METHODS FOR IDENTIFYING AND ANALYZING AMINO ACID SEQUENCES OF PROTEINS
The disclosure provides methods for determining the biosimilarity of a test protein in relation to a target biologic in which the test protein is digested by two distinct proteases, the resultant digested peptide fragments are analyzed by column chromatography-tandem mass spectrometry to achieve 100% of the amino acid sequence coverage and 100% amino acid sequence accuracy of the test protein.
1 . A method for determining the biosimilarity of a test protein in relation to a target biologic, the method comprising the steps of:
(a) digesting a first sample of a test protein for a first incubation time using a first protease and digesting a second sample of the test protein for a second incubation time using a second protease, wherein the first sample and the second sample are physically separated;
(b) applying column chromatography and tandem mass spectroscopy to the first sample under conditions sufficient to enhance binding of small peptides to the column, and generating a sequence of the test protein in the first sample;
(c) applying column chromatography and tandem mass spectroscopy to the second sample under conditions sufficient to enhance binding of small peptides to the column, and generating the sequence of the test protein in the second sample, wherein the first sample and second sample are physically separated;
(d) identifying the test protein as biosimilar to the target biologic when the test protein comprises 100% sequence identity to the target biologic; and
(e) identifying the test protein as not biosimilar to the target biologic when the test protein does not comprise 100% sequence identity to the target biologic.
2 . The method of claim 1 , wherein the monoclonal antibody comprises Adalimumab.
3 . The method of claim 1 , wherein the first protease is Trypsin.
4 . The method of claim 1 , wherein the second protease is Chymotrypsin.
5 . The method of claim 1 , wherein the first digestion period is about 0.1 to about 1.0 hour.
6 . The method of claim 5 , wherein the first digestion period is about 0.1 to about 0.5 hour.
7 . The method of claim 5 , wherein the first digestion period is about 0.6 to about 1.0 hour.
8 . The method of claim 5 , wherein the first digestion period is about 0.5 hours.
9 . The method of claim 1 , wherein the second digestion period is about 0.1 to about 2.0 hours.
10 . The method of claim 9 , wherein the second digestion period is about 0.1 to about 1.5 hours.
11 . The method of claim 9 , wherein the second digestion period is about 1.5 to about 2.0 hours.
12 . The method of claim 9 , wherein the second digestion period is about 1.5 hours.