Publications
2009
A. Tscheliessnig, A. Jungbauer
Journal of Chromatography A, 1216 (2009) 2676–2682
High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23–250 μg/ml. The calibration range of the method could be extended from 4 to 1000 μg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.
J. Krenkova, A. Gargano, N. A. Lacher, J. M. Schneiderheinze, F. Svec
Journal of Chromatography A, 1216 (2009) 6824–6830
Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to afford stationary phases for a strong and a weak cation exchange chromatography, respectively. Alternatively, poly(ethylene glycol) methacrylate was used for grafting in the first step in order to enhance hydrophilicity of the support followed by photografting with 2-acrylamido-2-methyl-1-propanesulfonic acid or acrylic acid in the second step. These new columns were used for the separation of proteins and peptides. A mixture of ovalbumin, α-chymotrypsinogen, cytochrome c, ribonuclease A and lysozyme was used to assess the chromatographic performance for large molecules while a cytochrome c digest served as a model mixture of peptides. All tested columns featured excellent mass transfer as demonstrated with very steep breakthrough curves. The highest binding capacities were found for columns prepared using the two step functionalization. Columns with sulfonic acid functionalities adsorbed up to 21.5 mg/mL lysozyme while the capacity of the weak cation exchange column functionalized with acrylic acid was 29.2 mg/mL.
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.
2007
E. Müller, C. Mann
Journal of Chromatography A, 1144 (2007) 30-39(2007) 30-39
The electro-acoustic effects, namely the ion vibration potential (IVP) and the colloidal vibration current (CVI), colloidal vibration potential (CVP) first described by P. Debye [P. Debye, J. Chem. Phys. 1 (1933) 13], are a result of charge separation of bound or free ions at different degrees by ultrasonic waves. Today commercial instruments are available to investigate liquid homogeneous and heterogeneous systems. In the present paper the application of this technique for the characterization of salts, protein solutions and resins for biochromatography is shown and valuable information about resins can be derived in a short time. Various resins were investigated with the following results: (1) the CVI magnitude is dependent of several parameters (such as particle size distribution, volume fraction, density difference); (2) the CVI is influenced by the surface modification of the resins. Polymeric modifications decrease the value of CVI. The CVI is generally lower for high capacity resins; (3) the measurement of the electro-acoustic effects can be used to detect small changes in resins. The CVI is dependent of the amount of adsorbed protein in “native” and denatured state.
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.
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
Y.-P. Lim, D. Josić, H. Callanan, J. Brown, D. C. Hixson
Journal of Chromatography A, 1065 (2005) 39–43(2005) 39–43
Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.
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2004
T. Hall, D. C. Wood, C. E. Smith
Journal of Chromatography A, 1041 (2004) 87–93(2004) 87–93
Monolithic media were compared with Q- and SP-Sepharose high performance chromatography for preparative purification and with Q- and SP-5PW chromatography for analysis of a pegylated form of myelopoietin (MPO), an engineered hematopoietic growth factor. The use of either monolithic or Sepharose based supports for preparative chromatography produced highly purified pegylated MPO with the monolithic media demonstrating peak resolution and repeatability at flow rates of 1 and 5 ml/min resulting in run times as much as five-fold shorter compared to Sepharose separations. The monolithic disks also resulted in 10-fold shorter run times for the analytical chromatography, however, their chromatographic profiles and peak symmetry were not as sharp compared to their Q-5PW and SP-5PW counterparts.
E. Vlakh, A. Novikov, G. Vlasov, T. Tennikova
Journal of Peptide Science, 10: 719–730 (2004)
Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing β-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods.
The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.
D. Ren, N. A. Penner, B. E. Slentz, H. D. Inerowicz, M. Rybalko, F. E. Regnier
Journal of Chromatography A, 1031 (2004) 87–92(2004) 87–92
Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and β-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.
E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova
Journal of Chromatography B, 810 (2004) 15–23
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova
Journal of Chromatography B, 810 (2004) 15–23
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova
Journal of Chromatography B, 810 (2004) 15–23
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
E. Vlakh, N. Ostryanina, A. Jungbauer, T. Tennikova
Journal of Biotechnology 107 (2004) 275–284
Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM® Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM® affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.
P. N. Nesterenko, M. A. Rybalko
Mendeleev Commun. 2004
The continuous flow gradient and its effect on chromatographic parameters were investigated for the separations of inorganic anions on a monolithic porous disk with bonded hydroxyproline residues.
2003
E. G. Vlakh, G. A. Platonova, G. P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T. B. Tennikova
Journal of Chromatography A, 992 (2003) 109–119
The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.