Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / hans, and is secreted into the bloodstream. It plays and important role in regulating the metabolic activities of the body, particularly the homeostasis of the blood glucose. Insulin glargine is a recombinant human insulin analogue produced by DNA technology using a strain of Escherichia coli and the insulin glargine differ only by three amino acids from human insulin. Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were developed and validated for the assessment of insulin glargine in biopharmaceutical formulations. A RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 30 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.5, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm x 7.8 mm i.d.), maintained at 25 ºC. The mobile phase consisted of 0.03 M MES acid buffer, pH 2.5, run isocratically at a flow rate of 0.6 mL/min. Chromatographic separation was obtained with retention times of 7.5 min, and 9.9 min, and was linear over the concentration range of 0.05 - 200 μg/mL (R2 = 0.9998) and 0.02 - 180 μg/mL (R2 = 0.9999), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm and 200 nm for RP-LC and SE-LC methods, respectively. The limits of detection and quantitation were 0.018 and 0.054 μg/mL, respectively, for the RP-LC and 0.009 and 0.027 μg/mL, for the SE-LC. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.13% and 99.38%, with bias lower than 0.85% and than 0.86%. The validated methods were applied for the determination of insulin glargine and related proteins and high molecular mass, in biotechnology-derived products, giving lower mean differences of the estimated content/potencies of 0.21% and 0.16% for the RP-LC and SE-LC related, compared to the in vitro cell culture assay. It is concluded that represents a contribution to establish alternatives to monitor stability, quality control and thereby assure therapeutic efficacy of the biotechnology-derived medicine. / A insulina humana é um hormônio secretado pelas células β, das ilhotas de Langerhans, e regula os níveis sanguíneos de glicose. A insulina glargina é produzida pela tecnologia do DNA recombinante, expressa em Escherichia coli, e difere da insulina humana pela modificação da aspargina na posição 21 da cadeia A por uma glicina, além da adição de dois resíduos de arginina C-terminais na cadeia B. No presente trabalho foram desenvolvidos e validados métodos por cromatografia líquida em fase reversa (CL-FR) e por exclusão molecular (CL-EM) para a avaliação de insulina glargina em formulações biofarmacêuticas. No método por CL-FR, foi utilizada coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 30 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,5, e a fase móvel B por acetonitrila, eluídas com fluxo constante de 0,5 mL/min. No método por CL-EM foi utilizada coluna BioSep-SEC-S 2000 (300 mm x 7.8 mm d.i.), mantida a 25 ºC. A fase móvel foi constituída de tampão MES 0,03 M, pH 2,5, eluída em vazão isocrática de 0,6 mL/min. Para ambos os métodos utilizou-se detector de arranjo de diodos (DAD) com detecções em 214 nm e 200 nm para CL-FR e CL-EM, respectivamente. A separação cromatográfica foi obtida nos tempos de 7,5 e 9,9 min, sendo linear na faixa de concentração de 0,05 - 200 μg/mL (R2 = 0,9998) e 0,02 - 180 μg/mL (R2 = 0,9999), respectivamente, para os métodos por CL-FR e CL-EM. Os limites de detecção e quantificação foram 0,018 e 0,054 μg/mL, respectivamente, para o método por CL-FR e 0,009 e 0,027 μg/mL por CL-EM. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,13 e 99,38%, com bias inferior a 0,85 e 0,86%, respectivamente, para os métodos por CL-FR e CL-EM. Os métodos propostos foram aplicados para avaliação da potência de insulina glargina, de proteínas relacionadas agregados de alta massa molecular em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio por cultura de células in vitro, observando-se diferenças das médias de teor/potência 0,21% e 0,16% inferiores para os métodos por CL-FR e CL-EM. Concluí-se que representa contribuição para estabelecer procedimentos para monitorar a estabilidade, o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biotecnológico.

9 Abstract In this Ph.D. thesis possibilities of using our proposed potential programs for a multiple-pulse amperometry and a fast scan differential pulse voltammetry in combination with flow systems are presented. The development of new sensitive amperometric and voltammetric methods for the determination of oxidisable biologically active organic compounds is another aim of this work. In the first part of the work, the flow injection system and multiple-pulse amperometric detection were employed to develop and optimize a simple, low-cost, and rapid method for the simultaneous determination of natural and synthetic antioxidants. This technique involves the application of an appropriate potential waveform consisting of a suitable sequence of pulses on a single working electrode, thus allowing distinguish the analytes in a mixture with no need of separation. Conditions for the determination of antioxidants and modelling of the potential program were tested and studied, respectively. Second part of the work describes and characterizes the application of the fast scan differential pulse voltammetry (FSDPV) in combination with the flow systems. FSDPV is the electroanalytical technique that use high scan rate to record voltammograms within several milliseconds and ensures high temporal resolution. This technique...

9 Abstract In this Ph.D. thesis possibilities of using our proposed potential programs for a multiple-pulse amperometry and a fast scan differential pulse voltammetry in combination with flow systems are presented. The development of new sensitive amperometric and voltammetric methods for the determination of oxidisable biologically active organic compounds is another aim of this work. In the first part of the work, the flow injection system and multiple-pulse amperometric detection were employed to develop and optimize a simple, low-cost, and rapid method for the simultaneous determination of natural and synthetic antioxidants. This technique involves the application of an appropriate potential waveform consisting of a suitable sequence of pulses on a single working electrode, thus allowing distinguish the analytes in a mixture with no need of separation. Conditions for the determination of antioxidants and modelling of the potential program were tested and studied, respectively. Second part of the work describes and characterizes the application of the fast scan differential pulse voltammetry (FSDPV) in combination with the flow systems. FSDPV is the electroanalytical technique that use high scan rate to record voltammograms within several milliseconds and ensures high temporal resolution. This technique...