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US 2021/0239663 A1Published application
Use of liquid chromatography and mass spectrometry to characterize oligonucleotides
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsMing HUANG, Haibo QIU, Xiaobin XU, Ning LI
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Abstract
The disclosure provides methods of characterizing a sample of oligonucleotides of interest using liquid chromatography and mass spectrometry.
Claims48
A method of characterizing a sample comprising a population of oligonucleotides of interest, comprising:
- a. providing a sample comprising a population of oligonucleotides of interest of identical sequence;
- b. subjecting the sample to liquid chromatography and mass spectrometry, thereby generating at least one mass spectrogram corresponding to the population of oligonucleotides of interest;
- and
- c. determining a percentage of total oligonucleotides in the sample corresponding to the population of oligonucleotides of interest.
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A method of characterizing a sample, comprising:
- a. providing a sample comprising a population of oligonucleotides of interest of identical sequence and/or modification, and at least one impurity comprising an additional population of oligonucleotides;
- b. subjecting the sample to liquid chromatography and tandem mass spectrometry (MS/MS),
- wherein the liquid chromatography comprises:
- i. hydrophilic interaction liquid chromatography (HILIC) comprising a mobile phase, wherein a first buffer comprises 15 mM ammonium formate or ammonium acetate in 70% acetonitrile (ACN), and a second buffer comprises 15 mM ammonium formate or ammonium acetate in 30% ACN, or
- ii. Ion-pairing Reversed-Phase Liquid Chromatography (IP-RPLC) comprising a mobile phase, wherein a first buffer comprises 50 mM Hexafluoroisopropanol (HFIP) and 5 mM N,N-Diisopropylethylamine (DIEA) in water and a second buffer comprises 50 mM HFIP and 5 mM DIEA in acetonitrile;
- wherein the MS/MS comprises fragmentation of the population of oligonucleotides of interest and the additional population of oligonucleotides in the sample using higher-energy collisional dissociation (HCD) comprising a normalized collisional energy (NCE) of 15% to 35%,
- thereby generating at least one mass spectrogram corresponding to the population oligonucleotides of interest and the additional population of oligonucleotides;
- and
- c. determining a percentage of total oligonucleotides in the sample corresponding to the population of oligonucleotides of interest.
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Dependencies are inferred from the claim text.