Publications
2021
Urh Černigoj, Jana Vidič, Ana Ferjančič, Urša Sinur, Klemen Božič, Nina Mencin, Anže Martinčič Celjar, Pete Gagnon, Aleš Štrancar
Electrophoresis, September 2021. https://doi.org/10.1002/elps.202100210
Abstract:
Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl, DEAE) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively-Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively-Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the DEAE-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than capillary electrophoresis, and resolution approaching capillary electrophoresis.
Rafael G. Ferreira, Neal F. Gordon, Rick Stock, Demetri Petrides
Processes 2021, 9(8), 1430
Abstract:
The COVID-19 pandemic has motivated the rapid development of numerous vaccines that have proven effective against SARS-CoV-2. Several of these successful vaccines are based on the adenoviral vector platform. The mass manufacturing of these vaccines poses great challenges, especially in the context of a pandemic where extremely large quantities must be produced quickly at an affordable cost. In this work, two baseline processes for the production of a COVID-19 adenoviral vector vaccine, B1 and P1, were designed, simulated and economically evaluated with the aid of the software SuperPro Designer.
In the purification process the retentate from the ultrafiltration step was subjected to anion-exchange chromatography (AEX), operating in capture mode, to remove protein and DNA impurities. A strong anion-exchange column with a monolithic structure (CIMmultus QA from Sartorius BIA Separations) is used in this step due to its high binding capacity for VPs.
Both processes were sized to produce 400 M/yr vaccine doses. The media and facility-dependent expenses were found to be the main contributors to the operating cost. The results indicate that adenoviral vector vaccines can be practically manufactured at large scale and low cost.
Pete Gagnon, Maja Leskovec, Sara Drmota Prebil, Rok Žigon, Maja Štokelj, Andrej Raspor, Sebastijan Peljhan, Ales Štrancar
Journal of Chromatography A, 2021
Abstract:
Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity ligand. A subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty capsids and debris. Empty and full capsid composition of chromatography fractions was evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with respect to capsid separation. Charging with cupric ions maintained resolution between early-eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete elimination of empty capsids. All metal ions bound contaminant subsets that required sodium hydroxide for removal. Columns charged with ferric iron and manganese bound more contaminants than all other metals. Columns charged with calcium, magnesium, barium, and copper bound the least. Contaminant binding on zinc-charged columns was intermediate between the two groups.
Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li, Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar, April Schubert, Akunna Iheanacho, and David Cetlin
BioProcess International, April 2021
Abstract
Over the past decade, adenoassociated virus (AAV) vectors have become established as leading gene-delivery vehicles. In 2017, the pipeline for gene therapies included 351 drugs in clinical trials and 316 in preclinical development. As those candidates advance, significant efforts are being made in process development and manufacturing for viral vectors, with the overall goal of reducing process impurities while maintaining the highest possible process yield.
Sartorius BIA Separations has developed and commercialized CIMmultus QA monoliths, which have been cited in several AAV downstream processes for their ability to separate empty and full virus particles effectively. Monolithic supports represent a unique type of stationary phase for liquid chromatography, bioconversion, and solid-phase synthesis. Aside from increased processing speed, monolithic flow-through pores (channels) also provide easy access for large molecules, which supports both purification and depletion of nanoparticles such as plasmid DNA (pDNA) molecules and AAV particles.
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
Sebastijan Peljhan, Maja Štokelj, Sara Drmota Prebil, Pete Gagnon and Aleš Štrancar
Cell & Gene Therapy Insights, March 2021
Abstract:
Ultracentrifugation (UC) is a well-known technique for fractionating adeno-associated virus (AAV) capsids according to their density, which is mainly a function of their encapsidated DNA mass. Empty capsids represent the lowest density subpopulation. Full capsids represent the highest density subpopulation, sometimes accompanied by partially full capsids of intermediate density. Fractions can be collected after sedimentation for analysis but the practice is laborious and discourages application of multiple monitoring techniques that might provide deeper insights into sample composition. Anion exchange chromatography (AEC) also achieves fractionation of empty and full capsids for many AAV serotypes. The degree of separation varies among serotypes and does not correlate strictly with UC. This is not surprising since separation by AEC is highly influenced by capsid surface charge, which is independent of the amount of DNA packaged within the capsids. Chromatography methods however present a significant analytical advantage in the ease of monitoring the column effluent, including with multiple detectors. UV absorbance at 260 nm and 280 nm permits estimation of empty and full capsid proportions in any given peak. Intrinsic fluorescence enables estimation of relative areas of empty capsid peaks and full capsid peaks. Light scattering does the same and permits the further determination of capsid size and mass. In this report, we merge UC with an HPLC monitoring array to simultaneously analyze dual wavelength UV, intrinsic fluorescence, and light scattering through cesium chloride density gradient strata. Limitations of each monitoring method are discussed. UC results are compared with chromatography profiles to highlight distinction between separation methods. Practical application of results for final product characterization is considered, along with potential to support development of better purification processes.
Attachments
Pete Gagnon, Maja Leskovec, Blaz Goricar, and Aleš Štrancar
BPI, December 17, 2020
Abstract:
With its first licensed therapeutic now marketed worldwide, adeno-associated virus (AAV) has become a preferred vector for gene therapy. However, unlocking its full potential still poses challenges, many of which are associated with purification. The first involves the transition from upstream to downstream processes. AAV-bearing lysates are laden with debris that foul filtration media and limit or prevent concentration. Another challenge involves reduction of soluble host-cell DNA, which is complicated by its strong association with nucleoproteins. A third involves elimination of empty capsids. Currently, ultracentrifugation meets that need, but scale-up issues make chromatographic alternatives attractive. A fourth challenge involves the need for rapid, accurate, and revealing analytical results to guide process development, support validation, document control, and enable reproducibility of manufacturing processes. The following article shares experimental data showing how those challenges can be addressed to advance the evolution of gene therapy with AAV.
Attachments
Petrović T, Alves I, Bugada D, Pascual J, Vučković F, Skelin A, Gaifem J, Villar-Garcia J, Vicente MM, Fernandes Â, Dias AM, Kurolt IC, Markotić A, Primorac D, Soares A, Malheiro L, Trbojević-Akmačić I, Abreu M, Sarmento E Castro R, Bettinelli S, Callegaro A, Arosio M, Sangiorgio L, Lorini LF, Castells X, Horcajada JP, Pinho SS, Allegri M, Barrios C, Lauc G.
Glycobiology. 2020 Nov.
Abstract:
A large variation in the severity of disease symptoms is one of the key open questions in COVID-19 pandemics. The fact that only a small subset of people infected with SARS-CoV-2 develop severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the IgG glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of inter-individual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting Nacetylglucosamine (GlcNAc) in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. To our knowledge, this is the first study exploring IgG N-glycome variability in COVID-19 severity.
Pete Gagnon, Blaz Goricar, Nina Mencin, Timotej Zvanut, Sebastijan Peljhan, Maja Lescovec and Ales Strancar
Pharmaceutics. 2021 Jan 17;13(1):113
Abstract:
HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.
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.
by Maribel Rios, Aleš Štrancar, J. Michael Hatfield and Pete Gagnon
BioProcess International, 2020
Abstract:
Adenoassociated viral (AAV) vectors have become synonymous with gene therapy delivery. However, because they are produced in such small quantities and because their upstream processes carry comparatively large amounts of host-cell DNA and other impurities, AAV purification can be challenging. Several researchers have applied different chromatographic strategies, but no universal method has been adopted in the biopharmaceutical industry.
This eBook features a discussion among several industry experts that explores challenges specific to AAV purification, shedding light on whether current strategies and separation technologies are up to the task. The conversation traverses issues relating to material handling at the upstream–downstream interface, removal of host-cell DNA, chromatographic separation of empty and full capsids, and a lack of fast and robust in-process analytics for downstream processes. Participants also explore whether the rise of AAV-based treatments will require downstream scientists to shift away from the antibody-centered conceptions of chromatography that have grown alongside the biotherapeutics industry.
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
U. Černigoj, A. Štrancar
DNA Vaccines. Methods in Molecular Biology, vol 2197, pp 167-192
Abstract
Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.
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.
Wang Chunlei, Mulagapati Sri Hari Raju, Chen Zhongying, Du Jing, Zhao Xiaohui, Xi Guoling, Chen Liyan, Linke Thomas, Gao Cuihua, Schmelzer Albert, Liu Dengfeng
Molecular Therapy Methods & Clinical Development, Volume 15, September 26 2019, Pages 257-263
Abstract
Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.
Keywords: AAV, AAV6.2, Chromatography, Anion exchange chromatography (AEC), Empty capsids, AUC, High-throughput
Katarina Marković, Radmila Milačič, Janja Vidmar, Stefan Marković, Katja Uršič, Martina Nikšić Žakeljc, Maja Cemazar, Gregor Sersa, Mojca Unk, Janez Ščančar
Journal of Trace Elements in Medicine and Biology, Volume 57, January 2020, Pages 28-39.
Abstract
Monolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run. Here, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. The data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. Low-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.
Keywords: low-pressure and high-pressure conjoint liquid chromatography, anion-exchange and affinity monolithic disks, inductively coupled plasma mass spectrometry, Pt-based chemotherapeutics, serum of cancer patients
Sofiya Fedosyuk, Thomas Merritt, Marco Polo Peralta-Alvarez, Susan J. Morris, Ada Lam, Nicolas Laroudie, Anilkumar Kangokar, Daniel Wright, George M. Warimwe, Phillip Angell-Manning, Adam J. Ritchie, Sarah C. Gilbert, Alex Xenopoulos, Anissa Boumlic, Alexander D. Douglas
Vaccine (2019).
Published online 30 April 2019.
A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.
Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype.
Keywords: Simian adenovirus, GMP, Clinical trials, Single-use, Biomanufacturing, Bioreactor, Purification
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