Publications
2020
P. Gagnon, B. Goričar, Š. Peršič, U. Černigoj, A. Štrancar
Cell & Gene Therapy Insights 2020; 6(7), 1035–1046
Abstract:
One of the barriers to development of industrial purification platforms for large mRNA has been an inadequate selection of high-performing capture-purification tools. Hybridization-affinity uses a polythymidine (Oligo dT) ligand to base-pair with the polyadenine tail of mRNA. It can be used for capture but it cannot discriminate dsRNA (double-stranded) from ssRNA (single-stranded) and it supports only brief cleaning with 100 mM sodium hydroxide. Traditional anion exchangers elute only mRNA smaller than about 500 bases unless the columns are heated to 50–70°C. Hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) separate ssRNA from dsRNA and short transcripts, but their sensitivity to fouling by proteins and aggregates makes them better suited for polishing than for capture. Better capture options are needed to meet the needs of large clinical trials, scale-up, and manufacture of vaccines. Beyond that, a new spectrum of gene therapy treatments await. This article introduces two new capture options that both eliminate dsRNA, DNA, and proteins in a wash step, then provide high-resolution polishing of ssRNA in an elution gradient at ambient temperature. One represents a new class of anion exchangers. The other exploits hydrogen bonding. Both support prolonged exposure to 1 M sodium hydroxide. Easy transition to either HIC or RPC provides high-resolution orthogonal polishing.
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
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.
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
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.
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.
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.
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.
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.
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.
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.
E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146
Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.
The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.
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
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.
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.
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.