Publications
2024
Marta Leban, Tina Vodopivec Seravalli, Martina Hauer, Ernst Böhm, Nina Mencin, Sandra Potušek, Andrej Thompson, Jurij Trontelj, Aleš Štrancar & Rok Sekirnik
Analytical and Bioanalytical Chemistry, March 2024
The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.
Klemen Božič, Ajda Sedlar, Špela Kralj, Urh Černigoj, Aleš Štrancar, Rok Sekirnik
Biotechnology and Bioengineering, 2024, 1–11
High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.
2023
Ana Ferjančič Budihna, Anže Martinčič Celjar, Sergeja Lebar, Andreja Gramc Livk and Aleš Štrancar
Cell & Gene Therapy Insights 2023; 9(9), 1231–1247 | DOI: 10.18609/cgti.2023.161
Rapid advancement of mRNA technology, as a response to the COVID-19 crisis, prompted an increased need for precise analytical methods to support the fast-paced mRNA process development. Accurate and robust analytics are required to support modifications in the mRNA production process, protocols, raw materials, in vitro transcription reaction, purification methods, scale-up, or final formulation processes, to ensure high quality and safety of the final product. This Innovator Insight demonstrates the application of an ion pair reverse phase chromatographic analytical method as a robust analytical tool to determine mRNA fragmentation while also separating in vitro transcription components from the main product. The method’s efficacy is assessed through a comprehensive stability study of a mRNA standard at different temperatures. The chromatographic analytical results are compared to the ones obtained by the capillary gel electrophoresis, a well-established method for the analysis of fragmented mRNA.
Špela Kralj, Špela Meta Kodermac, Ines Bergoč, Tomas Kostelec, Aleš Podgornik, Aleš Štrancar, Urh Černigoj
Electrophoresis. 2023; 1– 13
Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16 kbp in size.
Nejc Pavlin, Urh Černigoj, Mojca Bavčar, Tjaša Plesničar, Jan Mavri, Martin Zidar, Matevž Bone, Urška Kralj Savič, Tadej Sever, and Aleš Štrancar
Electrophoresis. 2023; 1– 11
High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.
Lucija Rebula, Andrej Raspor, Mojca Bavčar, Aleš Štrancar and Maja Leskovec
Journal of Chromatography B, Volume 1217, 15 February 2023
Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.
In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.
2022
- Can IVT yields be increased beyond 5-8 g/L?
- Does feeding nucleotides into the IVT reaction increase its yield?
- Is there a fast analytical method to quantify NTPs in IVT in real-time?
- Can production of mRNA be automated?
Transitioning from batch to fed-batch IVT can increase IVT yield to 12 g/L resulting in 50 % reduction in cost per gram of mRNA. Integrating HPLC monitoring of IVT reaction can allow real-time decisions on feed additions.
Janja Skok, Polona Megušar, Tina Vodopivec, Domen Pregeljc, Nina Mencin, Matevž Korenč, Andreja Krušič, Anže Martinčič Celjar, Nejc Pavlin, Jana Krušič, Matthias Mueller, Kevin McHugh, Aleš Štrancar, and Rok Sekirnik
Chemie Ingenieur Techik, October 2022
The COVID-19 pandemic triggered an unprecedented surge in development of mRNA-based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single-use bioreactors are traditionally used for cell-based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell-free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed-batch IVT approach, and demonstrate the feasibility of continuous fed-batch production, paving the way towards continuous manufacturing of mRNA.
Ana Ferjančič Budihna, Nejc Pavlin, Anže Martinčič Celjar, Andreja Gramc Livk and Aleš Štrancar
BioProcess International eBook, September 14, 2022
Robust and precise chromatographic analytical methods are key for the efficient development of the mRNA production process.
Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ analytical platform to support mRNA process development and product quantification and characterization.
2021
by Nejc Pavlin, Blaž Bakalar, Janja Skok, Špela Kralj, Andreja Gramc Livk, and Aleš Štrancar
BioProcess International, October 2021
Abstract:
Plasmid DNA (pDNA) has become a crucial component in the production of next generation therapeutics such as messenger RNA (mRNA) and viral vectors.
As companies ramp up their production capabilities and move towards clinical applications, obtaining cGMP grade pDNA has become a production bottleneck, leading to lengthy production delays.
There is a growing market demand for solutions that can streamline the production of cGMP pDNA and help optimize down-stream processes (DSP) for better yields & purity.
The key step in this process is having quantifiably reliable analytics that give rapid results
for process optimization and scale-up, as well as production runs.
Establishing and expanding inhouse pDNA production platform in a quick and efficient manner will be a key differentiator between more and less successful next generation therapeutics projects.
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.
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
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
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
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.
Feel free to download the eBook by clicking on the link to attachment below.
Attachments
2018
Miladys Limonta, Lourdes Zumalacarregui, Urska Vidic, Nika Lendero Krajnc
The main component of the Center for Genetic Engineering and Biotechnology (CIGB) candidate vaccine against Hepatitis C virus (HCV) is the pIDKE2 plasmid. The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharamceutical-grade plasmid DNA (pDNA)with 95% purity. The advantages of this procedure include high plasmid purity and the elimination of undesirable additives. such as toxic organic extractants and animal-derived enzymes. However, yields and consequently the productivity of the process are low. Previous work demonstrated that the most critical step of the process is the reverse phase chromatography, where conventional porous particle resins are used. Therefore, to increase the process productivity alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step. Here, a comparison between the behaviours of CIM® C4-HLD and Sartobind phenyl matrices was performed.