2D-DIGE As a Tool to Analyze Lot-to-Lot Consistency of Complex Therapeutic Products Such As BAX 855, a PEGylated Recombinant FVIII

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4360-4360
Author(s):  
Hanspeter Rottensteiner ◽  
Christina Monetti ◽  
Herbert Gritsch ◽  
Alfred Weber ◽  
Hartmut J. Ehrlich ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Hemophilia A ◽  
2D Dige ◽  
Straightforward Method ◽  
Difference Gel Electrophoresis ◽  
Identical Composition ◽  
Dimension 2 ◽  
Recombinant Fviii ◽  
And Control

Abstract Abstract 4360 2-Dimensional difference gel electrophoresis (2-D DIGE) is a method that circumvents the gel-to-gel variability inherent in conventional 2-dimensional gel electrophoresis (2-DE). We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The FVIII heterodimer is composed of heterogenous, strongly glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments and their identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII’s glycans due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate BAX 855, a PEGylated longer-acting variant of recombinant FVIII. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool to characterize and control quality of very complex pharmaceutically active ingredients such as PEGylated proteins. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment. Monetti:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

scholarly journals Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE)

2007 ◽  
Vol 45 (12) ◽  
pp. 2372-2380 ◽  
Author(s):  
D.J. Hobson ◽  
P. Rupa ◽  
G.J. Diaz ◽  
H. Zhang ◽  
M. Yang ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
2D Dige ◽  
Difference Gel Electrophoresis

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome

Platelets ◽  
2008 ◽  
Vol 19 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Anna Della Corte ◽  
Norma Maugeri ◽  
Agnieszka Pampuch ◽  
Chiara Cerletti ◽  
Giovanni de Gaetano ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Human Platelet ◽  
2D Dige ◽  
Difference Gel Electrophoresis

scholarly journals Calreticulin as A Novel Potential Metastasis-Associated Protein in Myxoid Liposarcoma, as Revealed by Two-Dimensional Difference Gel Electrophoresis

Proteomes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 13
Author(s):  
Takashi Tajima ◽  
Fusako Kito ◽  
Akihiko Yoshida ◽  
Akira Kawai ◽  
Tadashi Kondo
Keyword(s):  
Gel Electrophoresis ◽  
Expression Profiles ◽  
Myxoid Liposarcoma ◽  
Clinical Applications ◽  
Two Dimensional ◽  
2D Dige ◽  
Tumor Tissues ◽  
Difference Gel Electrophoresis ◽  
Protein Expression Profiles

Myxoid liposarcoma (MLS) is a mesenchymal malignancy. To identify innovate seeds for clinical applications, we examined the proteomes of primary tumor tissues from 10 patients with MLS with different statuses of postoperative metastasis. The protein expression profiles of tumor tissues were created, and proteins with differential expression associated with postoperative metastasis were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The validation was performed using specific antibodies and in vitro analyses. Using 2D-DIGE, we observed 1726 protein species and identified proteins with unique expression levels in metastatic MLS. We focused on the overexpression of calreticulin in metastatic MLS. The higher expression of calreticulin was confirmed by Western blotting, and gene silencing assays demonstrated that reduced expression of calreticulin inhibited cell growth and invasion. Our findings suggested the important roles of calreticulin in MLS metastasis and supported its potential utility as a prognostic biomarker in MLS. Further investigations of the functional properties of calreticulin and other proteins identified in this study will improve our understanding of the biology of MLS and facilitate novel clinical applications.

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