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.
Tomas Kostelec, Rok Sekirnik, Anže Martinčič Celjar, Kristina Šprinzar Nemec, Andreja Gramc Livk, Pete Gagnon, Aleš Štrancar
BioProcess International, June 2021
Abstract:
COVID-19 has focused a spotlight on the ability of mRNA technology to accelerate vaccine development and approval. That same technology can hasten development and approval of other therapeutic classes, including cancer immunotherapy, protein replacement, and gene therapy. Fulfilling those opportunities imposes significant challenges on process developers and manufacturers to improve existing processes. Scale-up to produce millions of doses (tens of kilograms) compounds those challenges. Furthermore, every step of the journey requires high-performance analytical methods, to ensure patient safety and maximize productivity.
Artaches A. Kazarian, Wesley Barnhart, Iain D.G. Campuzano, Jeremy Cabrera, Theodore Fitch, Jason Long, Kelvin Sham, Bin Wu, Justin K. Murray
Journal of Chromatography A,Volume 1634, 2020
Abstract:
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single stranded oligonucleotide in high purity (92%) under denaturing conditions.
The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
2020
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.
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.
Hietala V, Horsma-Heikkinen J, Carron A, Skurnik M, Kiljunen S.
Frontiers in microbiology vol. 10 1674. 23 Jul. 2019
Abstract
The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiM_fHyAci03, and Staphylococcus phage vB_SauM_fRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_fHoEco02 lysate from 3.5 × 104 Endotoxin Units (EU)/109 plaque forming units (PFU) to 0.09 EU/109 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/109 PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/109 PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.
Keywords: antibiotic resistance, bacteriophage, phage therapy, endotoxin, enterotoxin
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.
Attachments
2015
A.M. Almeida, J.A. Queiroz, F. Sousa, A. Sousa
Journal of Chromatography B, 978–979 (2015) 145–150
The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.
J-P Pirnay et al.
Pharm Res, Springer, 14 Jan 2015
The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge.
To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies.
This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.
Zunyang Ke, Yu Wang and Zhongming Li
Anion-exchange chromatography is a key capture step in downstream processing plasmid DNA (pDNA). Separation of pDNA using traditional particle-based anion-exchange supports is usually slow and has a low capacity for pDNA due to steric exclusion effects. Due to convective mass transfer properties, and large flow-through channels for binding large biomolecules, monoliths have been shown to provide a fast and efficient alternative for pDNA purification. This study describes the use of monoliths for purification of a therapeutic pDNA vaccine against multidrug resistant tuberculosis (MDR TB).
Attachments
Urh Černigoj, Urška Martinuč, Sara Cardoso, Rok Sekirnik, Nika Lendero Krajnc, Aleš Štrancar
Sample displacement chromatography (SDC) is a chromatographic technique that utilises different rela-tive binding affinities of components in a sample mixture and has been widely studied in the context ofpeptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoformsunder overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) orlinear isoform. Since displacement is more efficient when mass transfer between stationary and mobilechromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM)monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobic-ities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) weretested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoformseparation was shown to be dependent on column selectivity for individual isoform, column efficiencyand on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative modeelution often operate in parallel, therefore negative mode elution additionally influences the efficiencyof the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNAhomogeneity and a dynamic binding capacity of over 1 mg/mL at a relatively low concentration of AS.SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes,and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used.This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, whichis compatible with continuous, multicolumn chromatography systems, and could therefore be used toincrease productivity of pDNA production in the future.
Attachments
2013
E. Mota, A. Sousa, U. Černigoj, J. A. Queiroz, C. T. Tomaz, F. Sousa
Journal of Chromatography A (2013)
The demand for high-purity supercoiled plasmid DNA to be applied as a vector for new therapeutic strategies, such as gene therapy or DNA vaccination has increased in the last years. Thus, it is necessary to implement an analytical technique suitable to control the quality of the supercoiled plasmid as a pharmaceutical product during the manufacturing process. The present study describes a new methodology to quantify and monitor the purity of supercoiled plasmid DNA by using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows the separation of the plasmid isoforms by combining a NaCl stepwise gradient. The specificity, linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a supercoiled plasmid concentration up to 200 μg/mL. The main advantage achieved by using this monolithic column is the possibility to quantify the supercoiled plasmid in a sample containing other plasmid topologies, in a 4 min experiment. This column also permits the assessment of the supercoiled plasmid DNA present in more complex samples, allowing to control its quality throughout the bioprocess. Therefore, these findings strengthen the possibility of using this monolithic column associated with a powerful analytical method to control the process development of supercoiled plasmid DNA production and purification for therapeutic applications.
S. Haberl, M. Jarc, A. Štrancar, M. Peterka, D. Hodžić, D. Miklavčič
J Membrane Biol, DOI 10.1007/s00232-013-9580-5
The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.
B. Gabor, U. Černigoj, M. Barut, A. Štrancar
Journal of Chromatography A, 1311 (2013) 106-114
HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0 kbp, 5.2 kbp and 14.0 kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport.
M. Limonta, N. Lendero Krajnc, U. Vidic, L. Zumalacárregui
Biochemical Engineering Journal 80 (2013) 14-18
The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2. The designed downstream process of pIDKE2 plasmid produces up to 179 g/year. In order to conduct further improvements, modelling of the downstream process was performed. A methodology based on process analysis tools, such as experimental design and modelling was established to identify factors with the highest influence on production cost and the amount of annual plasmid. Taking into account that the pIDKE2 downstream process designed is in its initial stages of development, CIM technology was evaluated as a new manufacturing process on lab scale. Purity and recovery of CIM technology was better than porous particle matrix, thus SuperPro Designer was used in order to simulate the purification process. Cost efficiency optimization of the pIDKE2 downstream process was done with the simulation model.
A. Ghanem, R. Healey, F. G. Adly
Analytica Chimica Acta 760 (2013) 1-15
Abstract
Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cellmediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.
A. Romanovskaya, L. P. Sarina, D. H. Bamford, M. M. Poranen
Journal of Chromatography A (2013)
Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58–1810 bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25–27 bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved.
2012
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.
J. Ruščić, I. Gutierrez-Aguirre, L. Urbas, P. Kramberger, N. Mehle, D. Škorić, M. Barut, M. Ravnikar, M. Krajačić
Journal of Chromatography A, 1274 (2013) 129-136
Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only 356–360 nucleotides and no coding capacity. Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using Convective Interaction Media (CIM) monolithic columns. The ion-exchange chromatography on diethylamine (DEAE) monolithic analytical column (CIMac DEAE-0.1 mL) resulted in up to 30% PSTVd recovery whilst the hydrophobic interaction chromatography on C4 monolithic analytical column (CIMac C4-0.1 mL) improved it up to 60%. This was due to the fact that the binding of the viroid to the C4 matrix was less strong than to the highly charged anion-exchange matrix and could be easier and more completely eluted under the applied chromatographic conditions. Based on these preliminary results, a C4 HLD-1 (High Ligand Density) 1 mL monolithic tube column was selected for further experiments. One-litre-water samples were mixed with different viroid quantities and loaded onto the column. By using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the viroid RNA was quantified in the elution fraction (≈5 mL) indicating that 70% of the viroid was recovered and concentrated by at least two orders of magnitude. This approach will be helpful in screening irrigation waters and/or hydroponic systems’ nutrient solutions for the presence of even extremely low concentrations of PSTVd.
2011
M. J. Shin, L. Tan, M. H. Jeong, J.-H. Kim, W.-S. Choe
Journal of Chromatography A, 1218 (2011) 5273-5278
Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.