Sequential digestion of polypeptides for mass spectrometric analysis
AssigneeAmgen Inc.
InventorsDa Ren, Melissa Sato
Life of this patent
Estimating the term…
Abstract
The disclosed methods are directed to preparing polypeptides for multi-attribute analysis. The polypeptides are optionally denatured, reduced, and/or alkylated before being subjected to a first digestion. Following the first digestion the large and small fragments resulting from the digestion are separated from each other. A second digestion is then performed on the larger of the fragments. All of the fragments from the two digestions are then analyzed chromatographically, electrophoretically, or spectrometrically, or a combination of these methods. The methods are especially useful for the preparation of therapeutic polypeptides for analysis, especially those that are not easily cleaved.
Claims32
A method of preparing a therapeutic polypeptide for analysis, comprising:
- a. cleaving the polypeptide in a first digestion, wherein the cleaving produces at least two fragments of the polypeptide;
- b. separating the at least two fragments from each other to produce a first large peptide fragment-containing solution and a first small peptide fragment-containing solution;
- c. cleaving the large peptide fragments of the first large peptide fragment-containing solution in a second digestion, wherein at least one of the large peptide fragments of the first large peptide fragment-containing solution is cleaved to produce at least two fragments of the large peptide fragments of the first large peptide fragment-containing solution;
- d. combining the digested solution of c. and the small peptide fragment-containing solution of b;
- and
- e. analyzing the combined solution of d.
A method of preparing a therapeutic polypeptide for analysis, comprising:
- a. cleaving the polypeptide in a first digestion, wherein the cleaving produces at least two fragments of the polypeptide;
- b. separating the at least two fragments from each other to produce a first large peptide fragment-containing solution and a first small peptide fragment-containing solution;
- c. cleaving the large peptide fragments of the first large peptide fragment-containing solution in a second digestion, wherein at least one of the large peptide fragments of the first large peptide fragment-containing solution is cleaved to produce at least two fragments of the large peptide fragments of the first large peptide fragment-containing solution;
- d. separating the at least two fragments of the large peptide fragments from each other to produce a second large peptide fragment-containing solution and a second small peptide fragment-containing solution;
- e. cleaving the large peptide fragments of the second large peptide fragment-containing solution in a third digestion, wherein at least one of the large peptide fragments of the second large peptide fragment-containing solution is cleaved to produce at least two fragments of the large peptide fragments of the second large peptide fragment-containing solution;
- f. combining the digested solution of c., the first small peptide fragment-containing solution of b., and the second small peptide fragment-containing solution of d;
- and
- g. analyzing the combined solution of f.
A method of preparing a therapeutic polypeptide for analysis, comprising:
- a. denaturing, reducing, and alkylating the polypeptide;
- b. digesting the polypeptide with trypsin to produce large trypsin-cleaved polypeptide fragments and small trypsin-cleaved polypeptide fragments;
- c. separating the large and small trypsin-cleaved polypeptide fragments into a first aliquot and a second aliquot, respectively;
- d. digesting the large trypsin-cleaved polypeptide fragments of the first aliquot with neutrophil elastase;
- e. combining the first aliquot and second aliquot at about a 1:1 ratio;
- and
- f. analyzing the combined aliquots of step (e).
Dependencies are inferred from the claim text.