oligonucleotides have moved into focus as one of the preferred modalities modulating currently undruggable disease-associated targets. In the course of synthesis and storage of oligonucleotides a significant number of compound-related impurities can be generated. Purification protocols and analytical methods have become crucial for the therapeutic application of any oligonucleotides, be they antisense oligonucleotides (ASOs), small interfering ribonucleic acids (siRNAs) or conjugates. Ion-pair chromatography is currently the standard method for separating and analyzing therapeutic oligonucleotides. Although mathematical modeling can improve the accuracy and efficiency of ion-pair chromatography, its application remains challenging. Simple models may not be suitable to treat advanced single molecules, while complex models are still inefficient for industrial oligonucleotide optimization processes. Therefore, fundamental research to improve the accuracy and simplicity of mathematical models in ion-pair chromatography is still a necessity. In this study, we predict overloaded concentration profiles of oligonucleotides in ion-pair chromatography and compare relatively simple and more advanced predictive models. The experimental system consists of a traditional C18 column using (dibutyl)amine as the ion-pair reagent and acetonitrile as organic modifier. The models were built and tested based on three crude 16-mer oligonucleotides with varying degrees of phosphorothioation, as well as their respective n – 1 and (P = O)1 impurities. In short, the proposed models were suitable to predict the overloaded concentration profiles for different slopes of the organic modifier gradient and column load. 

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Adsorption modeling, Inverse method, Ion-pair chromatography, Oligonucleotides, Overloaded profiles
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-97500 (URN)10.1016/j.chroma.2023.464446 (DOI)001096330000001 ()37865023 (PubMedID)2-s2.0-85174499047 (Scopus ID)
Funder
Knowledge Foundation, 20210021
Available from: 2023-11-24 Created: 2023-11-24 Last updated: 2023-11-28Bibliographically approved
Lesko, M., Kaczmarski, K., Fornstedt, T. & Samuelsson, J. (2022). A closer study of overloaded elution bands and their perturbation peaks in ion-pair chromatography. Journal of Chromatography A, 1682, Article ID 463491.
Open this publication in new window or tab >>A closer study of overloaded elution bands and their perturbation peaks in ion-pair chromatography
2022 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1682, article id 463491Article in journal (Refereed) Published
Abstract [en]

There is strong renewed interest in ion-pair chromatography (IPC) because of its great importance for separating new-generation biosimilar pharmaceuticals such as oligonucleotides. Due to the complexity of the IPC process, its mathematical modeling is challenging, especially in preparative mode. In a recent study, Leśko et al. (2021) developed a mathematical model for predicting, with good accuracy, overloaded concentration profiles for sodium benzenesulfonate, describing how the overloaded solute concentration profiles change from Langmuirian to complicated U-shaped, and then back again to Langmuirian profiles, with increasing concentration of the ion-pair reagent in the mobile phase. This study identifies and explains the underlying mechanism generating these complex peak shapes and band-shape transformations; this was only possible by visualizing and modeling the underlying equilibrium perturbations that occur upon injection in preparative IPC. In the 2021 study, the model was derived based on the concentration profiles obtained using a conventional UV detector principle, so the concentration gradients and perturbation zones of the mobile-phase components were not visualized. In this study, the necessary mechanistic information was obtained via complementary experiments combining two detection principles, i.e., refractive index detection and UV detection, with modeling efforts. The models correctly described the invisible equilibrium perturbations and how these formed internal gradients of the mobile-phase components. The models also explained the complex overloaded solute-band deformations reported in the recent study. In addition, a rule of thumb was developed for predicting experimental conditions that could result in deformed solute elution profiles and/or for avoiding these deformations. The latter is crucial for the practical chromatographer, since such U-shaped solute-band profiles are undesirable in preparative separation due to the broader elution zones, resulting in lower productivity than that of normal band shapes.

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
Ion chromatography; Ions; Oligonucleotides; Refractive index, Band shapes; Concentration profiles; Ion-pair chromatography; Mobile phasis; Overloaded profile; Peak deformation; Perturbation peak; Phase component; U-shaped; U-shaped profile, Deformation
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-92180 (URN)10.1016/j.chroma.2022.463491 (DOI)000864596900003 ()36122513 (PubMedID)2-s2.0-85138054480 (Scopus ID)
Funder
Knowledge Foundation, 20210021
Available from: 2022-10-12 Created: 2022-10-12 Last updated: 2022-10-28Bibliographically approved
Gutgsell, A. R., Gunnarsson, A., Forssén, P., Gordon, E., Fornstedt, T. & Geschwindner, S. (2022). Biosensor-Enabled Deconvolution of the Avidity-Induced Affinity Enhancement for the SARS-CoV-2 Spike Protein and ACE2 Interaction. Analytical Chemistry, 94(2), 1187-1194
Open this publication in new window or tab >>Biosensor-Enabled Deconvolution of the Avidity-Induced Affinity Enhancement for the SARS-CoV-2 Spike Protein and ACE2 Interaction
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2022 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 94, no 2, p. 1187-1194Article in journal (Refereed) Published
Abstract [en]

Avidity is an effective and frequent phenomenon employed by nature to achieve extremely high-affinity interactions. As more drug discovery efforts aim to disrupt protein-protein interactions, it is becoming increasingly common to encounter systems that utilize avidity effects and to study these systems using surface-based technologies, such as surface plasmon resonance (SPR) or biolayer interferometry. However, heterogeneity introduced from multivalent binding interactions complicates theanalysis of the resulting sensorgram. A frequently applied practice is to fit the data based on a 1:1 binding model, and if the fit does not describe the data adequately, then the experimental setup is changed to favor a 1:1 binding interaction. This reductionistic approach is informative but not always biologically relevant. Therefore, we aimed to develop an SPR-based assay that would reduce the heterogeneity to enable the determination of the kinetic rate constants for multivalent binding interactions using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human receptor angiotensin-converting enzyme 2 (ACE2) as a model system. We employed a combinatorial approach to generate a sensor surface that could distinguish between monovalent and multivalent interactions. Using advanced data analysis algorithms to analyze the resulting sensorgrams, we found that controlling the surface heterogeneity enabled the deconvolution of theavidity-induced affinity enhancement for the SARS-CoV-2 spike protein and ACE2 interaction.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022
National Category
Clinical Medicine
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-88057 (URN)10.1021/acs.analchem.1c04372 (DOI)000739329600001 ()2-s2.0-85122750876 (Scopus ID)
Funder
Swedish Research Council, 2015-04627
Available from: 2022-01-13 Created: 2022-01-13 Last updated: 2022-02-03Bibliographically approved
Enmark, M., Häggstrom, J., Samuelsson, J. & Fornstedt, T. (2022). Building machine-learning-based models for retention time and resolution predictions in ion pair chromatography of oligonucleotides. Journal of Chromatography A, 1671, Article ID 462999.
Open this publication in new window or tab >>Building machine-learning-based models for retention time and resolution predictions in ion pair chromatography of oligonucleotides
2022 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1671, article id 462999Article in journal (Refereed) Published
Abstract [en]

Support vector regression models are created and used to predict the retention times of oligonucleotides separated using gradient ion-pair chromatography with high accuracy. The experimental dataset consisted of fully phosphorothioated oligonucleotides. Two models were trained and validated using two pseudo orthogonal gradient modes and three gradient slopes. The results show that the spread in retention time differs between the two gradient modes, which indicated varying degree of sequence dependent separation. Peak widths from the experimental dataset were calculated and correlated with the guanine cytosine content and retention time of the sequence for each gradient slope. This data was used to predict the resolution of the n - 1 impurity among 250 0 0 0 random 12-and 16-mer sequences; showing one of the investigated gradient modes has a much higher probability of exceeding a resolution of 1.5, particularly for the 16-mer sequences. Sequences having a high guanine-cytosine content and a terminal C are more likely to not reach critical resolution. The trained SVR models can both be used to identify characteristics of different separation methods and to assist in the choice of method conditions, i.e. to optimize resolution for arbitrary sequences. The methodology presented in this study can be expected to be applicable to predict retention times of other oligonucleotide synthesis and degradation impurities if provided enough training data.

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
Machine-learning, Support vector regression (SVR) model, Oligonucleotides, Ion-pair chromatography, Resolution
National Category
Analytical Chemistry
Research subject
Chemistry - Analytical Chemistry
Identifiers
urn:nbn:se:kau:diva-89822 (URN)10.1016/j.chroma.2022.462999 (DOI)000790442600004 ()35381559 (PubMedID)2-s2.0-85127304100 (Scopus ID)
Available from: 2022-05-19 Created: 2022-05-19 Last updated: 2022-09-07Bibliographically approved
Liangsupree, T., Multia, E., Forssén, P., Fornstedt, T. & Riekkola, M.-L. (2022). Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor. Biosensors & bioelectronics, 206, Article ID 114151.
Open this publication in new window or tab >>Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor
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2022 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 206, article id 114151Article in journal (Refereed) Published
Abstract [en]

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
Adaptive interaction distribution algorithm; Exomere; Exosome; Extracellular vesicle; Kinetics; Quartz crystal microbalance
National Category
Biophysics
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-89651 (URN)10.1016/j.bios.2022.114151 (DOI)000782658200006 ()35259607 (PubMedID)2-s2.0-85125643757 (Scopus ID)
Funder
Academy of Finland, 1311369Swedish Research Council, 20015-04627Knowledge Foundation, 20210021
Available from: 2022-04-28 Created: 2022-04-28 Last updated: 2022-12-01Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7123-2066

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