Author/Editor		Marinko, Petra; Byrne-Habič, Barbara B; Cvelbar, Martina; Krbavčič, Aleš
Title		Optimisation of polycrylamide gel electrophoresis as a method for the identifiaction and purity determination of recombinant human interferon alpha2 in pharmaceutical products
Translated title		Optimizacija metode poliakrilamidne gelske elektroforeze za identifikacijo in določanje čistoče rekombinantnega človeškega interferona alfa2 v farmacevtskih izdelkih
Type		članek
Source		Acta Pharm Zagreb
Vol. and No.		Letnik 50, št. 3
Publication year		2000
Volume		str. 209-18
Language		eng
Abstract		The SDSPage technique for determination of the commercially available therapeutic proteins interferon alfa 2a and 2b was studied. The aim of the study was to estimate whether the requirements of the European Pharmacopoeia for IFN-alpha2 concentrated solution could also be applied to finished products. Different staining procedures and gel concentrations were tested for maximum sensitivity and protein separation. A 14% acrylamide/bisacrylamide gel concentration was found to be optimal. The silver staining procedure according to Blum et al. (13) provided high sensitivity down to 1 ng on a colourless, transparent background. Detection with Coomassie Brilliant Blue staining achieved a sensitivity of approximately 17 ng, making the identification of IFN-alphfa2 in lyophilised products still possible. The molecular mass was determined by densitometry using a four-parameter regression analysis and compared to the pharmacopoeial standard. Solutions of IFN-alpha2 contain no human serum albumin which interferes with the detection of IFN-alpha2 impurities and can therefore be examined for purity as well as identity. However, strict pharmacopoeial requirements could only be followed when analysing solutions of 15.10 expression six IU mL-1 or higher concentrations, using sensitive silver staining. Impurities of molecular mass higher than that of IFN-alpha2 were detected in the samples under non-reducing conditions, whereas impurities of molecular mass lower than that of IFN-alpha2 were observed in almost all of the samples under reducing conditions. The findings of this study have been adopted for routine analysis of IFN-alpha2 products.
Summary		Proučevali smo metodo SDS-PAGE za določanje identitete in čistote komercialno dostopnih terapevtskih proteinov IFN-alfa2a in IFN-alfa2a. S študijo smo želeli ugotoviti, ali se zahtevam evropske farmakopeje za koncentrirano raztopino IFN-alfa2 lahko zadosti tudi pri kontroli končnega izdelka. Preizkušali smo različne postopke barvanja in različne koncentracije gela, da bi dosegli optimalno občutljivost in ločitev lis. Kot najprimernejši se je za ločbo izkazal 14% akrilamid/bisakrilamidni gel. Barvanje s srebrom po Blumu et al. (13) zagotavlja občutljivost do 1 ng na brezbarvnem, transparentnem ozadju. Občutljivost pri detekciji z briliantno modrim je približno 17 ng, kar omogoča identifikacijo IFN-alfa2 v liofiliziranih izdelkih. Molekulska masa IFN-alfa2 je bila določena denzitometrično glede na farmakopejski standard s štiri parametrsko regresijsko analizo. Raztopine IFN-alfa2 ne vsebujejo humanega serumskega albumina, ki moti zaznavanje IFN-alfa2 nečistot in jim zato lahko določamo poleg identitete tudi čistoto. Stroge farmakopejske zahteve lahko upoštevamo le pri elektroforezi raztopin jakosti vsaj 15.10 na 6 IU mL-1 barvanih s srebrom. Samo v omenjenih vzorcih smo v nereducirajočih pogojih zaznali nečistote z molekulsko maso višjo od IFN-alfa2. Nečistote z molekulsko maso nižjo od IFN-alfa2 pa smo v reducirajočih pogojih opazili v skoraj vseh vzorcih. Rezultate našega dela smo vključili v redno analizo IFN-alfa2 farmacevtskih izdelkov.
Descriptors		INTERFERON ALFA-2A INTERFERON ALFA-2B ELECTROPHORESIS, POLYACRYLAMIDE GEL DRUG CONTAMINATION SILVER STAINING