Pegylated Recombinant Granulocyte Colony-Stimulating Factor with Prolonged Mechanism of Action: a New Scheme for Active Pharmaceutical Substance Production

Автор: I.А. Puchkov, D.I. Bairamashvili, I.V. Miagkikh, and V.I. Shvets

Страница: 35-50

Pegylated Recombinant Granulocyte Colony-Stimulating Factor with Prolonged Mechanism of Action: a New Scheme for Active Pharmaceutical Substance Production

Biotekhnologiya, 2014, N 2, P. 35-50

UDC 577.112.083

Section:  “Biologicals Technology”

I.А. Puchkov ^{*, 1},  D.I. Bairamashvili ²,  I.V. Miagkikh ³,  and  V.I. Shvets ⁴

¹  The Russian Pharmaceutical Company Masterclone,  119019, Moscow Russia

²  The Shemyakin-and-Ovchinnikov Institute for Bioorganic Chemistry, Russ. Acad. Sci.,  117997, Moscow Russia

³  The Russian Innovative Pharmaceutical Company Generium,  123317, Moscow Russia

⁴  The Lomonosov Moscow University for Fine Chemical Technology,  119571, Moscow Russia

e-mail:  poutchkov@mail.ru

A production of an active pharmaceutical substance of the PEGylated recombinant granulocyte colony-stimulating factor, G-CSF (Filgrastim), with a prolonged mechanism of action has been carried out in four stages. The first (preliminary) stage included the protein solubilization from inclusion bodies, renaturation and ion exchange chromatography on SP-Sepharose FF at pH 6.0 to obtain a solution with hrG-CSF concentration 12—15 mg/ml. The second stage was performed by the modification of the protein via the selective covalent attachment of α-methyl-PEG-propionaldehyde to α-amino group of the N-terminal methionine in the recombinant G-CSF. The third step consisted of basic purification procedures of PEG-G-CSF by hydrophobic chromatography on Butyl-Sepharose 4 FF at pH 7.0, size-exclusion chromatography on Sephadex G-25 and ion-exchange chromatography on SP-Sepharose FF at pH of 4.5. At the final stage, the obtained preparation (pure according to the European Pharmacopoeia) was transferred to a final buffer by the size-exclusion chromatography on Sephadex G-25. As a result, a technological scheme for the recombinant PEG-G-CSF manufacturing allowing to produce a pharmacopoeia quality preparation with the yield of at least 35% of the initially taken protein was developed.

Kew words:  chromatography,  Filgrastim,  granulocyte colony-stimulating factor,  modification,  poly(ethylene) glycol.

The full English version of the article was published in “Biotechnology in Russia”, 2014, Issue 2, pp. 35-50 as I.А. Puchkov, D.I. Bairamishvili, I.V. Miagkikh, and V.I. Shvets “Pegylated Recombinant Granulocytic Colony-Stimulating Factor with Prolonged Mechanism of Action: a New Scheme for Active Pharmaceutical Substance Production”.

It is contained at the Russian Scientific Electron Library website:  http://elibrary.ru/item.asp?id=24072635

18.03.2015, 1694 просмотра.