# US 12,000,915 B2 · Method for conducting solid state NMR on macromolecule-containing solid state formulations

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,000,915 B2
- Document ID: US12000915B2
- Status: Granted
- Kind: B2
- Application number: 17/766,055
- Filed: 2020-10-02
- Published: 2022-11-17
- Granted: 2024-06-04
- Earliest claimed: 2019-10-03
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Marc A. Caporini; Ron C. Kelly; Mariana De Maillé
- Web page: https://patentgrep.com/patent/US12000915B2

## CPC

- G01R 33/448: {Relaxometry, i.e. quantification of relaxation times or spin density (G01R33/50 takes precedence)}
- G01N 24/08: by using nuclear magnetic resonance (G01N24/12 takes precedence)
- A61K 9/19: lyophilised {, i.e. freeze-dried, solutions or dispersions (lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00)}
- G01R: MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES (indicating correct tuning of resonant circuits H03J3/12)
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Disclosed herein is a method of conducting direct detection 1H solid state NMR (“ssNMR”) on a macromolecule-containing solid state formulation. The method includes conducting a 1H spin-lattice relaxation time (“T1”) experiment on the solid state formulation at various temperatures to generate a T1 value at each temperature, converting the T1 values to 1H spin-lattice relaxation rate (“R1”), and plotting R1 versus temperature to generate a relaxation rate curve for the solid state formulation. The relaxation rate curve can be analyzed to determine the molecular mobility of the macromolecule in the solid state formulation, the degree of aggregation in the solid state formulation, and/or the stability of the solid state formulation.

## Claims

### Claim 1

A method of conducting direct detection 1H solid state NMR (“ssNMR”) on a macromolecule-containing solid state formulation, the method comprising:
(a) equilibrating a solid state formulation comprising a macromolecule at a first temperature;
(b) conducting a 1H spin-lattice relaxation time (“T1”) experiment on the solid state formulation at the first temperature using direct detection 1H ssNMR to generate at least three free induction decay (“FID”) plots at the first temperature, wherein the T1 experiment comprises baseline suppression and a saturation recovery sequence having at least three variable delay times from which each FID plot is generated;
(c) equilibrating the solid state formulation at a second temperature, and repeating step (b) at the second temperature to generate at least three FID plots at the second temperature;
(d) equilibrating the solid state formulation at a third temperature, and repeating step (b) at the third temperature to generate at least three FID plots at the third temperature;
(e) generating a saturation recovery curve at each temperature; and
(f) fitting each saturation recovery curve to a nonlinear regression equation to generate a T1 value at each temperature.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
(g) converting each T1 value to 1H spin-lattice relaxation rate (“R1”), and
(h) plotting R1 versus temperature to generate a relaxation rate curve for the solid state formulation; and
analyzing the relaxation rate curve to determine the molecular mobility of the macromolecule in the solid state formulation, the degree of aggregation in the solid state formulation, and/or the stability of the solid state formulation.

### Claim 3 (depends on 1)

The method of claim 1, wherein the highest and lowest temperatures have a difference of at least about 25° C.

### Claim 4 (depends on 1)

The method of claim 1, wherein said method excludes one or both of retuning and recalibrating the ssNMR probe after equilibrating at the first temperature.

### Claim 5 (depends on 1)

The method of claim 1, wherein each T1 experiment comprises at least five variable delay times at each temperature to generate at least five FID plots at each temperature.

### Claim 6 (depends on 1)

The method of claim 1, wherein the saturation recovery curve is generated by:
(a) plotting the signal intensity of each of the at least three FID plots versus delay time; or
(b) Fourier transforming each of the at least three FID plots to generate a plot of intensity versus frequency; and
(i) plotting peak height versus delay time; or
(ii) plotting integral peak intensity versus delay time.

### Claim 7 (depends on 1)

The method of claim 1, wherein the T1 experiment comprises magic angle spinning.

### Claim 8 (depends on 1)

The method of claim 1, wherein all of the T1 experiments are conducted within a time period of up to 48 hours.

### Claim 9 (depends on 1)

The method of claim 1, wherein macromolecule is a biologic molecule.

### Claim 10 (depends on 1)

The method of claim 1, wherein the solid state formulation is a lyophilized formulation and the fitting of step (f) is monoexponential.

### Claim 11 (depends on 10)

The method of claim 10, wherein the highest and lowest temperatures have a difference of at least about 50° C.

### Claim 12 (depends on 10)

The method of claim 10, wherein the T1 experiment comprises a variable delay period in a range from about 0.01 seconds to about 60 seconds.

### Claim 13 (depends on 1)

The method of claim 1, wherein the solid state formulation is a frozen formulation and the fitting of step (f) is biexponential.

### Claim 14 (depends on 13)

The method of claim 13, wherein the highest and lowest temperatures have a difference of at least about 40° C.

### Claim 15 (depends on 13)

The method of claim 13, wherein each T1 experiment comprises nine variable delay times at each temperature to generate nine FID plots at each temperature.

### Claim 16 (depends on 13)

The method of claim 13, wherein the T1 experiment comprises a variable delay period in a range from about 0.1 seconds to about 240 seconds.

### Claim 17 (depends on 1)

The method of claim 1, wherein the solid state formulation is a lyophilized formulation and:
the saturation recovery curve is generated by plotting the signal intensity of each of the at least three FID plots versus delay time;
the T1 experiment comprises baseline suppression and magic angle spinning;
steps (d)-(f) are repeated at 15 or more temperatures;
each temperature is in a range from about −50° C. to about 150° C.;
the highest and lowest temperatures have a difference from about 75° C. to about 100° C.;
in each equilibrating step the solid state formulation is held at the temperature for a duration in the range of about one minute to about ten minutes before conducting the T1 experiment;
the method excludes retuning and recalibrating the ssNMR probe after equilibrating at the first temperature;
the variable delay period is in a range from about 0.1 seconds to about 60 seconds; and
each T1 experiment comprises six variable delay times at each temperature to generate six FID plots at each temperature.

### Claim 18 (depends on 1)

The method of claim 1, wherein the solid state formulation is a frozen formulation and:
the saturation recovery curve is generated by plotting the signal intensity of each of the at least three FID plots versus delay time;
the T1 experiment comprises baseline suppression and magic angle spinning;
steps (d)-(f) are repeated at 25 or more temperatures;
each temperature is in a range from about −50° C. to about 0° C.;
the highest and lowest temperatures have a difference from about 25° C. to about 40° C.;
in each equilibrating step the solid state formulation is held at the temperature for a duration in the range of about one minute to about ten minutes before conducting the T1 experiment;
the method excludes retuning and recalibrating the ssNMR probe after equilibrating at the first temperature;
the variable delay period is in a range from about 0.01 seconds to about 240 seconds; and
each T1 experiment comprises nine variable delay times at each temperature to generate nine FID plots at each temperature.

### Claim 19

A method of selecting a macromolecule-containing solid state formulation among a group of test macromolecule-containing solid state formulations, the method comprising:
(I) generating a relaxation rate curve for each macromolecule-containing solid state formulation in the group of test macromolecule-containing solid state formulations; wherein the relaxation rate curve for each macromolecule-containing solid state formulation is generated by:
(a) equilibrating the solid state formulation at a first temperature;
(b) conducting a 1H spin-lattice relaxation time (“T1”) experiment on the solid state formulation at the first temperature using direct detection 1H ssNMR to generate at least three free induction decay (“FID”) plots at the first temperature, wherein the T1 experiment comprises baseline suppression and a saturation recovery sequence having at least three variable delay times from which each FID plot is generated;
(c) equilibrating the solid state formulation at a second temperature, and repeating step (b) at the second temperature to generate at least three FID plots at the second temperature;
(d) equilibrating the solid state formulation at a third temperature and repeating step (b) at the third temperature to generate at least three FID plots at the third temperature;
(e) generating a saturation recovery curve at each temperature;
(f) fitting each saturation recovery curve to a nonlinear regression equation to generate a T1 value at each temperature;
(g) converting each T1 value to 1H spin-lattice relaxation rate (“R1”), and
(h) plotting R1 versus temperature to generate a relaxation rate curve for the solid state formulation;
(II) comparing the maximum R1 peak value of each curve, the temperature of the maximum R1 peak of each curve, the width of the maximum R1 peak of each curve, or a combination thereof; and
(III) selecting the solid state formulation which has the lowest maximum R1 peak value, the highest temperature of the maximum R1 peak, or the narrowest R1 peak width.

### Claim 20

A method of selecting a formulation excipient for use in a macromolecule-containing solid state formulation, the method comprising:
(I) generating a relaxation rate curve for each macromolecule-containing solid state formulation in a group of test macromolecule-containing solid state formulations, each formulation having a different composition of excipients, a different amount of one or more excipients, or both; wherein the relaxation rate curve for each macromolecule-containing solid state formulation is generated by:
(a) equilibrating the solid state formulation at a first temperature;
(b) conducting a 1H spin-lattice relaxation time (“T1”) experiment on the solid state formulation at the first temperature using direct detection 1H ssNMR to generate at least three free induction decay (“FID”) plots at the first temperature, wherein the T1 experiment comprises baseline suppression and a saturation recovery sequence having at least three variable delay times from which each FID plot is generated;
(c) equilibrating the solid state formulation at a second temperature, and repeating step (b) at the second temperature to generate at least three FID plots at the second temperature;
(d) equilibrating the solid state formulation at a third temperature, and repeating step (b) at the third temperature to generate at least three FID plots at the third temperature;
(e) generating a saturation recovery curve at each temperature;
(f) fitting each saturation recovery curve to a nonlinear regression equation to generate a T1 value at each temperature;
(g) converting each T1 value to 1H spin-lattice relaxation rate (“R1”), and
(h) plotting R1 versus temperature to generate a relaxation rate curve for the solid state formulation;
(II) comparing the maximum R1 peak value of each curve, the temperature of the maximum R1 peak of each curve, the width of the maximum R1 peak of each curve, or a combination thereof; and
(III) selecting an excipient that is present in the solid state formulation that has the lowest maximum R1 peak value, the highest temperature of the maximum R1 peak, or the narrowest R1 peak width.