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Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Published on: January 8, 2014
Purification and biochemical characterization of functional complement factor H from human plasma fractions
H Brandstätter1, P Schulz, I Polunic
1Octapharma PPGmbH, Research & Development, Vienna, Austria. hubert.brandstaetter@octapharma.com
Background And Objectives:
Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate.
Materials And Methods:
Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry.
Results:
Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays.
Conclusion:
This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies.
