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.
Feel free to download the eBook by clicking on the link to attachment below.
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.
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.
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.
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.
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.
P. Svete, R. Milačič, B. Mitrović, B. Pihlar
The Royal Society of Chemistry 2001, Analyst, 2001, 126, 1346–1354
Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 µg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100 : 1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm-3 NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min−1. The separated Zn species were determined in 1.0 cm3 eluate fractions “off line” by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm-3 NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min−1 and determination of the separated Zn species in 1.0 cm3 eluate fractions “off line” by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn–citrate was separated from both Zn–oxalate and from Zn–EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2–4%), tested for six parallel determinations (2 µg cm-3 Zn) of Zn–EDTA, Zn–citrate and Zn–oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm-3. The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.