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2021

Thanaporn Liangsupree, Evgen Multia, Marja-Liisa Riekkola

Journal of Chromatography A, Volume 1636, 2021

Abstract

Extracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation tech- niques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share sim- ilar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent de- velopment using an automated on-line system including selective affinity-based trapping unit and asym- metrical flow field flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma.

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2020

by Simon Staubach, Pete Gagnon, Katja Vrabec, Tjaša Lojpur, Sebastijan Peljhan, Bernd Giebel and Aleš Štrancar

BioProcess International, 2020

Abstract:

The traditional classification of extracellular vesicles (EVs) includes three types: exosomes, microvesicles, and apoptotic vesicles. Each type arises from a distinct origin and exhibits distinct characteristics. The problem is that their size ranges overlap and that the major surface proteins presented by exosomes also are present on the surfaces of microvesicles and apoptotic bodies. This makes it a challenge for process developers to identify the vesicle fraction that best serves a particular exosome therapy. Anion-exchange chromatography (AEC) can fractionate EVs into populations of different composition. This article highlights the complementarity of two analytical methods for characterizing distinctions among EV populations separated by AEC: imaging flow cytometry (IFCM) and size-exclusion chromatography.

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E. Multia, T. Liangsupree, M. Jussila, J. Ruiz-Jimenez, M. Kemell and M. Riekkola

Analytical Chemistry, 2020

Abstract:

An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography asymmetric flow field-flow fractionation (IAC-AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61-positive EVs and CD9-positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their sizebased subpopulations (e.g., exomeres and exosomes) for further analysis. Field-emission scanning electron microscopy elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in the ng/mL range using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size-based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37−80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84−6.6%.

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M. Morani, T.Duc Mai, Z. Krupova, P. Defrenaix, E. Multia, M. Riekkola, M. Taverna

Analytica Chimica Acta 1128 (2020) 45-51

Abstract

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles’ (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an ‘inorganic-species-free’ background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method method reached 8 × 10⁹ EVs/mL, whereas the calibration curve was acquired from 1.22 × 10¹⁰ to 1.20 × 10¹¹ EVs/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method.

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Pete Gagnon, Katja Vrabec, Tjaša Lojpur, and Aleš Štrancar

BioProcess International, 18 (4) April 2020

Abstract

Exosomes are a subject of rapidly growing therapeutic interest in the biopharmaceutical industry for two principal reasons. The first reason is that they are the primary communicators of instructions from source cells to target cells. Exosome surface features define their destination. They recognize complementary features on target cells, dock with them, and deliver their programmed instructions in the form of microRNA. The second reason is that exosomes are immunologically silent. As normal human cell products, and by contrast with gene therapy vectors such as virus particles, exosomes bypass the issue of triggering an immune response that might interfere with therapy.

Source cells include stem cells, which is why exosomes are of particular interest in the field of regenerative medicine. Recent research documenting the ability of exosomes to reverse the effects of severe strokes highlights their potential. It also underlines the need for scalable purification technology to advance these products through clinical trials and on to licensed manufacture. A platform approach was a major factor in the initial and continuing success of monoclonal antibodies. Exosomes likewise represent an extended family of individual products with similar properties. It stands to reason that a platform approach will prove equally valuable for exosomes. In this article we describe initial efforts toward that goal.

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2019

Calef Sánchez-Trasviña, Marco Rito-Palomares, and José González-Valdez

Advances in Polymer Technology, Volume 2019, December 12 2019, 10 pages

Abstract

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.

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Evgen Multia, Crystal Jing Ying Tear, Mari Palviainen, Pia Siljander, Marja-Liisa Riekkola

Analytica Chimica Acta (2019).
Published online 2019 Sep 11.

A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM®) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic light-scattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 min. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures.

Keywords: Immunoaffinity chromatography, Isolation, Monolithic disk column, Extracellular vesicles, Platelet-derived vesicles, CD61

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This discussion introduces new analytical approaches that enable in-line chromatographic detection of exosomes. One approach can discriminate extracellular vesicles from nonvesicle contaminants, and one potentially can discriminate exosomes from other vesicles. Examples illustrate how they enable development of more effective and better documented purification methods. The special qualifications of monolithic chromatography media for exosome purification are discussed. New process tools designed to accommodate some of the special challenges of exosome purification are introduced.

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2014

F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut

Protein Expression and Purification 95 (2014) 104–112

The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

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F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut
Protein Expression and Purification 95 (2014) 104–112

The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

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2013

M. Bartolini, I. W. Wainer, C. Bertucci, V. Andrisano

Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 77-81

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2 mm × 6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.

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F. Ibrahim, C. Andre, R. Aljhni, T. Gharbi, Y. C. Guillaume

Journal of Molecular Catalysis B: Enzymatic 94 (2013) 136-140

Acetylcholinesterase (AChE) is a serine protease that hydrolyzes the neurotransmitter acetylcholine. Here, the effects of hydroxyl radical (OH•) and nitric oxide (NO) on AChE activity were studied using a biochromatographic process. The enzyme was immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk. The AChE enzymatic mechanism was demonstrated from the chromatographic peak shape. A decrease in AChE activity was observed for each concentration of NO, while OH• dot radical formation led to an increase in the rate of enzymatic catalysis. Michaelis–Menten and Lineweaver–Burk plots were obtained in the presence or absence of the free radicals and their effects on Km and Vmax were evaluated. Our results indicated classical deactivation/activation kinetics without significant influence on the rate of substrate binding. The variation in transition state energies (ΔΔGES) induced by the free radicals indicated that a conformational change was occurring in the active site, while changes in the binding site were negligible. These results clearly demonstrate the direct role of OH• dot and NO on AChE activity and confirm the role they may play in Alzheimer's disease.

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H. G. Schwelberger, J. Feurle, F. Ahrens

Journal of Neural Transmission 120 (2013) 983-986

Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.

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E. A. Ponomareva, M. V. Volokitina, D. O. Vinokhodov, E. G. Vlakh, T. B. Tennikova

Anal Bioanal Chem (2013) 405:2195–2206

Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous
methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined.

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M. V. Volokitina, E. G. Vlakh, G. A. Platonova, D. O. Vinokhodov, T. B. Tennikova
J. Sep. Sci. 2013, 36, 2793-2805

Two ribonuclease A bioreactors based on lab-made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free-radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible-chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion-exchange HPLC on an ultra-short monolithic column (disk) using off- and on-line analytical approaches.

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2012

J. Subotič, K. Koruza, B. Gabor, M. Peterka, M. Barut, J. Kos, J. Brzin

Affinity Chromatography, Dr. Sameh Magdeldin (Ed.), ISBN: 978-953-51-0325-7, InTech

Proteolytic enzymes (also known as proteases, proteinases or peptidases) offer a wide range of applications. They are routinely used in detergent, leather, food and pharmaceutical industries, as well as in medical and basic research. Therefore, effective isolation procedures are of great importance. The chapter describes the use of recently discovered protease inhibitors from basidiomycetes as affinity chromatography ligands for isolating proteases. Affinity columns with serine and cysteine protease inhibitors immobilized to the natural polymer Sepharose have been prepared, the chromatography procedure optimized and used for isolating proteases from various bacterial, plant and animal sources. The cysteine protease inhibitor macrocypin showed superior characteristics as a ligand, so was selected for immobilization to CIM (Convective Interaction Media) monolithic disks. Different immobilization chemistries and process conditions were optimized to determine the best conditions for high capacity and selectivity. A very effective method for isolating cysteine proteases was developed using affinity chromatography with the fungal cysteine protease inhibitor macrocypin immobilized to a CIM monolithic disk.

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2010

F. Mancini, V. Andrisano

Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 355-361

A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant β-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1–IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the β-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1–IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt = 1.6 min), which was separated from the substrate and selectively detected at λexc = 320 and λem = 420 nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1–IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA.

The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (−)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1–IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1–IMER showed high stability during the analysis, permitting its use for more than three months without affecting enzyme activity.

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E.A. Ponomareva, V.E. Kartuzova, E.G. Vlakh, T.B. Tennikova

Journal of Chromatography B, 878 (2010) 567–574

The effect of different modes of α-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA–EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.

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2009

C. Delattre, M. A. Vijayalakshmi

Journal of Molecular Catalysis B: Enzymatic 60 (2009) 97–105

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.

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2008

C. Delattre, P. Michaud, M. A. Vijayalakshmi

Journal of Chromatography B, 861 (2008) 203–208

Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.

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