Methods directed to crystalline biomolecules
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.
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.