Methods directed to crystalline biomolecules

Patent metadata

Field	Value
Publication number	US-12473329-B2
Application number	US-202418603661-A
Country	US
Kind code	B2
Filing date	Mar 13, 2024
Priority date	Mar 14, 2017
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

Disclosed herein are methods of preparing a composition comprising crystalline biomolecules, for example, crystalline antibodies. In exemplary embodiments, the method comprises forming a fluidized bed of crystalline biomolecules using, for example, a counter-flow centrifuge to exchange buffer and/or to concentrate the crystalline biomolecules in a solution. Also provided are methods of detecting crystalline biomolecules and/or amorphous biomolecules in a sample.

First claim

What is claimed is:

1 . A method of detecting crystalline antibodies and/or amorphous antibodies in a sample, the method comprising:
a) obtaining a plurality of 1 H NMR Carr-Purcell-Meiboom-Gill (CPMG) spectra of the sample, and b) obtaining a 13 C NMR cross-polarization (CP) spectra of the sample.

2 . The method of claim 1 , comprising (i) operating a 1H resonance frequency of about 250 to about 1000 MHz during a) of the method, (ii) maintaining the temperature at about 250 to about 350 K, (iii) operating a magic angle spinning (MAS) probe, or (iv) any combination of the foregoing.

3 . The method of claim 2 , wherein the MAS probe comprises at least 2 RF channels.

4 . The method of claim 3 , wherein the MAS probe is tuned to 1 H and 13 C.

5 . The method of claim 3 , wherein the MAS probe is operated with a spinning frequency of about 2 kHz to about 8 kHz.

6 . The method of claim 1 , comprising using 90 degree pulses.

7 . The method of claim 6 , comprising using 1H 90 degree pulses of about 2.5 μs.

8 . The method of claim 1 , wherein the 1H CPMG spectra were obtained with about 5 to about 100 pi pulses of about 2 μs to about 20 μs in length.

9 . The method of claim 8 , wherein each of the 20 pi pulses were separated by about 10 μs to about 1 ms.

10 . The method of claim 9 , wherein the total time of CPMG pulses is about 500 μs to about 50 ms.

11 . The method of claim 1 , comprising obtaining a plurality of 1 H CPMG spectra while spinning at a frequency of up to 14 kHz.

12 . The method of claim 1 , wherein the contact time of the 13 C CP during the measuring is about 100 μs to about 10 ms.

13 . The method of claim 12 , wherein the measuring comprises an RF spin lock pulse on 13 C of about 20 kHz to about 100 kHz.

14 . The method of claim 13 , wherein the ramp pulse on 1 H is matched.

15 . The method of claim 1 , comprising quantifying the content of antibodies in the sample.

16 . The method of claim 1 , wherein the crystalline antibodies exhibit a spectroscopic signature different than amorphous antibodies or the crystalline antibodies exhibit a spectroscopic signature of higher molecular mobility than amorphous antibodies.

17 . The method of claim 1 , wherein the crystalline antibodies are bi-refringant.

18 . The method of claim 1 , wherein the crystalline antibodies do not diffract.

19 . The method of claim 1 , wherein the antibodies in the sample comprise one or more glycans.

Assignees

• Amgen Inc

Inventors

• Sharnez Rizwan
• Trieu William
• Caporini Marc A
• Kelly Ron C
• Burt Neill
• Nicholson Laura
• Christian Twinkle R

Classifications

• C30B7/10

  by application of pressure, e.g. hydrothermal processes · CPC title

• C30B7/00

  Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions (from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00) · CPC title

• G01R33/46

  NMR spectroscopy · CPC title

• G01R33/20

  involving magnetic resonance (medical aspects A61B5/055; magnetic resonance gyrometers G01C19/60) · CPC title

• G01N24/008

  by using resonance effects in zero field, e.g. in microwave, submillimetric region (by measuring absorption of microwaves by the material G01N22/00) · CPC title