Deglycosylation Reagents and Methods
Compositions and methods are provided for efficiently preparing a completely deglycosylated antibody where efficiency is measured in relative amounts of reagents in soluble or lyophilized form, and time and temperature of the reaction. Compositions and methods are also provided for separating substantially all N-linked glycans from a glycosylated antibody and for preserving functionality of the antibody. The methods are compatible with glycan labeling and protease digestion without the need for prior purification steps.
1 . A composition, comprising: (i) a bile salt or a detergent not including sodium dodecyl sulfate (SDS); (ii) one or more glycosidases; (iii) a completely deglycosylated antibody as determined by electrophoresis or by mass spectrometry; and (iv) glycan cleavage products.
2 . A composition according to claim 1 , wherein the detergent is a dialyzable non-cleavable carboxylated anionic surfactant.
3 . A composition according to claim 1 , wherein the completely deglycosylated antibody has antigen binding activity.
4 . A composition according to claim 3 , wherein the deglycosylated antibody is selected from deglycosylated human IgG1, human IgG2, human IgG3, human IgG4, human IgM, human IgA1, human IgA2, human IgE, murine IgG1, murine IgG2a and murine IgA.
5 . A composition according to claim 3 , wherein the completely deglycosylated antibody is suitable for use in immunoassays.
6 . A composition according to claim 1 , wherein the glycan cleavage products and/or the antibody are labeled with a fluorescent label, a radioisotope, methyl acetyl, an antibody or a combination thereof.
7 . A composition according to claim 1 , further comprising a protease.
8 . A composition according to claim 7 , wherein the protease is trypsin.
9 . A composition according to claim 7 , wherein the protease is selected from trypsin, GluC, AspN, proteinase K, Factor Xa, Enterokinase, LysC, Arg-C, LysN, IdeS, V-8 Protease, Papain, Alpha-Lytic Protease, Pyroglutamate Aminopeptidas, Leucine Aminopeptidase, Methionine Aminopeptidase, Aminopeptidase I, Aminopeptidase A, Carboxypeptidases (A, B, G, Y), pepsin, Cathepsins (B, C, D), α-Chymotrypsin, TEV, thrombin, IdeZ and IdeE.
10 . A composition according to claim 1 , wherein one or more of the glycosidases is a fusion protein.
11 . A composition according to claim 10 , wherein the glycosidase fusion protein is immobilized on a matrix.
12 . A composition according to claim 10 , wherein the fusion protein comprises a mutant O6-alkylguanine-DNA-alkyltransferase (AGT) and optionally is immobilized through affinity binding of the AGT to a matrix.
13 . A composition according to claim 1 , wherein the composition comprises an aqueous buffer.
14 . A composition, comprising: (a) a bile salt or a dialyzable non-cleavable carboxylate anionic surfactant; (b) one or more glycosidases; (c) a completely deglycosylated biologically active protein as determined by electrophoresis or by mass spectrometry; and (d) glycan cleavage products.
15 . A composition according to claim 14 , wherein the one or more glycosidase are a plurality of glycosidases.
16 . A composition according to claim 15 , wherein the plurality of glycosidases comprise exoglycosidases and/or endoglycosidases.
17 . A composition according to claim 14 , further comprising a protease.
18 . A composition according to claim 17 , wherein the protease is trypsin
19 . A method, comprising
(a) incubating a composition comprising a bile salt or a dialyzable non-cleavable carboxylate surfactant, not including sodium dodecyl sulfate (SDS); and one or more glycosidases with a glycosylated antibody for less than 60 minutes; and
(b) completely cleaving glycans from the glycosylated antibody to form a deglycosylated antibody and glycan cleavage products.
20 . The method according to claim 19 , further comprising isolating the deglycosylated antibody or cleaved glycan products.
21 . The method according to claim 19 , further comprising characterizing the antibody and/or the glycan cleavage products.
22 . The method according to claim 19 , further comprising determining the antigen binding activity of the deglycosylated antibody.
23 . The method according to claim 22 , comprising determining an activity of the antibody by an antibody-antigen binding assay selected from a radioimmune assay, an ELISA, an affinity binding assay, or an immunoprecipitation assay.
24 . The method according to claim 19 , further comprising (c) obtaining deglycosylated antibody for therapeutic use.
25 . The method according to claim 19 , further comprising (c) obtaining deglycosylated antibody for diagnostic use.
26 . The method according to claim 19 , wherein the composition further comprises a protease for forming a mixture of peptide fragments and glycan cleavage products.
27 . The method according to claim 19 , wherein the composition is lyophilized prior to incubating with glycosylated antibody wherein the glycosylated antibody is in an aqueous buffer.
28 . The method according to claim 19 , wherein the glycosidase is immobilized on a matrix.
29 . The method according to claim 19 , wherein the incubating is at a temperature of about 20° C.-60° C.
30 . A kit comprising a lyophilized glycosidase, an immobilized glycosidase, or a lyophilized buffer, or a combination thereof, wherein the lyophilized buffer comprises: a bile acid; or a dialyzable non-cleavable carboxylate anionic surfactant, excluding SDS; or a combination thereof.
31 . A kit according to claim 30 , further comprising a protease.