US 12,252,557 B2
Charged surface reversed phase chromatographic materials method for analysis of glycans modified with amphipathic, strongly basic moieties
Matthew A. Lauber, North Smithfield, RI (US); Scott A. McCall, Smithfield, RI (US); Babajide Okandeji, Providence, RI (US); and Pamela C. Iraneta, Brighton, MA (US)
Assigned to Waters Technologies Corporation, Milford, MA (US)
Appl. No. 16/095,919
Filed by Waters Technologies Corporation, Milford, MA (US)
PCT Filed Apr. 21, 2017, PCT No. PCT/US2017/028856
§ 371(c)(1), (2) Date Oct. 23, 2018,
PCT Pub. No. WO2017/189357, PCT Pub. Date Nov. 2, 2017.
Claims priority of provisional application 62/326,783, filed on Apr. 24, 2016.
Prior Publication US 2020/0332028 A1, Oct. 22, 2020
Int. Cl. C07B 37/00 (2006.01); B01D 15/32 (2006.01); B01D 15/36 (2006.01); B01D 15/38 (2006.01); C08B 37/00 (2006.01); G01N 33/58 (2006.01)
CPC C08B 37/006 (2013.01) [B01D 15/327 (2013.01); B01D 15/363 (2013.01); B01D 15/3847 (2013.01); G01N 33/58 (2013.01)] 22 Claims
 
1. A method for selectively isolating glycans from a sample based on the net charge of each glycan, the method comprising:
a) reacting a sample comprising glycans with a labeling reagent to produce labeled glycans;
b) loading the labeled glycans onto a chromatographic separation device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface is charged and comprises a hydrophobic surface group and one or more ionizable modifiers, such that the labeled glycans are selectively adsorbed onto the high purity chromatographic material;
c) selectively eluting the adsorbed labeled glycans from the high purity chromatographic material, the selective elution including:
exposing the labeled adsorbed glycans on the high purity chromatographic material to an aqueous mobile phase, the aqueous mobile phase comprising a concentration of a buffer and a concentration of an organic solvent, increasing the concentration each of the buffer and the organic solvent, thereby varying ionic strength of the aqueous mobile phase, and
selectively separating the labeled glycans from the high purity chromatographic material based on the net charge of each glycan and the ionic strength of the aqueous mobile phase;
wherein the organic solvent comprises water and acetonitrile and the buffer is selected from the group consisting of a first buffer comprising ammonium formate and formic acid, a second buffer comprising ammonium formate and acetic acid, and a third buffer comprising ammonium acetate and acetic acid.