Publications
2008
P. Gagnon
MSS2008
When monoclonal antibodies were first beginning to be commercialized, expression levels over 100 mg/L were considered outstanding, and cell culture was viewed as the bottleneck in manufacturing productivity. Antibody expression levels now commonly exceed 1 g/L and reports of 10 and 15 g/L have been recently announced. Downstream processing is now considered the bottleneck.
In one sense, the bottleneck is artificial. Cell culture production takes about two weeks (not counting preparation of seed stock) and purification takes about a week. In another sense, the bottleneck is real, and a genuine concern. Process time for the protein A capture step from 20,000 L of cell culture supernatant (CCS) commonly requires 72-96 hours. This represents multiple cycles. The long hold time for IgG produced in the early cycles increases the risk of degradation by proteolysis, deamidation, etc. It also increases the risk of contamination.
V. Frankovič, A. Podgornik, N. Lendero Krajnc, F. Smrekar, P. Krajnc, A. Štrancar
Journal of Chromatography A, 1207 (2008) 84–93(2008) 84 – 93
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. Jungbauer, R. Hahn
Journal of Chromatography A, 1184 (2008) 62–79(2008) 62 – 79
Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8 L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths’ inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 μm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.
E. S. Sinitsyna, E. N. Vlasova, E. G. Vlakh, T. B. Tennikova
Russian Journal of Applied Chemistry, 2008, Vol. 81, No. 8, pp. 1403–1409
Copolymers containing aldehyde, succinimidyl carbonate, and imidazolecarbamate groups were prepared by polymer-analogous transformations of epoxy groups of a macroporous monolithic polymeric support derived from glycidyl methacrylate and ethylene glycol dimethacyrlate. The effect of certain parameters on the course of the copolymer modification and immobilization of a protein on the surface of the polymeric support was studied. The possibility of using the matrices obtained for development of biorecognizing systems was examined.
M. Barut, A. Podgornik, L. Urbas, B. Gabor, P. Brne, J. Vidič, S. Plevčak, A. Štrancar
J. Sep. Sci. 2008, 31, 1867 – 1880
This review describes the novel chromatography stationary phase – a porous monolithic methacrylate-based polymer – in terms of the design of the columns and some of the features that make these columns attractive for the purification of large biomolecules. We first start with a brief summary of the characteristics of these large molecules (more precisely large proteins like immunoglobulins G and M, plasmid deoxyribonucleic acid (DNA), and viral particles), and a list of some of the problems that were encountered during the development of efficient purification processes. We then briefly describe the structure of the methacrylate-based monolith and emphasize the features which make them more than suitable for dealing with large entities. The highly efficient structure on a small scale can be transferred to a large scale without the need of making column modifications, and the various approaches of how this is accomplished are briefly presented in this paper. This is followed by presenting some of the examples from the bioprocess development schemes, where the implementation of the methacrylate-based monolithic columns has resulted in a very efficient and productive process. Following this, we move back to the analytical scale and demonstrate the efficiency of the monolithic column – where the mass transfer between the stationary and mobile phase is greatly enhanced – for the in-process and final control of the new therapeutics. The combination of an efficient structure and the appropriate hardware results in separations of proteins with residence time less than 0.1 s.
2007
R. Hahn, A. Tscheliessnig, P. Bauerhansl, A. Jungbauer
J. Biochem. Biophys. Methods 70 (2007) 87–94
Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.
R. Skudas, B. A. Grimes, E. Machtejevas, V. Kudirkaite, O. Kornysova, T. P. Hennessy, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 72-84(2007) 72 - 84
In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of ∼25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18 < C8 < C4. The resolution of proteins and peptides by reversed-phase gradient liquid chromatography on n-octadecyl, n-octyl, and n-butyl bonded monolithic silica columns is optimized. The results obtained imply the use of acetonitrile concentration gradient up to 75% for n-octadecyl and n-octyl bonded monolithic silica columns, and the use of acetonitrile concentration gradient up to 85% for n-butyl bonded monolithic silica columns. With the respect to the gradient times and flow rates, the optimum conditions are the best with n-octyl and n-butyl bonded monolithic silica columns, where the range of optimum gradient times is up to ∼30 min and mobile phase flow rates in the range of 0.5–1 ml/min. Consequently, the best performance towards peak resolution is obtained with n-octyl bonded monolithic silica column with the respect to low concentration of organic phase gradient, fast separations and low solvent consumptions due to low flow rates.
J. Vidič, A. Podgornik, J. Jančar, V. Frankovič, B. Košir, N. Lendero, K. Čuček, M. Krajnc, A. Štrancar
Journal of Chromatography A, 1144 (2007) 63–71(2007) 63 – 71
Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1 M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1 M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.
B. A. Grimes, R. Skudas, K. K. Unger, D. Lubda
Journal of Chromatography A, 1144 (2007) 14-29(2007) 14-29
In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.
I. Junkar, T. Koloini, P. Krajnc, D. Nemec, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1144 (2007) 48-54(2007) 48-54
Today, monoliths are well-accepted chromatographic stationary phases due to several advantageous properties in comparison with conventional chromatographic supports. A number of different types of monoliths have already been described, among them recently a poly(high internal phase emulsion) (PolyHIPE) type of chromatographic monoliths. Due to their particular structure, we investigated the possibility of implementing different mathematical models to predict pressure drop on PolyHIPE monoliths. It was found that the experimental results of pressure drop on PolyHIPE monoliths can best be described by employing the representative unit cell (RUC) model, which was originally derived for the prediction of pressure drop on catalytic foams. Models intended for the description of particulate beds and silica monoliths were not as accurate. The results of this study indicate that the PolyHIPE structure under given experimental condition is, from a hydrodynamic point of view, to some extent similar to foam structures, though any extrapolation of these results may not provide useful predictions of pressure versus flow relations and further experiments are required.
S. Laschober, M. Sulyok, E. Rosenberg
Journal of Chromatography A, 1144 (2007) 55-62(2007) 55-62
The present work aims at the optimisation of the synthesis of methyl-silsesquioxane monolithic capillary columns using a sol–gel based protocol. The influence of reaction conditions such as temperature, reaction mixture composition and catalyst concentration has been examined. The morphology of the products was studied by scanning electron microscopy and nitrogen adsorption. Monolithic capillary columns were obtained with a skeleton-like structure with open pores. Pore diameters vary from 0.8 to 15 μm, diameters of the xerogel network vary from 0.4 to 12 μm, respectively. Specific surface areas up to 334 m2/g have been observed, however, many materials did not possess areas above few m2/g which represents the limit of detection of the nitrogen porosimetry measurements. Excellent adhesion to the capillary wall was observed in all cases, and drying was possible at ambient conditions without the formation of cracks.
I. V. Kalashnikova, N. D. Ivanova, T. G. Evseeva, A. Yu. Menshikova, E. G. Vlakh, T. B. Tennikova
Journal of Chromatography A, 1144 (2007) 40–47(2007) 40–47
The subject of this paper is an investigation of the peculiarities of dynamic adsorption behavior of nanoparticles. For this purpose, virus-mimicking synthetic particles bearing different proteins at their outer surface were specially constructed using two approaches, e.g. the cross-linking of proteins and modification of polystyrene microsphere surface by proteins. Two chromatographic modes, namely ion-exchange and affinity liquid chromatography on ultra-short monolithic columns [Convective Interaction Media (CIM) DEAE and CIM QA disks] have been used as a tool for dynamic adsorption experiments. Such parameters as maximum adsorption capacity and its dependence on applied flow rate were established and compared with those obtained for individual proteins. Similarly to individual proteins, it was shown that the maximum of adsorption capacity was not changed at different flow rates. In addition, the permeability of porous space of used monolithic sorbents appeared to be sufficient for efficient separation of large particles and quite similar to the well-studied process applied for individual proteins.
N. Delmotte, U. Kobold, T. Meier, A. Gallusser, A. Strancar, C. G. Huber
Anal Bioanal Chem (2007) 389:1065–1074
Immunoadsorbers based on 2.0 × 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol μL-1. However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol μL−1 in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol μL-1.
D. Josić, J. G. Clifton
Journal of Chromatography A, 1144 (2007) 2-13
An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC–MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC–MS/MS protein identification will be discussed.
E. Machtejevas, S. Andrecht, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 97-101
The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 μm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the low column back pressures. Also, monolithic columns showed a lower clogging rate in long-term usage for biological samples as compared to particulate columns. The applied system set-up was tested to be useful for the routine peptide screening in search of disease biomarkers.
P. Brne, A. Podgornik, K. Benčina, B. Gabor, A. Štrancar, M. Peterka
Journal of Chromatography A , 1144 (2007) 120-125
Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20 mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.
C. K. Zacharis, E. A. Kalaitzantonakis, A. Podgornik, G. Theodoridis
Journal of Chromatography A, 1144 (2007) 126–134
In this study, sequential injection affinity chromatography was used for drug–protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy ΔG, enthalpy ΔH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 × 106 M-1 at pH 7.4 and 39 °C for a single binding site. The associated change in enthalpy (ΔH) was −27.36 kcal mol-1 and the change in entropy (ΔS) was −73 cal mol-1 K-1 for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (λext = 230 nm, λem = 350 nm) and spectrophotometric detection (λ = 280 nm).
T. Čerk Petrič, P. Brne, B. Gabor, L. Govednik, M. Barut, A. Štrancar, L. Zupančič Kralj
Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 243–249
In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immunoglobulin G represent more than 75% of all such proteins. In this paper, the characterization of short monolithic columns was performed followed by the optimization of a multidimensional approach, known as conjoint liquid chromatography, to deplete human serum albumin and immunoglobulin G from a human plasma sample. Two different chromatographic modes were used: ion-exchange chromatography and affinity chromatography. A monolithic stationary phase (convective interaction media disk) bearing strong anion-exchange groups and another immobilized with protein G were placed in series into one housing. The optimal binding conditions were found that removed a majority of human serum albumin and immunoglobulin G from the human plasma sample. This method was compared to the depletion using a combination of pseudo-affinity and affinity columns. The results of the human serum albumin and immunoglobulin G depletion were confirmed by 2D electrophoresis. It has been shown that anion-exchange and affinity chromatography using convective interaction media monolithic columns can represent an efficient complementary technique for human serum albumin and immunoglobulin G removal from human plasma.
2005
M. Barut, A. Podgornik, P. Brne, A. Štrancar
J. Sep. Sci. 2005, 28, 1876-1892
New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept – the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.
A. Podgornik, A. Štrancar
Biotechnology Annual Review, 11 (2005) 281-333
Modern downstream processing requires fast and highly effective methods to obtain large quantities of highly pure substances. Commonly applied method for this purpose is chromatography. However, its main drawback is its throughput since purification, especially of large molecules, requires long process time. To overcome this problem several new stationary phases were introduced, among which short layer monoliths show superior properties for many applications. The purpose of this review is to give an overview about short methacrylate monolithic columns commercialised under the trademark Convective Interaction Media® (CIM). Their unique properties are described from different perspectives, explaining reasons for their application on various areas. Approaches to prepare large volume methacrylate monolithic column are discussed and optimal solutions are given. Different examples of CIM monolithic column implementation are summarised in the last part of the article to give the reader an idea about their advantages.