The Effective Control of Hyperuricemia in Cancer Patients: A New Recombinant Conjugated Variant of Urate Oxidase.

OBJECTIVE: Management of hyperuricemia is crucial to controlling tumor lysis syndrome (TLS) during cancer therapy. Urate oxidase (UOX) that catalyzes the enzymatic oxidation of uric acid into allantoin, is effective in lowering plasma uric acid levels and controlling hyperuricemia. Recently, we developed a new recombinant conjugate variant of UOX therapeutic drug using PASylation technology. This study was designed to evaluate the stability, plasma half-life and immunogencity of PASylated UOX. METHODS: A recombinant variant of PASylated UOX from the Aspergillus flavus was manufactured using bioinformatics and experimental techniques. Ex vivo evaluation of stability of PASylated UOX was done in 50% human serum. For half-life test, recombinant PASylated UOX and rasburicase were administered at 1.5 mg/kg to 10 rats in two different groups and samples were collected after injection Production of antibodies against PASylated drug was also assayed. RESULTS: Residual activity of PASylated UOX in 50% human serum was higher than rasburicase and native UOX. Stability of PASylated UOX at 25°C and 37°C was also higher than rasburicase and native UOX. The PASylated half-life was ~32.1 hours, whereas half-life for rasburicase and native UOX was ~25.1 and ~22.8 hours, respectively. In immunogenicity examination, there is 33% and 36% decrease in the absorbance of native UOX and rasburicase, respectively when compared with that of PASylated UOX. CONCLUSION: Our data confirmed the efficacy and stability of PASylated UOX in comparison to the rasburicase. In summary, the results indicated that PASylated UOX drug is effective at lowering plasma uric acid levels with prolonged plasma half-life and decreased cost.
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An amperometric biosensor for specific detection of glycated hemoglobin based on recombinant engineered fructosyl peptide oxidase.

Here, we present a specific biosensor based on the detection of glycated hemoglobin (HbA1c) proteolytic digestion product, fructosyl valyl histidine (Fru-ValHis). A recombinant engineered fructosyl peptide oxidase (FPOX) enzyme with improved specificity was immobilized on the electrode surface modified by chitosan (CHIT), graphene oxide (GO) and gold nanoparticles (AuNPs). The biosensor exhibited a linear response toward different concentrations of Fru-ValHis ranging from 0.1 to 2 mM with a sensitivity of 8.45 µA mM-1 cm-2. Detection limit of the current biosensor for Fru-ValHis was 0.3 µM as the lowest quantity required giving a signal to a background. Analytical recovery of added Fru-ValHis in whole blood was 95.1-98.35% for FPOX/AuNPs/GO/CHIT/FTO electrode. For Fru-ValHis determination by FPOX-AuNPs-GO-CHIT/FTO electrode, within-run coefficient of variation (CV) was between 1.3% and 2.4% and between run CV was between 2.1% and 3.5%. A significant change in electron transfer resistance after the incubation of FPOX-modified electrode with Fru-ValHis was observed, while no response was achieved with control, indicating specific measurement of Fru-ValHis. Moreover, designed biosensor measured HbA1c in human blood samples and the results were well agreed with that obtained with NORUDIA™ N HbA1c diagnostic kit. Overall, suitable specificity of the engineered FPOX made the bioelectrode responded well to the Fru-ValHis level, which demonstrates a promising application for specific detection of HbA1c biomarker.

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.