Publications
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.
Attachments
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.
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%.
Attachments
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.
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.
2019
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
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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Attachments
2013
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.
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.
P. Fagan, C. Wijesundera
Journal of Separation Science, 10.1002/jssc.201201156
Eicosapentaenoic and docosahexaenoic acids are important bio-active fatty acids in fish oils. Monolithic HPLC columns both in the polymeric cation exchange (silver-ion) and RP formats were compared with corresponding packed columns for the isolation of these acids from tuna oil ethyl esters. Monolithic columns in both formats enabled rapid (typically 5–10 min) separations compared with packed columns (30 min). Polymeric monolithic silver-ion disc column rapidly furnished mixtures of eicosapentaenoic and docosahexaenoic esters (90% purity) within 5–10 min, but was unable to resolve individual esters. A preparative version of the same column (80 mL bed volume) enabled isolation (>88% purity) of 100 mg quantities of eicosapentaenoic and docosahexaenoic esters from esterified tuna oil within 6 min. Baseline separation of eicosapentaenoic and docosahexaenoic esters was achieved on all RP columns. The results show that there is potential to use polymeric monolithic cation exchange columns for scaled-up preparation of eicosapentaenoic and docosahexaenoic ester concentrates from fish oils.
J. A. Martin, P. Parekh, Y. Kim, T. E. Morey, K. Sefah, N. Gravenstein, D. M. Dennis, W. Tan
PLOS ONE, March 2013, Volume 8, Issue 3, e57341
Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.
2011
E. F. Maksimova, E. G. Vlakh, T. B. Tennikova
Journal of Chromatography A, 1218 (2011) 2425-2431
A series of macroporous monolithic methacrylate-based materials was synthesized by in situ free radical UV-initiated copolymerization of functional monomers, such as glycidyl methacrylate (GMA), butyl methacrylate (BuMA), 2-aminoethyl methacrylate (AEMA), 2-hydroxyethyl methacrylate (HEMA) and 2-cyanoethyl methacrylate (CEMA), with crosslinking agent, namely, ethylene glycol dimethacrylate (EDMA). The materials obtained were applied as the stationary phases in simple and robust technique – planar chromatography (PLC). The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized. The GMA–EDMA and BuMA–EDMA matrixes were successfully applied for the separation of low molecular weight compounds (the mixture of several dies), as well as poly(vinylpyrrolidone) and polystyrene homopolymers of different molecular weights using reversed-phase mechanism. The materials based on copolymers AEMA–HEMA–EDMA and CEMA–HEMA–EDMA were used for normal-phase PLC separation of 2,4-dinitrophenyl amino acids and polystyrene standards.
A. Trauner, M. H. Bennett, H. D. Williams
PLoS ONE 6(2): e16273. doi:10.1371/ journal.pone.0016273
We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.
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.
A. Čevdek, M. Franko
Analytical and Bioanalytical Chemistry 398 (2010), 555-562
This work presents a comparison of convective interaction media (CIM) and controlled pore glass (CPG) as solid supports for immunoglobulin antibodies used in bioanalytical detection of allergens in foodstuffs. A flow-injection manifold with highly sensitive thermal lens spectrometric detection was used for this purpose. Using beta-lactoglobulin, a milk allergen, as a model analyte, CIM disc supports had a higher linear range (0.2–3.5 μg L-1), better reproducibility (intra-day RSD = 1%, inter-day RSD = 10%), lower consumption of reagents, and better immunocolumn stability (1 month, over 240 injections of substrate), while providing comparable LODs (0.1 μg L-1). Application of CIM discs as solid supports in immunocolumns for allergen detection enables fast and sensitive screening of allergens in foodstuffs with sample throughput of up to eight samples per hour.
2007
E. Müller, C. Mann
Journal of Chromatography A, 1144 (2007) 30-39(2007) 30-39
The electro-acoustic effects, namely the ion vibration potential (IVP) and the colloidal vibration current (CVI), colloidal vibration potential (CVP) first described by P. Debye [P. Debye, J. Chem. Phys. 1 (1933) 13], are a result of charge separation of bound or free ions at different degrees by ultrasonic waves. Today commercial instruments are available to investigate liquid homogeneous and heterogeneous systems. In the present paper the application of this technique for the characterization of salts, protein solutions and resins for biochromatography is shown and valuable information about resins can be derived in a short time. Various resins were investigated with the following results: (1) the CVI magnitude is dependent of several parameters (such as particle size distribution, volume fraction, density difference); (2) the CVI is influenced by the surface modification of the resins. Polymeric modifications decrease the value of CVI. The CVI is generally lower for high capacity resins; (3) the measurement of the electro-acoustic effects can be used to detect small changes in resins. The CVI is dependent of the amount of adsorbed protein in “native” and denatured state.
M. Benčina, J. Babič, A. Podgornik
Journal of Chromatography A, 1144 (2007) 135–142
In gene therapy and DNA vaccination, RNA removal from DNA preparations is vital and is typically achieved by the addition of ribonuclease into the sample. Removal of ribonuclease from DNA samples requires an additional purification step. An alternative is the implementation of immobilized ribonuclease. In our work, ribonuclease was covalently coupled onto the surface of methacrylate monoliths via epoxy or imidazole carbamate groups. Various immobilization conditions were tested by changing immobilization pH. Ribonuclease immobilized on the monolith via imidazole carbamate groups at pH 9 was found to be six times more active than the ribonuclease immobilized on the monolith via epoxy groups. Under optimal immobilization conditions the Michaelis–Menten constant, Km, for cytidine-2,3-cyclic monophosphate, and turnover number, k3 were 0.52 mM and 4.6 s-1, respectively, and mirrored properties of free enzyme. Enzyme reactor was found to efficiently eliminate RNA contaminants from DNA samples. It was active for several weeks of operation and processed 300 column volumes of sample. Required residence time to eliminate RNA was estimated to be around 0.5 min enabling flow rates above 1 column volume per min.
2005
M. Benčina, K. Benčina, A. Štrancar, A. Podgornik
Journal of Chromatography A, 1065 (2005) 83–91(2005) 83–91
A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis–Menten constant, Kmapp, and turnover number, k3app, for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l-1 and 16 dA260nm min-1 mg-1 of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 °C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.
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2004
P. N. Nesterenko, M. A. Rybalko
Mendeleev Commun. 2004
The continuous flow gradient and its effect on chromatographic parameters were investigated for the separations of inorganic anions on a monolithic porous disk with bonded hydroxyproline residues.
2003
E. G. Vlakh, G. A. Platonova, G. P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T. B. Tennikova
Journal of Chromatography A, 992 (2003) 109–119
The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.