Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE
✍ Scribed by Christina Monetti; Hanspeter Rottensteiner; Herbert Gritsch; Alfred Weber; Friedrich Scheiflinger; Peter L. Turecek
- Publisher
- John Wiley and Sons
- Year
- 2011
- Tongue
- English
- Weight
- 558 KB
- Volume
- 32
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Abstract
2‐D DIGE is a method that circumvents the gel‐to‐gel variability inherent in conventional 2‐DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2‐D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily 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 whose 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 various PEGylated rFVIII conjugates. 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 for quality control of very complex pharmaceutically active ingredients.