E. S. Sinitsyna, J. G. Walter, E. G. Vlakh, F. Stahl, C. Kasper, T. B. Tennikova
Talanta 93 (2012) 139-146

Macroporous monoliths with different surface functionalization (reactive groups) were utilized as platforms for DNA analysis in microarray format. The slides based on a copolymer glycidyl methacrylate-co- ethylene dimethacrylate (GMA-EDMA) have been chosen as well known and thoroughly studied standard. In particular, this material has been used at optimization of DNA microanalytical procedure.

The concentration and pH of spotting solution, immobilization temperature and time, blocking agent and coupling reaction duration were selected as varied parameters. The efficiency of analysis performed on 3-D monolithic platforms was compared to that established for commercially available glass slides. As a practical example, a diagnostic test for detection of CFTR gene mutation was carried out. Additionally, the part of presented work was devoted to preparation of aptamer-based test-system that allowed successful and highly sensitive detection both of DNA and protein.

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A. G. Lopes

FBP-461, Food and Bioproducts Processing (2014)

As the biopharmaceutical industry matures, the trend towards increased flexibility and productivity, faster time tomarket and greater profitability are driving the replacement of traditional stainless steel equipment by single-use technology (SUT). The use of SUT in the biopharmaceutical industry can significantly impact the manufacturing process efficiency by reducing capital costs, improving plant flexibility, reducing start-up times and costs, and elim-inating both non-value added process steps and the risk of cross-contamination. In addition it significantly reduces process liquid waste, labour costs and on-site quality and validation requirements. This paper reviews the current status of the technology and the impact of SUT in the biopharmaceutical industry, with the aim of identifying the challenges and limitations that still need to be addressed for further adoption of these technologies. Even tough SUT has a multitude of systems available, its components and assemblies have little standardisation as well as alack of harmonised tests and procedures among suppliers, with an array of guidelines from a variety of sourcesand no critical limits have been established. In addition, the use of SUT has new validation requirements such as leachables and extractables, suppliers’ qualification and SUT lot-to-lot variability. The lack of expertise in these areas and the new training requirements when using SUT also need to be addressed. To date the majority of the avail-able literature regarding SUT is found in trade journals where typically suppliers are the main contributors. There is still a lack of engagement of the academic community, which contributes to very limited scientific proof from independent peer-reviewed research to support performance of SUT. This is particularly the case during operation and integrity testing of SUT, during for example on-site testing, transport and disposal. Another area where no work has been undertaken concerns conceptual approaches for facility clean-room requirement and appropriate layout design using SUT. Investment in novel technologies, research, standardisation and training is paramount for further development and implementation of SUTs across all sectors of the biopharmaceutical industry.

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F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut

Protein Expression and Purification 95 (2014) 104–112

The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

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M. M. Segura, M. Puig, M. Monfar, M. Chillon

HUMAN GENE THERAPY METHODS 23:182–197 (June 2012)

Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ul- tracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarifi- cation and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contami- nating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58– 69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.

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M. M. Segura, M. Puig, J. Piedra, S. Miravet

Adenovirus: Methods and protocols, Methods in Molecular Biology, vol. 1089

Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration–diafiltration. A Benzonase® digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38–45 %.

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M. M. St. Amand, B. A. Ogunnaike, A.S. Robinson

Published online in Wiley Online Library, 2013

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired posttranslational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2DHPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using
a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered
to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions.

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L. G. Berruex, R. Freitag, T. B. Tennikova

Journal of Pharmaceutical and Biomedical Analysis 24 (2000) 95–104

A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performance monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction media (CIM™)-disks, as stationary phase. An optimized procedure is described for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine IgG and a recombinant human antibody (type IgG1-κ) to all affinity disks is discussed. An essential feature of HPMAC is its compatibility to unusually high mobile phase flow rates (>4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. This makes HPMAC a promising tool for applications in fast process monitoring or screening. As an example for the former, the direct quantitative isolation of recombinant antibodies from serum-free culture supernatant is demonstrated.

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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.

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K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer

Biotechnology and Bioengineering 79 (2002) 733-740

Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.

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R. Hahn, E. Berger, K. Pflegerl, A. Jungbauer

Anal. Chem. 2003, 75, 543-548

When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the ε-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated.

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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.

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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.

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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).

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H. LeThanh, B. Lendl

Analytica Chimica Acta 422 (2000) 63–69

A fully automated method for the rapid determination of organic acids (citric-, malic- and tartaric acid) and sugars (glucose, fructose, and sucrose) in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy is presented. A convective interaction media (CIM) disc carrying quaternary amino moieties was added as a solid phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM disc whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution and recorded in their fully deprotonated form as a second flow injection peak. In both cases, the FTIR spectra corresponding to the peak maxima were selected for data evaluation. Two partial least squares models, one for sugars and the other for organic acids, were constructed based on the analysis of standards containing all six analytes. The developed method was applied to natural samples yielding results which were in good agreement with those obtained by an external reference method (enzymatic test kits). Deviations in the results were 3.4. and 4.1% for citric and malic acid and ranged from 4.7–5.1% for the sugars. The developed method is characterized by its short analysis time, experimental simplicity and its potential applications in routine analysis and process control.

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J. L. Ammerman, J. H. Aldstadt III

Microchim Acta (2009) 164:185-196

We describe the development and optimization of a sensitive and selective screening method for the measurement of trace levels of microcystins in surface waters. Several sample preparation techniques were compared, including solid-phase microextraction (SPME), particle-based solid-phase extraction (SPE), and monolith-based SPE. A flow-injection (FI) based approach employing a reversed-phase monolithic SPE column was found to be optimal. Quantification was performed by directly interfacing the FI-based SPE system to an electrospray ionization-mass spectrometer (ESI-MS). To more safely simulate peptidyl toxins such as the microcystins, a model peptide (i.e., angiotensin II) was used for method optimization. Sample loading flow rate and volume, eluent composition, and elution flow rate were optimized. Sample throughput was six samples per hour, a detection limit of 1.31 ng angiotensin II was demonstrated for a linear dynamic range from 1–1,000 ng and 3.4% relative standard deviation (n = 4, 100 ng sample). Sample volumes up to 1,000 ml of surface water could be loaded onto the monolithic SPE disk without exceeding the sorbent’s capacity. Unlike conventional particle-based SPE methods, the monolithic SPE disk does not need to be replaced between samples and could be used indefinitely. The FI-based SPE-ESI-MS method was successfully applied to the determination of microcystin-LR, the most common of the microcystins, in environmental samples and was demonstrated for the direct monitoring of chlorinated drinking water, with trends tracked over a period of eight months.

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R. Milačič, D. Ajlec, T. Zuliani, D. Žigon, J. Ščančar

Talanta 101 (2012) 203-210

In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in cow's milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk since analytical procedures reported were not quantitative. So, complex investigation was carried out to develop analytical method for quantitative determination of this biologically important molecule. Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation of Zn-citrate complexes ([Zn(Cit)]- and [Zn(Cit)2]4-; column recoveries 94–102%) and good repeatability and reproducibility of results with relative standard deviation (RSD±3.0%) were obtained. In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01 μg Zn mL-1 and 0.0005 μg Zn mL-1, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical tool for investigation of the percentage and the amount of Zn-citrate in human milk.

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H. Shirataki, C. Sudoh, T. Eshima, Y. Yokoyama, K. Okuyama

Journal of Chromatography A, 1218 (2011) 2381–2388

It is widely recognized that membrane adsorbers are powerful tools for the purification of biopharmaceutical protein products and for this reason a novel hollow-fiber AEX type membrane adsorber has been developed. The membrane is characterized by grafted chains including DEA ligands affixed to the pore surfaces of the membrane. In order to estimate the membrane performance, (1) dynamic binding capacities for pure BSA and DNA over a range of solution conductivity and pH, (2) virus reduction by flow-through process, and (3) HCP and DNA removal from cell culture, are evaluated and compared with several other anion-exchange membranes. The novel hollow-fiber membrane is tolerant of high salt concentration when adsorbing BSA and DNA. When challenged with a solution containing IgG the membrane has high impurity removal further indicating this hollow-fiber based membrane adsorber is an effective tool for purification of biopharmaceutical protein products including IgG.

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D. Josić, H. Schwinn, A. Štrancar, A. Podgornik, M. Barut, Y. P. Lim, M. Vodopivec

Journal of Chromatography A, 803 (1998) 61–71

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase IV, which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate–polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

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H. Podgornik, A. Podgornik

Enzyme and Microbial Technology 31 (2002) 855–861

The possibility of covalent attachment of LiP H2 and LiP H8 to CIM monoliths was studied. Due to negligible diffusional resistance, they can be useful tools to study characteristics of the immobilized lignin peroxidase (LiP). Immobilization to epoxy groups was performed using alkaline conditions (borate-phosphate buffer; pH 7.5). Characteristics of immobilized LiP were compared and factors that influence their biologic activity were evaluated using flow through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol (VA) into veratraldehyde (Vald). While VA oxidation rate increased by increasing flow rate (up to 1.5 ml/min) for LiP H2, it was almost constant in a wide flow rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison to free enzyme. The catalytic constant was found to be approximately 0.5 s-1 for both isoforms. Immobilized LiP H8 was used for decolorization of azo dye Mahogany.

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F. Mancini, V. Andrisano

Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 355-361

A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant β-amyloid precursor protein cleaving enzyme (hrBACE1). The approach is based on the use of an immobilized enzyme reactor (IMER) containing the target enzyme (hrBACE1–IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the β-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1–IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt = 1.6 min), which was separated from the substrate and selectively detected at λexc = 320 and λem = 420 nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1–IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA.

The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (−)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1–IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1–IMER showed high stability during the analysis, permitting its use for more than three months without affecting enzyme activity.

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