Can aptameric ligands specific to plasma coagulation factor VII bind the recombinant form with high affinity: affinity measurement by fluorescence method

Background: among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven[TM], has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products. Aptamers as nucleic acid based affinity ligands are more promising than monoclonal antibodies. In addition, DNA aptamers are more acceptable than RNA ones in this regard

Methods: in this study, two of the aptameric DNA oligonucleotides that showed acceptable affinities for purification of coagulation factor VIIa from plasma, were selected to evaluate their affinity against Aryoseven. A serial dilution of fluorescence labeled aptamers was incubated against the concentration of 1 nM from Aryoseven. Then, a fluorescence index was calculated according to the fluorescence intensity data measured from test and control samples. The dissociation constant of aptamers was calculated using according to the fluorescence index Prism5 software

Results: results showed that the binding affinity of the 44 nucleotide aptamer was more than 81 nucleotide aptamer sequence. As a result, this aptamer could be optimized in order to develop aptamer based affinity chromatography process for this form of recombinant coagulation factor VIIa

Discussion: aptamers with shorter length of sequence could show higher affinity in target binding, as they could adapt more easily to suitable conformation according to target interaction. However, it should be considered that the selectivity of affinity ligands is also important for target purification and analytical applications

Periplasmic expression of TNF related apoptosis inducing ligand [TRAIL] in E.coli

Tumor necrosis factor-related apoptosis-inducing ligand [TRAIL], a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 [DE3] and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space

Formulation development and toxicity assessment of triacetin mediated nanoemulsions as novel delivery systems for rapamycin

The aim of this investigation was to design and develop nanoemulsions [NEs] as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin [as oil], various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance [TEER] and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments

Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b [rhIFN alpha 2b] has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFN alpha 2b. The protein was incubated for 1 h at 40-70 [degree sign]C and for up to 240 h at 50 [degree sign]C and its aggregation tendency was then studied using optical density [at 350 nm], SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH [5, 6 and 7] and buffer concentrations [10, 55 and 100 mM] on the aggregation of protein following incubation at 50 [degree sign]C for 72 h was also evaluated. The results obtained for samples incubated at 50 [degree sign]C for up to 240 h showed that OD[350] and the amount of higher molecular weight aggregates [HMW] increased and the monomer content decreased significantly [p<0.05] as the incubation time increased. Following incubation at various temperatures, a significant increase in OD[350], drop in monomer content and increase in the amount of HMW aggregates were observed [p<0.05]. Data obtained from incubation of samples at 50 [degree sign]C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 [p<0.05]. At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFN alpha 2b

Challenges to improve the stability and efficacy of an intravesical BCG product

The aim of this investigation was to improve the storage stability and survival rate of an intravesical BCG product, manufactured with an attenuated strain of Mycobacterium bovis [Pasteur strain 1173P2 of BCG] in the presence of sodium glutamate. Formulations with various concentrations of trehalose [a known protectant] were developed as liquid and lyophilized forms. Formulations were evaluated by different methods, including optical density measurement, safety assessment, skin reaction test, moisture content determination, viability assay, bacterial and fungal contaminations and the results were compared with those obtained for sodium glutamate-containing formulations. The stability tests were also carried out in various storage durations and different temperatures. To develop the lyophilization protocol, glass transition temperatures in the presence of both stabilizers were determined using differential scanning calorimetry. In general, results showed that trehalose could considerably increase the stability of the product against freezing and drying processes, increase the survival rate even in the liquid formulations, as well as the production of an acceptable cake. However, further studies are required to optimize the product characteristics

reversed phase high performance liquid chromatographic method for determination of rapamycin

Easily degradating and various isomeric forms of rapamycin [Sirolimus] face the determination of this compound to many challenges. In this study, we developed and validated the isocratic reversed phase high performance liquid chromatographic [RP-HPLC] method for rapamycin. Separation was performed on a C[8] column [MZ, 15 × 4.6 mm, 5 [micro]m particle size] using methanol:water [80:20 v/v] as the mobile phase with the flow rate of 1 mL/min. The column temperature was set at 57degreeC and the detection was carried out at the wavelength of 277 nm. The method was linear over a concentration range of 0.025-2 [micro]g/mL. The coefficient of variation of intra- and inter-day, assessed at three concentration levels of 0.075, 0.3 and 0.900 [micro]g/mL, was less than 2%. Limit of quantification [LOQ] was found 25 ng/mL. The method with high percent recovery and short retention time of rapamycin, was found to be simple, rapid and reproducible

Halotolerant ability and alpha-amylase activity of some saltwater fungal isolates

Four halotolerant fungal isolates originating from the saltwater Lake Urmia in Iran were selected during a screening program for salt resistance and alpha-amylase activity. The isolates were identified based on sequencing the ITS region and a part of the [beta]-tubulin gene, as Penicillium chrysogenum [isolate U1; CBS 132820], Fusarium incarnatum [isolate U2; CBS 132821], and Penicillium polonicum [isolate U3; CBS 132822, and isolate U4; CBS 132823]. The growth of these isolates was determined by measuring the colony diameter and mycelia dry weight in Sabouraud dextrose agar and yeast nitrogen base medium supplemented with NaCl, KCl, and LiCl.Isolate U4 showed a growth up in 15% NaCl and U1 was the only isolate that could grow in 20% KCl. None of the strains grew in a media containing LiCl. The salt supplemented medium did not increase the size of colony diameter in all isolates [p > 0.05]. The ability of the selected isolates for amylase production was quantitatively tested and showed that P. polonicum isolate U4 was the most potent producer of amylase with a yield of 260.9 U/L after 60 h, whereas P. polonicum isolate U3 was the lowest one with a production level of 97.9 U/L after 48 h. P. polonicum isolate U4 could be a suitable candidate for production of amylase on an industrial scale after optimization

Evaluation of the effect of pegylated single-walled carbon nanotubes on viability and proliferation of jurkat cells

Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes [CNTs], regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs [SWCNTs] with phospholipid-PEG [Pl-PEG] conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO[2] humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility

Separation of somatropin and its degradation products by high-performance liquid chromatography using a reversed-phase polymeric column

The accurate prediction of protein stability is one of the most challenging goals in protein formulation and delivery. In this study, a gradient RP-HPLC method is described for the separation of human growth hormone [hGH] variants as deamidated and oxidized forms. The methodology employed a polymeric poly [styrene-co-divinylbenzene] column and a 1mL/min flow rate of a linear gradient of 0.1% v/v TFA/acetonitrile and TFA/Water [pH=2.0] mixture as the mobile phase. The overall run time of this method was 12 min and the average retention times were about 8.7 min for the native somatropin, 7.2 min for the deamidated form and 1.6 and 5.3 min for oxidized variants. The method was also validated in terms of selectivity, linearity, intra- and inter-day variations. In conclusion the method was found to have the potential for being applied as an initial and rapid evaluation method for assessing the quality and quantity of hGH during downstream processing, formulation and storage