► Application of short monolithic columns for analytics of macromolecules and nanoparticles – recent overview. ► Impact of macromolecule interactions on peak spreading and resolution in IEX. ► ...Overview of binding sites number for different class of macromolecules and nanoparticles.
Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control.
Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate ...chelates between phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO2 nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO2). We demonstrate the specificity of CIM-OH-TiO2 column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation.
Display omitted
•Simple immobilisation of rutile TiO2 nanoparticles onto polymethacrylate monolith.•Stable and homogeneous monolayer of TiO2 nanoparticles was proven.•Application of MOAC in combination with chromatographic monolith.•Chromatographic separation of phosphorylated peptides from nonphosporylated.•Enrichment of phosphorylated peptides from digested model protein.
•pA ligand utilization decreases with increase of ligand density.•pA ligand utilization is preserved for monoliths with different pore sizes.•IgG binding capacity on pA ligand exceeded theoretical ...monolayer capacity.•Binding capacity for monoliths bearing IgG ligands increases with antigen size.
We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher.
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.
•Testing HIC ligands on chromatographic monoliths for pDNA downstream process.•Pyridine ligand was the optimal for pDNA isoform separation at preparative loading.•Pyridine and commercial C4 monolith ...for pDNA polishing step were compared.•Higher recovery and homogeneity of eluted sc pDNA from pyridine compared to C4.•Both ligands showed comparable efficiencies for the removal of other impurities.
Due to stringent requirements for the purity and efficacy of the plasmid DNA (pDNA) as a pharmaceutical product, chromatography is often used in the downstream process. Monolithic stationary phases exhibit several advantages over the conventional beaded supports mainly due to the convective flow and higher surface accessibility and are efficient chromatographic supports for separation of large biomolecules such as pDNA. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process in CIM™ HiP2 Plasmid Process Pack and the goal of the present work was a development of a hydrophobic methacrylate monolith with improved resolution for pDNA isoforms separation and removal of host contaminants. After a detailed search for an appropriate ligand, a pyridine-modified monolithic support was chosen and tested under descending ammonium sulfate linear gradient. The purification process was optimized according to the most efficient pDNA isoforms separation and the quantification of the main impurities during the purification steps was performed, as well as the purity and the recovery of eluted supercoiled (sc) pDNA isoform. Usage of pyridine-modified monoliths resulted in a more efficient separation between pDNA isoforms, with a similar dynamic binding capacity and recovery as C4 HLD monoliths (over 3mg/mL and 90%, respectively). Pyridine and C4 HLD monoliths were equally efficient for the removal of the main process impurities, but pyridine exhibited higher purity in terms of sc pDNA homogeneity (98%) comparing with C4 HLD (95%), showing to be a suitable alternative to C4 HLD for this polishing step.
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 pharmaceutical-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 behaviors of CIM
C4-HLD and Sartobind phenyl matrices was performe
Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf) glycosylation changes are common marker for ...congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific highthroughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC) analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. Keywords: high-throughput, immunoaffinity chromatography, monoliths, N-glycosylation, oriented antibody immobilization, transferrin.
Monoliths are today probably the most studied chromatographic supports. There are plethora of publications dealing with different aspects of their preparation, characterization, and applications. The ...reason for this interest is their inherent properties related to their particular structure, like ease of preparation in various volumes, fast analytics at low pressure and room temperature, and high productivity as a consequence of flow‐unaffected properties, especially important for isolation of large biological molecules. Because of that, structure of several monoliths was optimized for analytics and purification of biologic nanoparticles like viruses, virus‐like particles (VLPs), cells structures, or even intact cells. In this review, some recent applications of monoliths in the field of bioparticle isolation are described and results are discussed in terms of particular monolith properties.
•Determination of mechanical properties as a function of monolith pore size and porosity.•Correlation between compression modulus and permeability.•Determination of monolith structure having minimal ...compressibility.
Chromatographic monoliths have several interesting properties making them attractive supports for analytics but also for purification, especially of large biomolecules and bioassemblies. Although many of monolith features were thoroughly investigated, there is no data available to predict how monolith mechanical properties affect its chromatographic performance. In this work, we investigated the effect of porosity, pore size and chemical modification on methacrylate monolith compression modulus. While a linear correlation between pore size and compression modulus was found, the effect of porosity was highly exponential. Through these correlations it was concluded that chemical modification affects monolith porosity without changing the monolith skeleton integrity. Mathematical model to describe the change of monolith permeability as a function of monolith compression modulus was derived and successfully validated for monoliths of different geometries and pore sizes. It enables the prediction of pressure drop increase due to monolith compressibility for any monolith structural characteristics, such as geometry, porosity, pore size or mobile phase properties like viscosity or flow rate, based solely on the data of compression modulus and structural data of non-compressed monolith. Furthermore, it enables simple determination of monolith pore size at which monolith compressibility is the smallest and the most robust performance is expected. Data of monolith compression modulus in combination with developed mathematical model can therefore be used for the prediction of monolith permeability during its implementation but also to accelerate the design of novel chromatographic monoliths with desired hydrodynamic properties for particular application.
Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the ...context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/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, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future.
PDF
