Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Abstract

Background: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and posttranslational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for largescale expression of cytokines typically results in much lower volumetric productivity.

O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

“…O-linked glycosylated IFN α-2b produced in a human kidney (HEK293) cell expression system was produced as already described (8). Protein concentrations were measured using a BCA assay kit from Sigma (St. Louis, USA).…”

“…Recently, Loignon and coworkers developed a high-yield process for the production of an O-linked glycosylated human recombinant IFN α-2b in the human embryonic kidney cell line HEK293 (8). This platform offers advantages, since naturally occurring IFN α-2 is O-glycosylated, whereas recombinant IFN α-2b produced in a bacterial expression system is not (9).…”

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

“…Interferon-α2b has only a single glycan, an O-linked T-antigen structure which is disialylated [11]. The linkage of the Galβ1,3GalNAc disaccharide to the inner region of the ganglioside in GM1a is β1,4 whereas in the O-linked structure (T-Ag) on glycoproteins it is attached through an α1,O linkage.…”

“…ST3Gal1 and CMP-Leg5Ac7Ac were produced as previously described [1] as was interferonα2b in HEK293 cells [11]. The α 1 -antitrypsin was purchased from r-Peptide (Bogart GA) and the GM1a was a gift from Dr Shawn Defrees (Neose Technologies Inc.).…”

Expressão gênica empregando pseudopartículas em células de mamíferos (HEK 293T e Huh 7.0) cultivadas em diferentes meios de cultura livres de soro.

“…A célula HEK 293, é uma linhagem derivada de células embrionárias humanas de rim, transformada com adenovírus tipo 5 humano, foi desenvolvida há 37 anos (GRAHAM et al, 1977), mas apenas nos últimos anos tem sido extensivamente usada, sendo a linhagem humana provavelmente mais utilizada para pesquisas relacionadas a expressão de proteínas recombinantes (ADAM et al, 2008;DUMONT et al, 2012;FISCHER et al, 2012;LOIGNON et al, 2008;SUN et al, 2006;SWIECH et al, 2011;WAJIH;OWEN;WALLIN, 2008). Esta linhagem exibe uma morfologia epitelial e uma das razões para sua larga aplicação é o fato destas células crescerem facilmente em suspensão e em SFM disponíveis comercialmente (DUROCHER; BUTLER, 2009;LIU et al, 2006;MEI et al, 2006;ZHU, 2012 VAN DER VALK et al, 2004).…”