# US 2021/0239663 A1 · Use of liquid chromatography and mass spectrometry to characterize oligonucleotides

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- Number: US 2021/0239663 A1
- Document ID: US20210239663A1
- Status: Published application
- Kind: A1
- Application number: 17/161,803
- Filed: 2021-01-29
- Published: 2021-08-05
- Earliest claimed: 2020-01-31
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Ming HUANG; Haibo QIU; Xiaobin XU; Ning LI
- Web page: https://patentgrep.com/patent/US20210239663A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 30/88: Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- H01J 49/004: {Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn}
- G01N 2030/8813: {biological materials}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)

## Abstract

The disclosure provides methods of characterizing a sample of oligonucleotides of interest using liquid chromatography and mass spectrometry.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample further comprises at least one impurity comprising at least one additional population of oligonucleotides.

### Claim 3 (depends on 2)

The method of claim 2, wherein the additional population of oligonucleotides comprises a fragmentation product of or synthesis byproduct of the oligonucleotides of interest.

### Claim 4 (depends on 2)

The method of claim 2, further comprising generating a mass spectrogram corresponding to the at least one additional population of oligonucleotides and determining the percentage of total oligonucleotides in the sample corresponding to the at least one additional population of oligonucleotides.

### Claim 5 (depends on 1)

The method of claim 1, wherein the oligonucleotides of interest are deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or DNA-RNA hybrids.

### Claim 6 (depends on 1)

The method of claim 1, wherein the oligonucleotides of interest are single stranded or double stranded.

### Claim 7

(canceled)

### Claim 8 (depends on 1)

The methods of claim 1, wherein the oligonucleotides of interest comprise a hairpin or stem-loop structure.

### Claim 9 (depends on 1)

The methods of claim 1, wherein the oligonucleotides of interest are between 15 and 100 nucleotides in length.

### Claim 10 (depends on 1)

The method of claim 1, wherein the oligonucleotides of interest are therapeutic oligonucleotides.

### Claim 11 (depends on 10)

The method of claim 10, wherein the therapeutic oligonucleotides comprise antisense oligonucleotides (ASO), dsRNAs, siRNAs, aptamers or microRNAs.

### Claim 12 (depends on 1)

The method of claim 1, wherein the oligonucleotides of interest comprise at least one modification.

### Claim 13 (depends on 12)

The method of claim 12, wherein the at least one modification is at the 5′ end, the 3′ end, an internal nucleobase, or a combination thereof, of the oligonucleotide of interest.

### Claim at position 14

14-15. (canceled)

### Claim 16 (depends on 12)

The method of claim 12, wherein the at least one modification comprises a locked nucleic acid (LNA), a phosphorothioate (PS) linkage, a terminal 5′ or 3′ phosphate (PO), a 5′ methyl (5-Me) modification, a 2′-O-Methyl (2′-O-Me) modification, a 2′-O-methoxyethyl (2′-MOE) modification, a constrained ethyl (cET) nucleoside analog, polyethylene glycol (PEG) or a combination thereof.

### Claim 17 (depends on 1)

The method of claim 1, wherein the liquid chromatography comprises hydrophilic interaction liquid chromatography (HILIC) or Ion-pairing Reversed-Phase Liquid Chromatography (IP-RPLC).

### Claim 18 (depends on 17)

The method of claim 17, wherein the HILIC comprises a mobile phase buffer comprising ammonium acetate or ammonium formate.

### Claim 19 (depends on 18)

The method of claim 18, wherein the mobile phase comprises a first buffer comprising 15 mM ammonium acetate in 70% acetonitrile (ACN) and a second buffer comprising 15 mM ammonium acetate in 30% (ACN).

### Claim 20

(canceled)

### Claim 21 (depends on 18)

The method of claim 18, wherein the mobile phase comprises a first buffer comprising 15 mM ammonium formate in 70% acetonitrile (ACN) and a second buffer comprising 15 mM ammonium formate in 30% (ACN).

### Claim 22 (depends on 17)

The method of claim 17, wherein HILIC separation comprises a column temperature of between 23 and 50° C.

### Claim 23

(canceled)

### Claim 24 (depends on 17)

The method of claim 17, wherein the HILIC comprises a column with a solid phase with a mean nominal particle size of 3 μm, a median particle pore size of 200 Å, a 2 mm inner diameter, and a 150 mm length column.

### Claim 25 (depends on 17)

The method of claim 17, wherein the IP-RPLC comprises a mobile phase buffer comprising Hexafluoroisopropanol (HFIP) and 5 mM N,N-Diisopropylethylamine (DIEA).

### Claim 26 (depends on 25)

The method of claim 25, wherein the mobile phase comprises a first buffer comprising 50 mM HFIP and 5 mM DIEA in water and a second buffer comprising 50 mM HFIP and 5 mM DIEA in acetonitrile.

### Claim 27 (depends on 17)

The method of claim 17, wherein the IP-RPLC comprises a column with a mean nominal particle size of 1.7 μm, a median particle pore size of 130 Å, a column length 100 mm and a 2.1 mm inner diameter.

### Claim 28

(canceled)

### Claim 29 (depends on 1)

The method of claim 1, wherein the mass spectrometry comprises electrospray ionization (ESI).

### Claim at position 29

30-31. (canceled)

### Claim 32 (depends on 1)

The method of claim 1, wherein the mass spectrometry is tandem mass spectrometry (MS/MS).

### Claim 33

(canceled)

### Claim 34 (depends on 32)

The method of claim 32, wherein the MS/MS comprises fragmentation of the population of oligonucleotides of interest, the at least additional population of oligonucleotides, or a combination thereof.

### Claim 35 (depends on 34)

The method of claim 34, wherein the fragmentation comprises higher-energy collisional dissociation (HCD) comprising a normalized collisional energy (NCE) of 15% to 35%.

### Claim at position 34

36-37. (canceled)

### Claim 38 (depends on 1)

The method of claim 1, wherein step (c) comprises determining the intact mass of the oligonucleotides of interest.

### Claim 39 (depends on 2)

The method of claim 2, wherein step (c) further comprises determining the intact mass of the at least one additional population of oligonucleotides.

### Claim 40 (depends on 1)

The method of claim 1, wherein step (c) comprises determining the structure of the oligonucleotides of interest using mass spectrometry.

### Claim 41 (depends on 2)

The method of claim 2, wherein step (c) further comprises determining the structure of the at least one additional population of oligonucleotides.

### Claim 42

(canceled)

### Claim 43 (depends on 1)

A method of making a composition comprising an oligonucleotide of interest comprising:
a. synthesizing the oligonucleotide of interest; and
b. characterizing the oligonucleotide of interest using the methods of of claim 1.

### Claim 44 (depends on 43)

The method of claim 43, wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% of the total oligonucleotides in the composition are the oligonucleotide of interest.

### Claim 45

(canceled)

### Claim 46 (depends on 43)

A composition, comprising oligonucleotides of interest made by the methods of claim 43.

### Claim 47 (depends on 46)

A method of treating a subject in need thereof, comprising administering the composition of claim 46.

### Claim at position 45

48-49. (canceled)

### Claim 50

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.

### Claim at position 47

51-70. (canceled)

### Claim 71 (depends on 50)

A method of making a composition comprising an oligonucleotide of interest comprising:
a. synthesizing the oligonucleotides of interest thereby generating a sample comprising a population of the oligonucleotides of interest; and
b. characterizing the sample using the methods of claim 50.