Publications
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
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.
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.
2010
F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Raspor, A. Štrancar, M. Peterka
Vaccine 28 (2010) 2039–2045
Plasmid DNA (pDNA) used in vaccination and gene therapy has to be highly pure and homogenous, which point out necessity to develop efficient, reproducible and scalable downstream process. Convective Interaction Media (CIM) monolithic chromatographic supports being designed for purification of large molecules and nanoparticles seem to be a matrix of choice for pDNA purification. In present work we describe a pDNA purification process designed on two different CIM monolithic columns, based on anion-exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) chemistry. HIC monolith enabled separation of supercoiled (sc) pDNA from open circular (oc) pDNA, genomic DNA (gDNA) and endotoxins regardless to flow rates in the range at least up to 380 cm/h. Dynamic binding capacity of new HIC monolith is up to 4 mg of pDNA per milliliter of support. Combination of both chromatographic steps using optimized CaCl2 precipitation enabled production of pure pDNA, satisfying all regulatory requirements. Process was found to be reproducible, scalable, and exhibits high productivity. In addition, in-line monitoring of pDNA purification process is shown, using CIM DEAE disk monolithic columns.
N. Lendero Krajnc, F. Smrekar, A. Štrancar, A. Podgornik
Journal of Chromatography A, 1218 (2011) 2413-2424
The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4 M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5 M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing “car-parking problem” phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.
M. Peterka, P. Kramberger, A. Štrancar
Wang, Perry G. (ur.). Monolithic chromatography and its modern applications. St Albans: ILM publications, 2010, pg. 489-508
Downstream processing (DSP) for purification can become a significant bottleneck in the production of novel biotherapeutics, such as viral vectors and vaccines (viral or DNA). Although different techniques can be used for the purification of large molecules and particles, liquid chromatography is the preferred method as it achieves the purity required by regulatory agencies. Despite the popularity of conventional chromatographic media, the diffusional mass transfer of large molecules and relatively small pore size remain limiting factors for the efficient separation of large biomolecules and particles.
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.
2009
S. Yamamoto, M. Nakamura, C. Tarmann, A. Jungbauer
Journal of Chromatography A 1216 (2009) 2616-2620
Our previous study has shown that there is a good correlation between the number of charges of DNA (from trimer to 50-mer) and the number of binding sites B in electrostatic interaction chromatography (ion-exchange chromatography, IEC). It was also found that high salt (NaCl) concentration is needed to elute large DNAs (>0.6 M). In this paper we further performed experiments with large DNAs (up to 95-mer polyT and polyA) and charged liposome particles of different sizes (ca. 30, 50 and 100 nm) with a monolithic anion-exchange disk in order to understand the binding and elution mechanism of very large charged biomolecules or particles. The peak salt (NaCl) concentration increased with increasing DNA length. However, above 50-mer DNAs the value did not increase significantly with DNA length (ca. 0.65–0.70 M). For liposome particles of different sizes the peak salt concentration (ca. 0.62 M) was similar and slightly lower than that for large DNAs (ca. 0.65–0.70 M). The binding site values (ca. 25–30) are smaller than those for large DNAs. When arginine was used as a mobile phase modulator, the elution position of polyA and polyT became very close whereas in NaCl gradient elution polyT appeared after polyA eluted. This was mainly due to suppression of hydrophobic interaction by arginine.
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.
2008
M. Ćurković Perica, I. Šola, L. Urbas, F. Smrekar, M. Krajačić
Journal of Chromatography A 1216 (2009) 2712-2716
A procedure based on Sartorius BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.
J. Ivancic-Jelecki, M. Brgles, M. Šantak, D. Forčić
Journal of Chromatography A 1216 (2009) 2717-2724
Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a Sartorius BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.
C. Delattre, P. Michaud, M. A. Vijayalakshmi
Journal of Chromatography B, 861 (2008) 203–208
Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.
2007
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1144 (2007) 85-89(2007) 85-89
A convection interaction media (trade name CIM, Sartorius BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.
I.Vovk, B. Simonovska
Journal of Chromatography B, 849 (2007) 337-343
The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; Mr 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with Mr 82 kDa) and a basic protein with Mr 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with Mr 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.