Preparation of a Monolith with Covalently Bound Bovine Serum Albumin for Capillary Electrochromatography

Capillary electrochromatography (CEC) is important for applications in enantiomer separation. The problems associated with column fabrication bring a challenge in developing monoliths with ease of preparation, robustness of separation, enhanced mass transfer, and lower pressure drop. In this research, the covalent binding of proteins on to a monolithic matrix was investigated to overcome the drawback of loss and/or denaturing of the biomolecules from physical adsorption and encapsulation method. A chitosan/silica hybrid monolith was prepared and a protein, bovine serum albumin, was covalently immobilized on the column. The prepared monolith was evaluated using the enantioseparation of D,L-tryptophan by CEC. It was found that separation of tryptophan enantiomers with a resolution of 2.44 was achieved by using 20 mmol L^?1 phosphate buffer at pH 7.5. A higher chitosan concentration was also proven to be of possible use in the synthesis with the aid of acetic acid as the solvent. The much shorter retention time and increased separation ability demonstrate the advantages of capillary column under investigation.     

Fabrication of an ionic‐liquid‐based polymer monolithic column and its application in the fractionation of proteins from complex biosamples

An ionic‐liquid‐based polymer monolithic column was synthesized by free radical polymerization within the confines of a stainless‐steel column (50 mm × 4.6 mm id). In the processes, ionic liquid and stearyl methacrylate were used as dual monomers, ethylene glycol dimethacrylate as the cross‐linking agent, and polyethylene glycol 200 and isopropanol as co‐porogens. Effects of the prepolymerization solution components on the properties of the resulting monoliths were studied in detail. Scanning electron microscopy, nitrogen adsorption–desorption measurements, and mercury intrusion porosimetry were used to investigate the morphology and pore size distribution of the prepared monoliths, which showed that the homemade ionic‐liquid‐based monolith column possessed a relatively uniform macropore structure with a total macropore specific surface area of 44.72 m²/g. Compared to a non‐ionic‐liquid‐based monolith prepared under the same conditions, the ionic‐liquid‐based monolith exhibited excellent selectivity and high performance for separating proteins from complex biosamples, such as egg white, snailase, bovine serum albumin digest solution, human plasma, etc., indicating promising applications in the fractionation and analysis of proteins from the complex biosamples in proteomics research.     

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H₂O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.     

Ice-templated porous polymer/UiO-66 monolith for Congo Red adsorptive removal

Chitosan/MOF composite porous monolith used in water remediation as adsorbent can realize high-efficient removal of pollutant in water and facile recycling from water. However, dissolution of chitosan (without crosslinking) in acidic aqueous solution will cause breakage of composite monolith. Herein, we report a chitosan/UiO-66 monolith prepared by ice-templating method. Specially, a pre-crosslinking treatment (by glutaraldehyde) is employed before the monolith formation, which obviously boosts its stability in aqueous solution. The composite monolith is evaluated by SEM, N₂ adsorption, XRD, and batch adsorption tests for Congo Red (CR). The results show that the composite monolith possesses a typical ice-templating structure with hierarchical (mirco- / meso- and macro-) pores. UiO-66 particles are embedded on the surface of chitosan matrix, and the crystal structure of UiO-66 is not changed obviously by the crosslinking and freezing process. The composite monolith exhibits high adsorption efficiency (90% of CR was removed from its aqueous solution in 60 min) and the maximum adsorption capacity of 246.21 mg/g (derived from Langmuir model) can be reached. After adsorption, the monolith is collected by a facile procedure and recovered using ethanol for evaluating its reusability. After 4 cycles, the CR removal efficiency of the composite monolith still remains ~90% of the initial efficiency. This work demonstrates that the simple crosslinking procedure before monolith formation can ensure the intact shape of the chitosan/MOF monolith during adsorption.     

Application of Supermacroporous Monolithic Hydrophobic Cryogel in Capturing of Albumin

Supermacroporous poly{2-hydroxyethyl methacrylate-co-[N,N-bis(2,6-diisopropylphenyl)-perylene-3,4,9,10-tetracarboxylic diimide]} [poly(HEMA-co-DIPPER)] monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with DIPPER as functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50 μm size. Poly(HEMA-co-DIPPER) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA-co-DIPPER) cryogel was 14.7 g H₂O/g dry cryogel. Poly(HEMA-co-DIPPER) cryogel was used in the adsorption/desorption of albumin from aqueous solutions. The nonspecific adsorption of albumin onto plain poly(HEMA) cryogel was very low (3.36 g/g polymer). The maximum amount of albumin adsorption from aqueous solution in acetate buffer was 40.9 mg/g polymer at pH 5.0. It was observed that albumin could be repeatedly adsorbed and desorbed with the poly(HEMA-co-DIPPER) cryogel without significant loss of adsorption capacity.     

Design and evaluation of synthetic silica-based monolithic materials in shrinkable tube for efficient protein extraction

Sample pretreatment is a required step in proteomics in order to remove interferences and preconcentrate the samples. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transport compared with more common packed beds. These features are due to the micro-structure within the monolithic silica column which contains both macropores that reduce the back pressure, and mesopores that give good interaction with analytes. The aim of this work was to fabricate a continuous porous silica monolithic rod inside a heat shrinkable tube and to compare this with the same material whose surface has been modified with a C(18) phase, in order to use them for preconcentration/extraction of proteins. The performance of the silica-based monolithic rod was evaluated using eight proteins; insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, ovalbumin, hemoglobin, and bovine serum albumin at a concentration of 60 μM. The results show that recovery of the proteins was achieved by both columns with variable yields; however, the C(18) modified silica monolith gave higher recoveries (92.7 to 109.7%) than the non-modified silica monolith (25.5 to 97.9%). Both silica monoliths can be used with very low back pressure indicating a promising approach for future fabrication of the silica monolith inside a microfluidic device for the extraction of proteins from biological media.     

High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format

Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples.     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

Characterisation of grafted weak anion-exchange methacrylate monoliths

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 90nm. 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 280cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using beta-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 17mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1M 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.     

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.     

γ-irradiated chitosan-polyvinyl pyrrolidone hydrogels as pH-sensitive protein delivery system

The effects of pH of the buffer solution and the composition of the hydrogel system on the bovine serum albumin (BSA) adsorption capacity of chitosan (CS)–polyvinyl pyrrolidone (PVP) (CSPVP) hydrogels and release of BSA were investigated. Poly-electrolyte CSPVP hydrogels with different compositions were prepared by irradiating CS/PVP/water mixtures with γ-rays at ambient temperature. The adsorption capacity of hydrogels was found to increase from 0 to 350 mg BSA/g dry gel, by changing external stimuli and hydrogel composition. The adsorption of BSA within CSPVP hydrogels increased with increase in CS content in the hydrogels. When the irradiation doses of hydrogel increased, the adsorption of BSA decreased. The maximum adsorption of BSA was observed at pH 5. A significant amount of the adsorbed BSA (up to 95%) was eluted in the phosphate medium containing 0.1 M NaCl at pH 7.4.     

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.     

Dye attached poly(hydroxyethyl methacrylate) cryogel for albumin depletion from human serum

Cibacron Blue F3GA was immobilized on poly(hydroxyethyl methacrylate) cryogel and it was used for selective and efficient depletion of albumin from human serum. The poly(hydroxyethyl methacrylate) was selected as the basic component because of its inertness, mechanical strength, chemical and biological stability, and biocompatibility. Cibacron Blue F3GA was covalently attached to the poly(hydroxyethyl methacrylate) cryogel to produce poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel affinity column. The poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was characterized with respect to gelation yield, swelling degree, total volume of macropores, Fourier Transform Infrared spectroscopy, and scanning electron microscopy. It was found that the maximum amount of adsorption (343 mg/g of dry cryogel) obtained from experimental results is very close to the calculated Langmuir adsorption capacity (345 mg/g of dry cryogel). The maximum adsorption capacity for poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel column was obtained as 950 mg/g of dry cryogel for nondiluted serum. The adsorption capacity decreased with increasing dilution ratios while the depletion ratio of albumin remained as 77% in serum sample. Finally, the poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was optimized for using in the fast protein liquid chromatography system for rapid removal of the high abundant proteins from the human serum.     

Biocompatible polymeric monoliths for protein and peptide separations

The concept of biocompatibility with reference to chromatographic stationary phases for separation of biomolecules (including proteins and peptides) is introduced. Biocompatible is a characteristic that indicates resistance to nonspecific adsorption of biomolecules and preservation of their structures and biochemical functions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide‐ and poly(meth)acrylate‐based monoliths] used for protein and peptide separations are reviewed in detail, with emphasis on size exclusion, ion exchange, and hydrophobic interaction chromatographic modes. Biocompatible monoliths for enzyme reactors are also included. The two main synthetic approaches to produce biocompatible monoliths are summarized, i. e., surface modification of a monolith that is not inherently biocompatible and direct copolymerization of hydrophilic monomers to form a biocompatible monolith directly. Integration of polyethylene glycol into the poly(meth)acrylate monolith network is becoming popular for reduction of non‐specific protein interactions.     

Iodide fluorescence quenching of sol-gel immobilized BSA

Several potential applications of sol-gel immobilized proteins may require drying the gel before use. The attendant shrinkage may sterically restrict the penetration of solutes to the entrapped protein. In this study, collisional quenching of intrinsic bovine serum albumin (BSA) fluorescence by dissolved iodide was used to quantitatively compare the accessibility of protein molecules entrapped in hydrated and dried sol-gel monoliths. The results show that iodide penetration to immobilized BSA is sterically restricted by approximately 25% in hydrated gels and 50% in dried gels, relative to the quenching behavior of dissolved BSA. The further decrease upon drying is consistent with the overall degree of monolith shrinkage.     

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.     

Polymer monoliths synthesized by radiation co-polymerization in solution

Hydrophilic co-polymer monoliths were prepared by irradiating alcoholic solutions containing diethyleneglycol dimethacrylate (DEGDMA) and 2-hydroxyethylacrylate (HEA) monomers. The effect of monomer ratio, solvent properties and radiation dose on the porous properties of the monoliths was studied in detail and compared to the monolith prepared from DEGDMA. Increase of the HEA content in the co-monomer mixture (up to 18 vol%) resulted in monoliths with increased pore size and hydrophilic character. The biggest pores were obtained when methanol was used as solvent.The use of the monoliths as chromatographic columns for separation of proteins, amino and nucleic acids is also reported.     

Single and binary adsorption of proteins on ion-exchange adsorbent: The effectiveness of isothermal models

Simultaneous and sequential adsorption equilibria of single and binary adsorption of bovine serum albumin and bovine hemoglobin on Q Sepharose FF were investigated in different buffer constituents and initial conditions. The results in simultaneous adsorption showed that both proteins underwent competitive adsorption onto the adsorbent following greatly by protein-surface interaction. Preferentially adsorbed albumin complied with the universal rule of ion-exchange adsorption whereas buffer had no marked influence on hemoglobin adsorption. Moreover, an increase in initial ratios of proteins was benefit to a growth of adsorption density. In sequential adsorption, hemoglobin had the same adsorption densities as single-component adsorption. It was attributed to the displacement of preadsorbed albumin and multiple layer adsorption of hemoglobin. Three isothermal models (i.e. extended Langmuir, steric mass-action, and statistical thermodynamic (ST) models) were introduced to describe the ion-exchange adsorption of albumin and hemoglobin mixtures. The results suggested that extended Langmuir model gave the lowest deviation in describing preferential adsorption of albumin at a given salt concentration while steric mass-action model could very well describe the salt effect in albumin adsorption. For weaker adsorbed hemoglobin, ST model was the preferred choice. In concert with breakthrough data, the research further revealed the complexity in ion-exchange adsorption of proteins.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

A facile and efficient strategy for one-step in situ preparation of hydrophobic organic monolithic stationary phases by click chemistry and its application on protein separation

A simple one-step in situ “click” modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 “click” monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the “clicked” modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.