► Single step purification of pDNA on miligram scale. ► Characterization of grafted HIC groups. ► Multidimensional purification of pDNA. ► Very fast separation of pDNA using monoliths.
Purification ...of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7mg/ml under IEX conditions and 2.1mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5mg ofpDNA/ml of monolith from cell lysate.
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.
Binding of three different bacteriophages (phages), namely T7, lambda and M13 on methacrylate monoliths was investigated. Phage M13 exhibited the highest dynamic binding capacity of 4.5
×
10
13
...pfu/mL while T7 and lambda showed capacity of 1
×
10
13
pfu/mL, all corresponding to values of around 1
mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2
M while there was a constant capacity decrease for T7 and M13 phages. Under optimal conditions, recovery for all three phages approached 100%. Measurement of a pressure drop increase during loading enabled estimation of adsorbed phage layer thickness. At a maximal capacity it was calculated to be around 50
nm for T7 phage and 60
nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80
nm layer thickness was estimated for M13 phage showing its orientation along the pore.
Abstract 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.
A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The ...thickness of the grafted layer was determined by measuring permeability and found to be approximately 90
nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280
cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using β-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17
mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1
M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein
z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.
A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When ...optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on‐line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.
The most important prerequisite for the application of PCR-based methods, among them the detection and quantifi cation of genetically modifi ed organisms (GMOs) is the ability to extract significant ...amounts of DNA of adequate quality from the sample under investigation. The sample of interest in our work was soybean lecithin with expected low DNA content. The aim of this study was to set up a fast and effective HPLC (High Performance Liquid Chromatography) method using CIM® (Convective Interaction Media, BIA Separations d.o.o., Ljubljana, Slovenia) DEAE (DiEthylAminoEthyl) anion-exchange disk monolithic columns (disks) for the isolation of DNA from soybean lecithin samples. As the reference isolation procedure we used CTAB (CethylTrimethylAmmonium Bromide) method, which is widely used in GMO detection. It was demonstrated, that CIM® DEAE disks allow effi cient isolation of DNA from soybean lecithin. Furthermore, in comparison with the CTAB method, the method was less time-consuming and reduced the use of some aggressive chemicals. The quality of isolated DNA was tested with spectrophotometric analysis, agarose gel electrophoresis and by amplification of soybean specifi c lectin gene with qualitative and real-time PCR. The isolated soybean DNA was of adequate quantity and quality for PCR analysis, even though mostly degraded, present in small amounts and contaminated with some impurities, among them potential PCR inhibitors. The study expanded the applicability of monolithic columns in the isolation of biomolecules from highly processed food materials and their potential use for nucleic acids detection.
Geom. Topol. 20 (2016) 353-388 We establish the Hodge conjecture for the top dimensional cohomology group
with integer coefficients of any $q$-complete complex manifold $X$ with $q<\dim
X$. This ...holds in particular for the complement
$X=\mathbb{C}\mathbb{P}^n\setminus A$ of any complex projective manifold
defined by $q<n$ independent equations.
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