Dissertação
Desenvolvimento e validação de métodos cromatográficos para avaliação de teor/potência de insulina glargina
Fecha
2016-03-30Registro en:
SCHRAMM, Vanessa Grigoletto. Development and validation of chromatographic methods for the content/potency evaluation of insulin glargine. 2016. 61 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal de Santa Maria, Santa Maria, 2016.
Autor
Schramm, Vanessa Grigoletto
Institución
Resumen
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.