mL Of Matrix Research Articles

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification

Bevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein A affinity chromatography (AC). However, protein A is a very expensive ligand, thereby increasing the cost of purification. Furthermore, the harsh elution conditions required to recover bevacizumab from the AC column can damage both the mAb and protein A. In contrast, short peptides show higher stability, easier synthesis and lower cost and are therefore ideal ligands for AC. In the present study, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment that binds bevacizumab, was synthesized and immobilized on agarose. The peptidyl-agarose showed affinity for bevacizumab, with an equilibrium dissociation constant value of 2.2±0.5 x 10−7 M under optimal conditions. Samples of CHO cell filtrate producing bevacizumab were loaded on the peptidyl-agarose chromatography column. Bevacizumab was recovered from the elution fraction with a yield of 94% and a purity of 98%. The maximum capacity (qm) 38±2 mg of bevacizumab per mL of matrix was comparable to that of commercial protein A matrices. Moreover, the peptide ligand showed greater stability and a lower cost than protein A. Unlike peptides previously reported for IgG purification, the ligand described herein allows mAb elution under mild conditions, thereby favoring the integrity of bevacizumab. The lack of Trp, Met or Cys in the peptide prevents its oxidation and extends the useful life of the chromatographic matrix.

The development of a monolith-based purification process for Orthopoxvirus vaccinia virus Lister strain

The purification of large viruses remains an important field of research and development. The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8μm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1×109pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.

High throughput analysis and capture of benzo[a]pyrene using supermacroporous poly(4-vinyl pyridine-co-divinyl benzene) cryogel matrix

A novel supermacroporous cryogel matrix, poly(4-vinyl pyridine-co-divinyl benzene) has been synthesized by free radical polymerization using 4-vinyl pyridine and divinyl benzene as monomers and poly(ethylene glycol) diacrylate as crosslinker. Hydrophobic property of such synthesized matrix allows capture of polyaromatic hydrocarbons like benzo[a]pyrene. The cryogel matrix produced is highly porous (porosity more than 80%) having large and interconnected pores in the range of 10–100μm that allows easy passage of particulate matter and viscous liquids. The newly synthesized cryogel minicolumns (0.5ml; 0.9cmdia.×1.2cm height) were used to develop a high throughput analysis system in a miniaturized platform (open ended 96-well microtitre plate). The performance of the cryogel columns in 96-well microtitre plate was reproducible in different wells having approximately 72% binding. This system is drainage- and leakage- protected and can be used for the easy, fast and parallel analysis of many samples. The 96-well format also allows efficient analysis of benzo[a]pyrene in the spiked sample. The cryogel matrix was then used for column chromatography, which showed that the matrix has a binding capacity of 2μg per ml of matrix for benzo[a]pyrene, thus providing a system for remediation/separation of benzo[a]pyrene. The chromatographic column could bind and partially separate the spiked mixture of benzo[a]pyrene and anthracene.

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Aim Therapies exist for the bulk removal of antibodies in sensitized transplant patients, but there are no means to specifically deplete antibodies that recognize a specific HLA molecule. Current bulk antibody removal procedures may leave a patient compromised immunologically and more susceptible to opportunistic infections. The goal of this project was to create specific HLA-A∗02:01 removal matrices and demonstrate their ability to capture defined anti-HLA antibodies. Methods Soluble class I HLA-A∗02:01 molecules were produced in a native conformation in mammalian cells, purified by affinity chromatography, coupled to a Sepharose matrix, and loaded into a column enclosure. Various columns were created and tested with an automated chromatography system by initial injection of various HLA antibodies under different loading conditions. After an extensive column wash, the bound antibodies were released by a pH shift and quantitated. Loading and elution profiles were recorded. Results Initial tests show that HLA columns maintain their structural integrity and function. Multiple passes of the antibody W6/32, recognizing only intact HLA molecules, resulted in consistent and repeatable binding patterns. During the entire performance process, several parameters were identified, determining column saturation, capacity and efficiency values. Specificity was shown by removing a typical A∗02:01 recognition pattern from an A2 sera. In addition, treatment conditions were established to reuse the device. Conclusions Anti-HLA antibody removal devices are highly efficient in selectively depleting HLA antibodies. Capable of removing up to 1 mg per ml of matrix depending on the avidity of the antibody makes this device very efficient for research studies of HLA antibodies and provides promise for therapeutic applications. Furthermore, this device might also find an application in HLA diagnostics, allowing for the deconvolution of serologic samples and providing a new way to evaluate high PRA patients.

Affinity sorbent based on tryptophyl-threonyl-tyrosine for binding of the immunoglobulins G: Sorption characteristics and aspects of practical application

New chromatographic material based on tryptophil-threonil-tirosine was prepared. This sorbent effectively binds human, sheep, goat and cow immunoglobulins G. New sorbent shows high selectivity for removing immunoglobulins from blood plasma. Effective sorption capacity is 15-25 mg of immunoglobulin G per ml of matrix. Optimal method of covalent attachment ligand to polysaccharide matrix allows achieving high stability of the sorbents in terms of use and storage. This sorbent can be used in medicine and biotechnology.

RNA interference in human foreskin fibroblasts within the three-dimensional collagen matrix

The technique of RNA interference (RNAi) was trialed in primary human foreskin fibroblasts, both in monolayer culture and in the fibroblast-populated collagen matrix. Knockdown of lamin A/C, p53, and FAK was possible with low-confluency (<50%) monolayer fibroblasts, a transfection vehicle concentration of 1%, and an siRNA concentration of 25-50 nM. Knockdown also was possible in the collagen matrix using similar reagent concentrations and a cellular density of one million fibroblasts per ml of matrix. Optimization of transfection conditions appeared to be important to increase knockdown efficiency. Consistent with prediction, knockdown of FAK induced apoptosis in the fibroblast-populated collagen matrix.

Cross-linked Leucaena Seed Gum Matrix: An Affinity Chromatography Tool for Galactose-specific Lectins

A cross-linked leucaena (Leucaena leucocephala) seed gum (CLLSG) matrix was prepared for the isolation of galactose-specific lectins by affinity chromatography. The matrix was evaluated for affinity with a known galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina). The matrix preparation was simple and inexpensive when compared to commercial galactose-specific matrices (i.e. about 1.5 US dollars/100 ml of matrix). The current method is also useful for the demonstration of the affinity chromatography technique in laboratories. Since leucaena seeds are abundant and inexpensive, and the matrix preparation is easy, CLLSG appears to be a promising tool for the separation of galactose-specific lectins.

A Sensitive Method for the Simultaneous Determination of Caffeine and its Dimethylxanthine Metabolites in Human Plasma: Application to CYP1A2 Phenotyping

A sensitive high performance liquid chromatographic assay was developed for the determination of caffeine and its dimethylxanthine metabolites in human plasma. Caffeine, paraxanthine, theophylline, and theobromine were extracted from 0.5 mL of matrix with methylene chloride. Separation of the analytes was achieved using a mobile phase consisting of 0.1 M sodium acetate pH 4.5, methanol, tetrahydrofuran, and distilled water (10:6.5:1.4:82.1, v/v) and a Microsorb MV C18 analytical column with UV detection at 274 nm. Excellent linearity was observed for each analyte over the concentration range of 20 – 8000 ng/mL. The within- and between-day coefficients of variation were less than 4.1% at all concentrations examined. This method is suitable for pharmacokinetic or pharmacogenetic studies utilizing caffeine as a probe for CYP1A2 activity whether given alone or in combination with other CYP probe drugs.

Hydrodynamics and performance in fluidized bed adsorption

The performance of fluidized bed adsorption is strongly influenced by the hydrodynamics of the fluidization process. Especially axial mixing in the liquid and solid phase may lead to reduced capacity and resolution. In this article axial mixing in the liquid phase of a classified fluidized bed based on porous glass granules is presented. Axial mixing was analyzed by measurements of residence time distributions in a fluidized bed, showing a reduction of mixing at increased ratio of bed height to diameter as well as at increased linear velocity of the liquid stream. These results were transferred to two real adsorption systems on two different scales: In a bench scale (up to 15 mL of adsorbent) the purification of monoclonal antibodies from hybridoma supernatant was performed with a cation exchanger, in a larger scale (up to 750 mL of matrix) the adsorption of bovine serum albumin (BSA) on the same matrix was investigated. The results showed an increase of capacity at increased bed height-to-diameter ratio; with regard to linear velocity a broad range of only slightly changed capacity was found. A shift from dispersion controlled to diffusion controlled adsorption at intermediate linear velocity was proposed by isolating the effect of pore diffusion from the effect of dispersion.

Affinity Immobilization of Escherichia coli : Catalysis by Intact and Permeable Cells Bound to Starch

The binding of viable Escherichia coli cells to an immobilized ligand of a surface receptor for maltodextrins has recently been demonstrated (T. Ferenci and K. S. Lee, J. Mol. Biol. 160:431-444, 1982). The interaction of bacteria and ligand immobilized in a chromatographic column was investigated over a wide range of applied cell densities, temperatures, eluant pH values, osmotic concentrations, and flow rates. Over 95% retention of bacteria applied to starch-Sepharose was found at cell densities up to 10 per ml of matrix, between pH 5.5 and 8.0, between 8 and 55 degrees C, in the presence of 0 to 0.5 M NaCl, and at elution flow rates up to 37 column volumes per h. The catalytic capability and stability of affinity-immobilized cells was demonstrated with the cytoplasmic beta-galactosidase activity of starch-bound cells. Intact immobilized bacteria exhibited slowly increasing beta-galactosidase activity over several days with a plateau after 6 days. Bacteria made permeable by treatment with toluene were also bound to starch-Sepharose but showed maximum beta-galactosidase activity within 1 day and exhibited no loss of enzyme activity in 8 days of continuous elution at ambient temperatures.