Method of analyzing enzyme compositions with lipolytic, proteolytic and amylolytic activity
AssigneeSolvay Pharmaceuticals GmbH
InventorsAndreas Potthoff, Andreas Koerner, Bernd Thumbeck
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Abstract
A method for analyzing the identity, protein and/or peptide pattern and also the stability of samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, particularly mixtures of digestive enzymes such as pancreatin, for use in manufacturing medicinal products comprising such enzyme mixtures, e.g. precipitated pancreatin or pancreatin mini-microspheres.
Claims22
A method for analyzing a protein sample containing a physiologically acceptable digestive enzyme mixture with lipolytic, proteolytic and amylolytic activity by two-dimensional gel electrophoresis, said method comprising:
- (a) preparing the protein sample by dissolving the sample in a gel electrophoresis solvent composition comprising a protein solving solvent, an internal standard for quantifying proteins, and a protease inhibitor;
- (b) defining the first dimension of the gel electrophoresis by isoelectric focusing, and applying a gradient to separate protein fractions;
- (c) re-buffering the protein fractions;
- (d) transferring the protein fractions from (c) to the second dimension of the gel electrophoresis and separating components of the fractions by SDS-PAGE;
- (e) fixing and staining SDS-PAGE gels resulting from (d);
- and
- (f) evaluating the gels densitometrically by fluorescence scanning.
A solvent composition for two-dimensional gel electrophoresis analysis of enzyme mixtures with lipolytic, proteolytic and amylolytic activity, said solvent composition comprising:
- (a) a protein solving gel electrophoresis solvent comprising a lysis buffer comprised of 7M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte at pH 3-10;
- (b) an internal standard for quantifying proteins;
- and
- (c) a protease inhibitor.
Dependencies are inferred from the claim text.