Performance of a non-grafted monolithic support for purification of supercoiled plasmid DNA

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.     

Naphthyl methacrylate-phenylene diacrylate-based monolithic column for reversed-phase capillary electrochromatography via hydrophobic and π interactions

A neutral naphthyl methacrylate-phenylene diacrylate-based monolith (NPM) was introduced for RP-CEC of various neutral and charged solute probes via hydrophobic and π interactions. The NPM column was prepared by the in situ polymerization of naphthyl methacrylate as the functional monomer and 1,4-phenylene diacrylate (PDA) as the crosslinker in a ternary porogenic solvent containing cyclohexanol, dodecanol and water. The NPM column exhibited cathodal EOF despite the fact that it was devoid of any fixed charges. NPM exhibited stronger EOF than its counterpart naphthyl methacrylate monolith (NMM) made from the in situ polymerization of naphthyl methacrylate and trimethylolpropane trimethacrylate (TRIM). As for NMM, it is believed that the EOF arises from the adsorption of mobile phase ions onto the monolith surface. The higher EOF exhibited by NPM may be attributed to the acrylate nature of PDA as compared to the methacrylate nature of TRIM, and therefore PDA has a higher binding capacity for mobile phase ions due to its higher polarity than TRIM. The adsorption of mobile phase ions together with the additional π interactions offered by the aromatic rings of the NPM matrix modulated solute retention and separation selectivity. The applications of NPM were demonstrated by the separation of a wide range of small and large solutes including peptides, tryptic peptide maps and proteins.     

Fabrication of high-permeability and high-capacity monolith for protein chromatography

A novel approach for the fabrication of macroporous poly(glycidyl methacrylate-ethylene glycol dimethacrylate) monolith is presented. The method involved the use of sodium sulfate granules and organic solvents as co-porogens. Compared with the conventional monoliths [ML-(1-3)] using organic solvents only as a porogen, the improved monoliths [MLS-(1-3)] showed not only higher column efficiency and dynamic binding capacity (DBC) for protein (bovine serum albumin, BSA), but also higher column permeability and lower back pressure. It is considered that the superpores introduced by the solid granules played an important role for the improvement of the monolith performance. Moreover, poly(glycidyl methacrylate-diethylamine) tentacles were grafted onto the pore surface of MLS-3 monolith. This has further increased the DBC of BSA to 74.7 mg/ml, about three times higher than that of the monoliths without the grafted tentacles. This grafting does not obviously decrease the column permeability, so a new monolith of high column permeability and binding capacity has been produced for high-performance preparative protein chromatography.     

Preparation and characterization of polyethyleneimine modified ion-exchanger based on poly(methacrylate-co-ethylene dimethacrylate) monolith

A polyethyleneimine (PEI) modified ion-exchanger was prepared based on poly(methacrylate-co-ethylene dimethacrylate) monolith cast in 100 mm x 4.6 mm I.D. stainless steel tube with heptane as the porogenic solvent at 65 degrees C for 12 h. The pores larger than 500 nm presented 85% of total pore volume of PEI monolith and provided the better permeability for separation. Bovine serum albumin (BSA) binding capacity on the column was enhanced with increasing the molecular weight of PEI, indicated that the brush ligand emanated from the surface and captured more protein by multiple binding sites. Titration experiment as well as BSA retention versus the pH of mobile phase showed that the monolith exhibited weak ion-exchange property, and recovered BSA on the monolith reached 97% when NaCl content in mobile phase was higher than 0.5 M. Frontal analysis and gradient elution of BSA indicated that PEI monolith provided the rapid mass transfer in chromatographic procedure, which made the dynamic binding capacities as well as column efficiency keep as constants at high operating flow rate. Fast separation of three mode proteins mixture (lysozyme, hemoglobin and BSA) on the monolith was achieved within 3 min at velocity of 1445 cm/h. This demonstrated the potential of PEI monolith for the rapid analysis and separation of proteins.     

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

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 CuCl(2)-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.     

Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Preparation of a Hybrid Cation-Exchange Monolith and Its Application for the Separation of Proteins by High Performance Liquid Chromatography

A hybrid cation-exchange monolith for high performance liquid chromatography (HPLC) was prepared by a one-pot synthesis with reverse-atom transfer radical polymerization, in which sodium bisulfite was used as the inorganic material, methyl methacrylate was used as organic material, 2, 2'-Azobisisobutyronitrile as initiator, and ferric trichloride as the inorganic catalyst. Moreover, sodium bisulfite was used to provide the sulfonic group. The conditions of polymerization were optimized. The chemical groups of the monolith were assayed by Fourier transform infrared spectroscopy; the morphology of monolithic material was investigated by scanning electron microscopy; the pore size distribution was determined by a mercury porosimeter; and the mechanical stability and permeability of the monolith were investigated by the back-pressure drop at several linear velocities. Finally, the monolith was used to separate human serum albumin from human plasma in conjunction with HPLC in 5 min, and the influences of buffer concentration and pH value on the elution of human serum albumin were investigated. In addition, the monolith was applied to separate lysozyme from egg whites in a short time (4 min) and separate a mixture of aromatic compounds.     

Preparation and application of hydrophobic hybrid monolithic columns containing polyhedral oligomeric silsesquioxanes for capillary electrochromatography

Hydrophobic organic-inorganic hybrid monolithic columns were synthesized via thermally initiated free radical polymerization with the confines of 75 μm id capillary using a polyhedral oligomeric silsesquioxane (POSS) reagent containing eight or more methacrylate groups as the crosslinker. Three organic functional monomers, butyl methacrylate (BuMA), lauryl methacrylate (LMA) and methacrylic acid (MAA), were selected and copolymerized with the POSS in the presence of 1-propanol and 1,4-butanediol to prepare the poly(POSS-co-BuMA), poly(POSS-co-LMA), and poly(POSS-co-MAA) monoliths, respectively. The 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) was copolymerized as ionizable monomer into the poly(POSS-co-BuMA) and poly(POSS-co-LMA) for the generation of EOF in capillary electrochromatography (CEC). A hybrid poly(POSS-co-LMA-co-MAA) monolith was also similarly prepared by copolymerizing ternary monomers of POSS, LMA, and MAA, and compared with poly(POSS-co-BuMA), poly(POSS-co-LMA), and poly(POSS-co-MAA) monoliths. The resulting four kinds of POSS-contained hybrid monoliths exhibited good permeability and mechanical stability. Their column efficiencies were evaluated by the separation of alkylbenzene homologues and polar compounds in CEC. The results indicated that the highest efficiencies of 194,100 and 102,100 theoretical plates per meter for thiourea and benzene were obtained on the poly(POSS-co-LMA-co-MAA) monolith. Additionally, the poly(POSS-co-LMA-co-MAA) monolith exhibited better selectivity for separation of polar compounds than those of other hybrid monoliths.     

Fast preparation of photopolymerized poly(benzyl methacrylate-co-bisphenol A dimethacrylate) monoliths for capillary electrochromatography

A novel porous polymer monolith was prepared in situ in a fused-silica capillary using photoinitiated polymerization. Bisphenol A dimethacrylate (BPADMA) was selected as a crosslinker, copolymerized with benzyl methacrylate (BMA) in the presence of a binary porogenic solvent consisting of cyclohexanol and 1-decanol in ≤10 min. The resulting poly(BMA-co-BPADMA) monoliths exhibited good permeability and mechanical stability. Mixtures of alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) or phenolic compounds were successfully separated by CEC. A similar monolith was also prepared with ethylene dimethacrylate (EDMA) as the crosslinker instead of BPADMA to compare the separation ability of the resulting monoliths. The results indicated that poly(BMA-co-BPADMA) monoliths have better selectivity for aromatic analytes and greater chromatographic stability in higher aqueous mobile phase.     

Variation in the chromatographic,material, and chemical characteristics of methacrylate‐based polymer monoliths during photoinitiated low‐temperature polymerization

Both the separation behavior and the structure of a polymer monolith column depends on both the reaction solution composition and the polymerization conditions. In photoinitiated low‐temperature polymerization, polymerization temperature, irradiation intensity, and polymerization time were key factors to control the monolith characteristics. In this study, the effect of polymerization time on the chromatographic, material, and chemical characteristics of poly(butyl methacrylate‐co‐ethylene dimethacrylate) monoliths was studied using pyrolysis‐gas chromatography, Raman spectroscopy, inverse size exclusion chromatography, scanning electron microscopy, and chromatographic methods. Both butyl methacrylate and ethylene dimethacrylate monomers were incorporated into the monolith as the polymerization time increased, and it resulted in increases in both the flow resistance (decrease in both permeability and total/through pore porosities) and retention factors. The longer polymerization time led to lower relative amounts of free methacrylate functional groups in the monolith, i.e. cross‐linking was enhanced. The increase of the polymerization time from 8 to 12 min significantly reduced the separation efficiency for the retained analyte, whereas an increase in the fraction of the mesoporosity was observed.     

High performance DNA separation media using synergistic radiation grafting

Simultaneous radiation grafting technique was carried out using 2-diethylaminoethyl methacrylate as monomer to introduce positively charged groups into polyethylene and polyamide microcarriers. Special selection of diverse solvents and comonomers like N-vinylpyrrolidone, 2-hydroxyethyl methacrylate leads to high grafting yields. The functionality of the cationic carriers were examined with regard to their DNA binding properties using a high molecular weight phage λ DNA. Comparative examinations with commercial DNA and cationic separation gels revealed an up to 50% higher DNA binding capacity of the grafted carriers.     

Preparation of high loading PolyHIPE monoliths as scavengers for organic chemistry

A novel microcellular polyHIPE monolith of high functional group capacity has been prepared by a two-step process including the synthesis of the scaffold by polymerization of a highly concentrated emulsion and then the in situ surface polymerization of methacrylate monomers. Application of the resulting functionalized monolith is demonstrated in a scavenging reaction of poly(glycidyl methacrylate)-grafted polyHIPE with 1-hexylamine. The open-cellular structure of the core combined with the good accessibility of the grafted functional polymer chains allows total scavenging of the amine in a relatively short period.     

Preparation of a novel polymer monolith with functional polymer brushes by two‐step atom‐transfer radical polymerization for trypsin immobilization

Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]‐co‐glycidyl methacrylate} brushes were fabricated via two‐step atom‐transfer radical polymerization and used as a trypsin‐based reactor in a continuous flow system. This is the first time that atom‐transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface‐initiated atom‐transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.     

Adsorption behavior of large plasmids on the anion-exchange methacrylate monolithic columns

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 0.4M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5M 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.     

Poly(N-isopropylacrylamide)-grafted dual stimuli-responsive filter paper for protein separation

Thermal and salt dual stimuli-responsive filter-paper-based membranes were prepared by UV-induced grafting of NIPAM-based polymers on paper surface. The grafting ratio could be controlled by monomer concentration during grafting polymerization. The results from pressure drop measurement of the mobile phase flowed cross the membrane demonstrate that an appropriate grafting ratio would be 8%-10%. Protein adsorption on the membrane through hydrophobic interaction could be promoted by increasing temperature and lyotropic salt concentration. The effect of grafted polymer structure on protein binding performance was studied. Filter paper grafted with NIPAM-based branched copolymer consisting of hydrophobic monomer moieties shows ten times higher protein binding capacity than that of the original filter paper. The separation of plasma proteins using the dual stimuli-responsive membrane was examined to demonstrate feasible application for hydrophobic interaction chromatographic separation of proteins.     

Integrated electrokinetically driven microfluidic devices with pH‐mediated solid‐phase extraction coupled to microchip electrophoresis for preterm birth biomarkers

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low‐abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH‐mediated solid‐phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed‐phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH‐mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50‐fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15‐fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.     

Silica nanoparticle-templated methacrylic acid monoliths for in-line solid-phase extraction–capillary electrophoresis of basic analytes

Polymeric ion-exchange monoliths typically exhibit low capacities due to the limited surface area on the globules of the monoliths. The ion-exchange binding of protonated weakly basic analytes on deprotonated carboxylate sites on methacrylate polymer monoliths has been increased by templating the monoliths with silica nanoparticles. The templating method is achieved by adding the nanoparticles as a suspension to the polymerisation mixture. After polymerisation, the nanoparticles are removed by washing the monolith with strong base. Monolithic columns prepared using this procedure have exhibited a 33-fold increase in ion-exchange capacity when compared to untemplated monoliths prepared and treated under similar conditions. The templating procedure does not alter the macroporous properties of the polymer monolith, confirmed through scanning electron microscopy and BET surface area analysis, but provides increased capacity predominantly through the re-orientation of more carboxylic acid groups. The resulting increase in ion-exchange capacity has proven to be useful for the preconcentration and separation of neurotransmitters by in-line solid-phase extraction–capillary electrophoresis. The increased capacity of the templated monolith allowed the injection time to be increased 10 times over that of an untemplated monolith, allowing 10 times more sample to be injected with the efficiencies and recoveries remaining unaffected. The enhancement in sensitivity for the test mixture of neurotransmitter (dopamine, norepinephrine and metanephrine) ranged 1500–1900 compared to a normal hydrodynamic injection in capillary electrophoresis. Efficiencies obtained for the neurotransmitters were 100 000–260 000 plates, typical of those obtained in capillary zone electrophoresis. The applicability of the increased capacity silica nano-templated polymer monolith was demonstrated by analysing trace levels of caffeine in biological, food and environmental samples.     

Facile Fabrication of Polymeric Ionic Liquid Grafted Porous Polymer Monolith for Mixed‐Mode High Performance Liquid Chromatography

A novel polymeric ionic liquid grafted porous polymer monolith has been facilely fabricated for mixed‐mode chromatography. The column is prepared from poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) monolith through hydrolyzation of the epoxy moieties into hydroxyl groups, followed by "grafting from" polymerization of ionic liquid of 1‐vinyl‐3‐butylimidazolium chloride. Successful modification is characterized by scanning electron microscope, infrared spectroscopy, elemental analysis and mercury intrusion porosimetry. The HPLC performance of developed column is evaluated by separating acidic vitamin B analytes, neutral steroids and basic aromatic amines in mixed‐mode chromatography on a single column, respectively. The ionic liquid affords the monolith with both enhanced separation ability and improved column efficiency.     

“Smart” molecularly imprinted monoliths for the selective capture and easy release of proteins

A new thermally switchable molecularly imprinted monolith for the selective capture and release of proteins has been designed. First, a generic poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) monolith reacted with ethylenediamine followed by functionalization with 2‐bromoisobutyryl bromide to introduce the initiator for atom transfer radical polymerization. Subsequently, a protein‐imprinted poly(N‐isopropylacrylamide) layer was grafted onto the surface of the monolithic matrix by atom transfer radical polymerization. Scanning electron microscopy and energy‐dispersive X‐ray spectroscopy of the cross‐sections of imprinted monoliths confirmed the formation of dense poly(N‐isopropylacrylamide) brushes on the pore surface. The imprinted monolith exhibited high specificity and selectivity toward its template protein myoglobin over competing proteins and a remarkably large maximum adsorption capacity of 1641 mg/g. Moreover, this “smart” imprinted monolith featured thermally responsive characteristics that enabled selective capture and easy release of proteins triggered only by change in temperature with water as the mobile phase and avoided use of stronger organic solvents or change in ionic strength and pH.