Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

BACKGROUND PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating Nterminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. RESULTS: Selected linear 5 kDa and 20 kDa mPEG ­ potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the diPEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods.

Produção e purificação integrada de protease fibrinolítica de Mucor subtilissimus UCP 1262 / Integrated production and purification of fibrinolytic protease from Mucor subtilissimus UCP 1262

As enzimas fibrinolíticas podem ser obtidas de micro-organismos por meio de processos fermentativos. O presente trabalho teve como objetivo avaliar a produção e extração integrada da protease fibrinolítica de Mucor subtilissimus UCP 1262 usando sistema de duas fases aquosas (SDFA). O processo integrado foi realizado para avaliar a produção, partição e recuperação da protease fibrinolítica, segundo planejamento experimental 23, utilizando como variáveis independentes a massa molar do polietileno glicol (PEG), a concentração do PEG e a concentração do sulfato de sódio. A maior atividade fibrinolítica (15,40U/mL) foi obtida na fase rica em sulfato de sódio no ensaio composto por 10% de sal e 18% de PEG 8000 (g/mol). Recuperações superiores a 80% foram obtidas. A protease fibrinolítica apresentou pH ótimo 7,0, estabilidade entre os pH 6,0 e 8,5, temperatura ótima 50°C, sendo estável de 10°C a 50°C. A enzima foi classificada como uma serino protease, com massa molecular de 52kDa. Como resultado, o processo é notavelmente eficaz para pré-purificar a protease fibrinolítica com baixo custo e rapidez significativa. Quando comparada a outras técnicas de produção e purificação isoladas, a fermentação extrativa é um processo digno a ser substituto das etapas iniciais de separação convencionais.(AU)

Fibrinolytic enzymes can be obtained from microorganisms through fermentative processes. The study aimed to evaluate the fibrinolytic protease production and integrated extraction from Mucor subtilissimus UCP 1262 by extractive fermentation using Aqueous Two-Phase Systems (ATPS). The integrated process was carried out to assess the production, partition and fibrinolytic enzyme recovery, according to a 2 3 -experimental design, using as independent variables Polyethylene glycol (PEG) molar mass, PEG and sodium sulphate concentration, concentration. The highest fibrinolytic activity (15.40U/mL) was obtained in sodium sulfate rich phase in the assay comprising of 10% of salt and 18% of PEG 8000 (g/mol). Yield greater than 80% was obtained. The fibrinolytic protease presented optimum pH 7.0 and stability between pH 6.0 and 8.5, and optimum temperature 50°C, stable between 10°C to 50°C. The enzyme was classified as a serine-protease with 52kDa of molecular weight. As a result, the process is remarkably effective to pre-purify the fibrinolytic protease with a low cost and significantly faster processing time. When compared to other isolated production and purification techniques the extractive fermentation is worthy of being a candidate to replace the initial stages of conventional separation processes.(AU)

Isolation of polysaccharides from tea and their hypoglycemic activity / 中草药

Objective To isolate water-soluble neutral polysaccharides(NTPS) and acidic polysaccharides(ATPS) from tea,investigate their hypoglycemic effects,and elucidate the relationship between structure and activity.Methods Weak base macroporous anion-exchange resin D315 was used to isolate two kinds of tea polysaccharides NTPS and ATPS based on electric charge of tea polysaccharides.Their monosaccharide compositions were analyzed by gas chromatography and ion chromatography.NTPS and ATPS were further isolated by DEAESepharose FF.Molecular weights of the main fractions were analyzed by HPGPC.The model of diabetic mellitus was established by ip alloxan in mice.The blood samples were collected with tail vein puncture to determine blood glucose.The hypoglycemic activity between neutral and acidic polysaccharides was compared after ig tea polysaccharides for 12 d successively.Results Both NTPS and ATPS were(heteropolysaccharides.) NTPS was mainly composed of neutral polysaccharide and its main monopolysaccharide was D-galactose.NTPS contained 6.82% galacturonic acid.Whereas,ATPS was mainly composed of acidic polysaccharide and contained 33.02% D-galactose,whose neutral polysaccharide was mostly(L-)rhamnose,D-arabinose and D-galactose. The molecular weights of both two kinds of tea polysaccharides were less than 3?10~4.Continuous ig administration of NTPS and ATPS [200 and 400 mg/(kg?d)] for 12 d was found to depress glucose increases in alloxan-dependent diabetic model mice.Conclusion Two different structures of tea polysaccharides NTPS and ATPS are isolated by anion-exchange resin D315 firstly.Both tea polysaccharides show good hypoglycemic effect.