RESUMO
The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity. Microchip capillary electrophoresis-mass spectrometry (CE-MS) was subsequently utilised for a more detailed characterization. It was observed that panitumumab is subject to partial N-terminal pyroglutamate formation. Incomplete conversion is uncharacteristic for N-terminally exposed glutamines and in case of panitumumab gives rise to forms which show successive mass offsets of 17 Da, respectively. If not separated before mass spectrometric analysis, e.g. by capillary electrophoresis, such near isobaric species coalesce into single MS peaks, which subsequently hampers or prevents their assignment. With a total of 42 panitumumab isoforms assigned by CE-MS, these observations highlight a potential pitfall of commonly applied rapid identity testing workflows and demonstrate that even low complexity biopharmaceuticals can require separation strategies which offer high separation selectivity for species close in mass.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Anticorpos Monoclonais/química , Panitumumabe , Espectrometria de Massas/métodos , Eletroforese Capilar/métodos , Receptores ErbBRESUMO
Espécies de Acacia são tradicionalmente utilizadas para o tratamento de diversas patologias. Estudos quimiotaxonômicos têm relacionado o gênero Acacia como fonte de compostos fenólicos, os quais apresentam propriedades biológicas diversas. Este trabalho destaca a atividade antioxidante de Acacia podalyriifolia. Esta planta é originária da Austrália e amplamente cultivada no Sul do Brasil como ornamental. O material vegetal (flores) foi seco à sombra, submetido à extração com etanol, seguido de partição com hexano, diclorometano e acetato de etila. A triagem fitoquímica realizada no extrato etanólico bruto e nas suas frações, demonstrou a presença de compostos fenólicos e flavonóides. A determinação do conteúdo fenólico foi realizada com o reativo Folin Ciocalteau, verificando ser a fração acetato de etila (FAE) a que apresentou a maior concentração (338,5 mg de ácido gálico/g de FAE). Na avaliação da atividade captadora de radical, empregando o radical livre DPPH, a fração acetato de etila demonstrou atividade antioxidante mais elevada (IC50 = 3,22 µg/mL de FAE).
Acacia species are usually used as treatment for several diseases. Chemotaxonomic studies have related the Acacia genus as a source of phenolic compounds, which present several biological properties. This work emphasizes the antioxidant activity of Acacia podalyriifolia. This plant is natural from Australia and vastly cultivated in the South of Brazil as ornamental feature. The plant material (flowers) was shadow-dried and submitted to the extraction with ethanol, followed by partition with hexane, dichloromethane and ethyl acetate. The phytochemical screening performed in the ethanol crude extract and in its fractions showed the presence of phenolic compounds and flavonoids. Determination of the phenolic content was performed with the Folin Ciocalteau reagent. It was found out that the ethyl acetate fraction (FAE) was the one that displayed the biggest concentration (338,5 mg of gallic acid/g of FAE). In the evaluation of the radical scavenging activity, using the free radical DPPH, the ethyl acetate fraction showed the highest antioxidant activity (IC50 = 3,22 µg/mL from FAE).
