Method of analyzing enzyme compositions with lipolytic, proteolytic and amylolytic activity
AssigneeSolvay Pharmaceuticals GmbH
InventorsAndreas Potthoff, Andreas Koerner, Bernd Thumbeck
Life of this patent
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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, wherein the mixture can be used in the manufacture of a pharmaceutical preparation, 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);
- (f) evaluating the gels densitometrically by fluorescence scanning;
- and
- (g) determining the identity and absolute quantity of one or more proteins in the digestive enzyme mixture.
A method of comparing a first protein sample to a second protein sample by two-dimensional gel electrophoresis, wherein each protein sample contains a physiologically acceptable digestive enzyme mixture with lipolytic, proteolytic and amylolytic activity and wherein one of the mixtures can be used in the manufacture of a pharmaceutical preparation, said method comprising:
- (a) preparing the first protein sample and second protein sample by separately dissolving each 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 the protein fractions in the first sample and the second sample;
- (c) re-buffering the protein fractions of the first sample and the second sample;
- (d) transferring the protein fractions of the first sample from (c) to the second dimension of the gel electrophoresis and separating components of the fractions by SDS-PAGE;
- (e) transferring the protein fractions of the second sample from (c) to the second dimension of the gel electrophoresis and separating components of the fractions by SDS-PAGE;
- (f) fixing and staining a first SDS-PAGE gel resulting from (d) and a second SDS-PAGE gel resulting from (e);
- and
- (g) comparing the first gel resulting from (f) to the second gel resulting from (f).
Dependencies are inferred from the claim text.