Ml Of Beads Research Articles

Loading, release and stability of epirubicin-loaded drug-eluting beads.

The aim of this study was to determine the loading efficiency, physico-chemical stability and release of epirubicin-loaded DC Bead™ (Biocompatibles UK Ltd, a BTG International group company) (bead size 70-150 µm (=DC BeadM1™) and 100-300 µm) after loading with epirubicin solution (2 mg/ml) or reconstituted powder formulation (25 mg/ml) and controlled storage. DC Bead™ were loaded with 76 mg epirubicin solution (Epimedac™, Medac GmbH) or 75 mg epirubicin powder formulation (Farmorubicin™, Pharmacia Pfizer GmbH) per 2 ml of beads. Drug loading efficiency and stability were determined by measuring the epirubicin concentration in the excess solution after predetermined intervals (maximum 24 h) and different agitation conditions.Syringes with loaded beads were stored protected from light at room temperature. At predetermined intervals the beads were transferred into 200 ml phosphate buffered solution (pH 7.2) as elution medium and stirred automatically for 2 h not followed or followed by addition of 200 ml of 20% sodium chloride (=NaCl) solution and stirred for another 2 h to analyse the drug release and integrity of the epirubicin-loaded beads. Elution experiments were performed and samples taken periodically over a four-week period (day 0, 7, 14 and 28). A reversed-phase high-performance liquid chromatography assay with ultraviolet detection was utilized to analyse the concentration and purity of epirubicin. The loading procedure for DC Bead™ with epirubicin drug solutions resulted in a loading percentage of 95-99% within 6 h dependent on the bead size, epirubicin concentration in the loading solution and loading conditions. Loading levels remained stable and no epirubicin degradation products were observed over the period of 28 days, while the loaded beads were stored light protected at room temperature.Release of epirubicin into 200 ml phosphate buffered solution elution medium and additionally followed by release into the admixture with 200 ml 20% NaCl solution amounted to 5% and about 20% of the loaded epirubicin, respectively. Integrity of loaded epirubicin was proven over 28 days. Epirubicin can be loaded into DC Bead™ of different sizes using the epirubicin powder formulation (25 mg/ml) or epirubicin injection concentrate (2 mg/ml). Physico-chemical stability is maintained over a period of at least 28 days when stored light protected at room temperature. Elution of epirubicin is dependent on the volume and cation exchange capacity of the elution medium.

Bioengineering the Liver: Scale-Up and Cool Chain Delivery of the Liver Cell Biomass for Clinical Targeting in a Bioartificial Liver Support System

Acute liver failure has a high mortality unless patients receive a liver transplant; however, there are insufficient donor organs to meet the clinical need. The liver may rapidly recover from acute injury by hepatic cell regeneration given time. A bioartificial liver machine can provide temporary liver support to enable such regeneration to occur. We developed a bioartificial liver machine using human-derived liver cells encapsulated in alginate, cultured in a fluidized bed bioreactor to a level of function suitable for clinical use (performance competence). HepG2 cells were encapsulated in alginate using a JetCutter to produce ∼500 μm spherical beads containing cells at ∼1.75 million cells/mL beads. Within the beads, encapsulated cells proliferated to form compact cell spheroids (AELS) with good cell-to-cell contact and cell function, that were analyzed functionally and by gene expression at mRNA and protein levels. We established a methodology to enable a ∼34-fold increase in cell density within the AELS over 11–13 days, maintaining cell viability. Optimized nutrient and oxygen provision were numerically modeled and tested experimentally, achieving a cell density at harvest of >45 million cells/mL beads; >5×1010 cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7–1]×1011). A short-term storage protocol at ambient temperature was established, enabling transport from laboratory to bedside over 48 h, appropriate for clinical translation of a manufactured bioartificial liver machine.

Co-Immobilization of Glucose Oxidase-Catalase: Optimization of Immobilization Parameters to Improve the Immobilization Yield

Co-immobilization of glucose oxidase (EC 1.1.3.4) and catalase (EC 1. 11.1.6) on non-porus glass surfaces using ?-aminopropyltriethoxysilane and polyethyleneimine as an activator and gluteraldehyde as cross linking agent has been carried out for its potential use. Polyethyleneimine was found to be a superior immobilization activator than ?-aminopropyltriethoxysilane. In present study, the effects of rough and smooth beads and optimization of the ratio of enzyme concentrations, activator material and concentration of the cross linking agent were investigated using response surface technology. With optimized concentration of glucose oxidase to catalase ratio (0.97), polyethyleneimine (150 mg/l) and gluteraldehyde (15 ml), the effect of glass bead concentration for maximum immobilization yield was investigated. Central composite optimization strategy with 16 experiments increased immobilization yield from 82.63% to 92.74%. It was observed that 0.3 ml of beads per 120 U of glucose oxidase were necessary for higher immobilization yield.

Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System

Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.

Enzymatic Redesigning of Biologically Active Heparan Sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Characterization and optimization of a chromatographic process based on ethylenediamine- N, N, N′, N′-tetra(methylphosphonic) acid-modified zirconia particles

The primary objective of work was to characterize, optimize and model a chromatographic process based on ethylenediamine- N, N, N′, N′-tetra(methylphosphonic) acid (EDTPA)-modified zirconia particles. Zirconia particles were produced by spray-drying colloidal zirconia. Zirconia spheres produced were further classified, calcined and modified with EDTPA to yield a solid-phase support for use in bio-chromatography (r_PEZ). Specifically, the ability of r_PEZ to selectively bind and enrich IgG, IgA, and IgM from biological fluids was evaluated and demonstrated. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium disassociation constant ( K d) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. The maximum static binding capacity ( Q max) was found to be in the range 55–65 mg IgG per ml of beads, and unaffected by temperature. The maximum dynamic binding capacity ( Q x ) was found to be in the range 20–12 mg IgG per ml of beads. The adsorption rate constant ( k a) was determined by a split-peak approach to be between 982 and 3242 l mol −1 s −1 depending on the linear velocity. The standard enthalpy and entropy values were estimated for this interaction of IgG with this novel support.

Interaction of immunoglobulin G with N,N,N′,N′-ethylenediaminetetramethylenephosphonic acid-modified zirconia

Zirconia beads (25–38 μm in diameter) were modified with N,N,N′,N′-ethylenediaminetetramethylenephosphonic acid to generate a pseudo-biospecific support, r_PEZ. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium dissociation constant ( K d) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. Temperature had no significant impact on the maximum static binding capacity ( Q max) and the equilibrium-binding constant ( K d), whereas pH and the salt concentration had a noticeable impact on both Q max and K d values. Q max was found to be in the range of 55–65 mg IgG per ml of beads and unaffected by temperature. The maximum dynamic binding capacity ( Q x) was found to be in the range of 20–12 mg IgG per ml of beads. The adsorption rate constant ( k a) was determined by a split-peak approach to be between 982 and 3242 l mol −1 s −1 depending on the linear velocity. Adsorption rate of IgG on r_PEZ was studied as a function of both feed concentration and linear velocity. The standard enthalpy and entropy values were estimated for the interaction of IgG with this novel support. The binding constants were also determined by modeling the batch protein-uptake data.

DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9 x 10(7) cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 micrograms per 10(6) viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.

Calcium alginate immobilized hybridomas grown using a fluidized-bed perfusion system with a protein-free medium

Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2 x 10(7) cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.

Technical note: Application of immobilized cells of Kluyveromyces marxianus for continuous hydrolysis to fructose of fructans in Jerusalem artichoke extracts

Dead cells of Kluyveromyces marxianus having inulase (β‐D‐fructo fructanohydrolase E.C.3.2.1.7) activity were immobilized in alginate beads and used as a biocatalyst in a packed bed reactor and a stirred batch reactor. Fructosans of Jerusalem artichoke tubers, after extraction, were utilized for continuous or semi‐continuous production of fructose. In a bed reactor packed with 100 ml of beads, a volumetric productivity of 36 g/l/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a half life of 28 days was observed for the biocatalyst. Using artichoke extract containing 20% fructan solution, 98% conversion could be achieved in a batch reactor in 20 hr. Repeated cycling of beads resulted in considerable loss of catalyst from the reactor and subsequent loss in catalytic activity, thus giving a half life of only 14 days.

Androgen receptor affinity chromatography: Synthesis and properties of 17α-epoxypropyl- dihydrotestosterone sepharose

We have prepared a new affinity chromatography reagent, 17α-epoxypropyl-dihydro-testosterone linked to Thiopropyl-Sepharose, with potential for use in purification of androgen receptor and other specific androgen binding proteins. The linkage is stable, and the ligand has reasonably high affinity for the receptor. Starting with 5α-androstane-3β-ol-17-one, we synthesized in two steps 17α-allyl-dihydrotestosterone, which was then oxidized to 17α-epoxypropyl-DHT yielding 2 diastereomers in about a 4:1 ratio. The 17α-allyl-DHT had about 50% of DHT's affinity for rat uterine androgen receptor, while the affinity of the major epoxide isomer was 9% and that of the minor isomer was 4%. Reaction of the epoxides with Thiopropyl-Sepharose-6B gave about 7 μ mol of covalently bound DHT per ml of beads. These beads took up 83% of the androgen receptor from a rat uterine cytosol in a preliminary study, which more than equalled the performance of identically prepared estradiol beads successfully used for estrogen receptor purification. The use of the new DHT beads in purifications of the androgen receptor and other binding proteins is now being explored by other laboratories.

Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

ABSTRACTNon‐viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß‐2,1‐fructan fructano‐hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.