Comparison of NMR and Dynamic Light Scattering for measuring diffusion coefficients of formulated insulin: implications for particle size distribution in drug products
Sharadrao M. Patil1, David A. Keire2, Kang Chen1*
Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, 1Silver Spring, Maryland and 2St. Louis, Missouri, United States.
Table S-1. DOSY measured diffusion coefficients for 1 lot of human insulin formulation Novolin R®.
DOSY Exp. No. / Peak No. / 1H Chemical Shift(ppm) / Diffusion
coefficient
(cm2/s) / DOSY
Diffusion
coefficient
(cm2/s) / Averaged Diffusion coefficient
(cm2/s)a / Standard Deviation (St. Dev.) (cm2/s) / Relative
St. Dev.
(RSD)
1 / 1 / 1.07 / 1.04E-06 / 1.10E-06 / 1.08E-06 / 1E-08 / 1%
2 / 1.00 / 1.09E-06
3 / 0.88 / 1.11E-06
4 / 0.68 / 1.12E-06
5 / 0.51 / 1.12E-06
2 / 1 / 1.06 / 1.03E-06 / 1.07E-06
2 / 1.00 / 1.07E-06
3 / 0.88 / 1.09E-06
4 / 0.68 / 1.09E-06
5 / 0.51 / 1.09E-06
3 / 1 / 1.06 / 1.02E-06 / 1.07E-06
2 / 1.00 / 1.07E-06
3 / 0.88 / 1.06E-06
4 / 0.68 / 1.09E-06
5 / 0.51 / 1.09E-06
4 / 1 / 1.07 / 1.02E-06 / 1.06E-06
2 / 1.00 / 1.06E-06
3 / 0.87 / 1.07E-06
4 / 0.68 / 1.09E-06
5 / 0.51 / 1.08E-06
5 / 1 / 1.07 / 1.03E-06 / 1.08E-06
2 / 1.00 / 1.07E-06
3 / 0.87 / 1.10E-06
4 / 0.68 / 1.10E-06
5 / 0.51 / 1.10E-06
a. The DOSY diffusion coefficient of 1.08e-6 cm2s-1 is slightly larger than the 1.07e-6 cm2s-1 value in Table 1 due to the time period (1 year) between the two data collections.
Table S-2. Diffusion coefficients measured from 2D DOSY-NMR spectra.
LotNo. / DOSY Peak Diffusion coefficients (cm2/s) / DOSY Diffusion coefficients (D) (cm2/s) / Inter Lot <D> (cm2/s) / Inter lot St. Dev. (cm2/s) / Relative St. Dev. (RSD)
Peak 1 / Peak 2 / Peak 3 / Peak 4 / Peak 5
Apidra®
Chemical shift (ppm)è / 1.22 ppm / 1.15 ppm / 0.98 ppm / 0.76 ppm / 0.51 ppm
1 / 1.07E-06 / 1.14E-06 / 1.18E-06 / 1.17E-06 / 1.16E-06 / 1.14E-06 / 1.15E-06 / 2E-08 / 2%
2 / 1.11E-06 / 1.18E-06 / 1.21E-06 / 1.20E-06 / 1.19E-06 / 1.18E-06
3 / 1.08E-06 / 1.16E-06 / 1.17E-06 / 1.14E-06 / 1.16E-06 / 1.14E-06
Humalog®
Chemical shift (ppm)è / 1 ppm / 0.89 ppm / 0.79 ppm / 0.74 ppm / 0.57 ppm
1 / 1.04E-06 / 1.08E-06 / 1.08E-06 / 1.09E-06 / 1.09E-06 / 1.08 E-06 / 1.10E-06 / 6E-08 / 6%
2 / 1.15E-06 / 1.18E-06 / 1.18E-06 / 1.18E-06 / 1.18E-06 / 1.17E-06
3 / 1.03E-06 / 1.07E-06 / 1.07E-06 / 1.07E-06 / 1.07E-06 / 1.06E-06
Humulin R®
Chemical shift (ppm)è / 1.07 ppm / 0.89 ppm / 0.69 ppm / 0.64 ppm / 0.52 ppm
1 / 9.73E-07 / 9.89E-07 / 1.00E-06 / 9.82E-07 / 1.01E-06 / 9.91E-07 / 1.04E-06 / 5E-08 / 5%
2 / 9.95E-07 / 1.02E-06 / 1.04E-06 / 1.01E-06 / 1.05E-06 / 1.02E-06
3 / 1.07E-06 / 1.11E-06 / 1.10E-06 / 1.09E-06 / 1.11E-06 / 1.10E-06
Novolin R®
Chemical shift (ppm)è / 1.21 ppm / 1.07 ppm / 1 ppm / 0.88 ppm / 0.51 ppm
1 / 1.04E-06 / 1.05E-06 / 1.08E-06 / 1.09E-06 / 1.10E-06 / 1.07E-06 / 1.07E-06 / 1E-09 / 0.1%
2 / 9.96E-07 / 1.05E-06 / 1.10E-06 / 1.10E-06 / 1.10E-06 / 1.07E-06
3 / 1.01E-06 / 1.05E-06 / 1.09E-06 / 1.11E-06 / 1.10E-06 / 1.07E-06
Novolog®
Chemical shift (ppm)è / 1.23 ppm / 0.98 ppm / 0.88 ppm / 0.81 ppm / 0.62 ppm
1 / 1.14E-06 / 1.22E-06 / 1.26E-06 / 1.22E-06 / 1.19E-06 / 1.21E-06 / 1.18E-06 / 2E-08 / 2%
2 / 1.11E-06 / 1.18E-06 / 1.19E-06 / 1.20E-06 / 1.16E-06 / 1.17E-06
3 / 1.12E-06 / 1.17E-06 / 1.20E-06 / 1.19E-06 / 1.15E-06 / 1.17E-06
Table S-3. DLS measured diffusion coefficients for 1 lot of human insulin formulation Novolin R®.
Well No. / Range 1Diffusion Coefficient (cm²/s) / % Pd / %
Intensity / % Mass / Hydrodynamic Radius (nm) / Range 2
Diffusion Coefficient (cm²/s) / % Pd / %
Intensity / % Mass / Hydrodynamic Radius (nm) / Range 3
Diffusion Coefficient (cm²/s) / % Pd / %
Intensity / % Mass / Hydrodynamic Radius (nm) / Normalized Diffusion Coefficient
(cm2/s) / Averaged Diffusion Coefficient
(cm2/s) / Relative
St. Dev.
(RSD)
1 / 1.30E-06 / 4.1 / 84.2 / 100.00 / 1.9 / 9.29E-08 / 12.40 / 6.3 / 3.50E-03 / 26 / 2.20E-08 / 11.80 / 9.5 / 4.60E-03 / 109.5 / 1.30E-06 / 1.31 ± 0.02 E-06 / 2%
2 / 1.28E-06 / 5.4 / 85.4 / 100.00 / 1.9 / 7.53E-08 / 12.30 / 8.2 / 2.70E-03 / 32.1 / 1.77E-08 / 9.94 / 6.4 / 7.70E-03 / 136.1 / 1.28E-06
3 / 1.30E-06 / 3.1 / 86.1 / 100.00 / 1.9 / 6.62E-08 / 7.00 / 9.9 / 2.20E-03 / 36.5 / 8.58E-09 / 7.53 / 4.0 / 2.10E-03 / 281.6 / 1.30E-06
4 / 1.33E-06 / 7.6 / 86.7 / 100.00 / 1.8 / 8.90E-08 / 11.00 / 7.8 / 3.30E-03 / 27.1 / 1.40E-08 / 8.76 / 5.5 / 1.50E-03 / 172.6 / 1.33E-06
5 / 1.32E-06 / 6.2 / 86.2 / 100.00 / 1.8 / 1.05E-07 / 5.90 / 7.0 / 4.60E-03 / 23 / 2.13E-08 / 12.50 / 6.8 / 4.10E-03 / 113.5 / 1.32E-06
Table S-4. DLS Regularization fitting results.a
Lot No. / Range1 Diffusion Coefficient (cm²/s) / % Pd / % Intensity / % Mass / Hydrodynamic Radius (nm) / Range2 Diffusion Coefficient (cm²/s) / % Pd / % Intensity / % Mass / Hydrodynamic Radius (nm) / Range3 Diffusion Coefficient (cm²/s) / % Pd / % Intensity / % Mass / Hydrodynamic Radius (nm) / Normalized Diffusion Coefficient (cm²/s) / DLS Diffusion Coefficient (cm²/s)Apidra®
1 / 8.72E-07 / 11.9 / 73.8 / 100 / 2.8 / 3.74E-08 / 11.5 / 26.2 / 1.20E-02 / 64.5 / NA / NA / NA / NA / NA / 8.72E-07 / 9.01 ± 0.4 E-07
9.29E-07 / 8 / 80.1 / 100 / 2.6 / 5.73E-08 / 12 / 19.9 / 1.10E-02 / 42.1 / NA / NA / NA / NA / NA / 9.29E-07
2 / 9.70E-07 / 5.8 / 97.4 / 100 / 2.5 / 2.94E-08 / 11.4 / 2.6 / 1.20E-03 / 82.1 / NA / NA / NA / NA / NA / 9.70E-07 / 9.31 ± 0.6 E-07
8.92E-07 / 11.2 / 94.2 / 100 / 2.7 / 1.86E-08 / 9.82 / 5.8 / 1.70E-02 / 130.1 / NA / NA / NA / NA / NA / 8.92E-07
3 / 9.22E-07 / 10.2 / 92.4 / 100 / 2.6 / 3.29E-08 / 8.7 / 7.6 / 2.60E-03 / 73.4 / NA / NA / NA / NA / NA / 9.22E-07 / 8.85 ± 0.5 E-07
8.48E-07 / 12.2 / 93.6 / 100 / 2.8 / 3.50E-08 / 11.9 / 6.4 / 2.60E-03 / 69 / NA / NA / NA / NA / NA / 8.48E-07
Humulin R®
1 / 1.36E-06 / 15.3 / 60.6 / 100 / 1.8 / 8.99E-08 / 11.9 / 25.7 / 1.40E-02 / 26.9 / 1.86E-08 / 12.39 / 13.7 / 1.10E-02 / 129.9 / 1.36E-06 / 1.35 ± 0.01 E-06
1.35E-06 / 14.1 / 59.9 / 100 / 1.8 / 1.28E-07 / 20.3 / 15.2 / 2.70E-02 / 18.8 / 3.67E-08 / 20.98 / 22.8 / 4.00E-03 / 65.7 / 1.35E-06
2 / 1.31E-06 / 9 / 30.1 / 99.9 / 1.8 / 1.92E-07 / 12.4 / 7.5 / 8.70E-02 / 12.6 / 4.38E-08 / 15.21 / 55.3 / 2.00E-02 / 55.2 / 1.31E-06 / 1.34 ± 0.04 E-06
1.37E-06 / 12.2 / 30.8 / 99.9 / 1.8 / 9.18E-08 / 19.7 / 36.9 / 5.00E-02 / 26.3 / 2.27E-08 / 17.96 / 32.3 / 5.00E-02 / 106.4 / 1.37E-06
3 / 1.27E-06 / 20.9 / 39.5 / 99.9 / 1.9 / 1.54E-07 / 25.8 / 7.6 / 4.00E-02 / 15.7 / 4.63E-08 / 24.45 / 46.5 / 1.40E-02 / 52.1 / 1.27E-06 / 1.26 ± 0.01 E-06
1.26E-06 / 19.1 / 46.4 / 99.9 / 1.9 / 5.04E-08 / 56.8 / 48.8 / 4.40E-02 / 48 / 6.23E-09 / 18.16 / 4.7 / 1.10E-02 / 387.9 / 1.26E-06
Novolin R®
1 / 1.32E-06 / 11.4 / 92.9 / 100 / 1.8 / 2.58E-08 / 8.6 / 7.1 / 2.20E-03 / 93.7 / NA / NA / NA / NA / NA / 1.32E-06 / 1.31 ± 0.02 E-06
1.29E-06 / 10 / 89.8 / 100 / 1.9 / 3.63E-08 / 12.2 / 7.3 / 8.50E-04 / 66.5 / 1.01E-08 / 11.1 / 3 / 2.90E-03 / 238.8 / 1.29E-06
2 / 1.45E-06 / 12.9 / 85.2 / 100 / 1.7 / 1.84E-07 / 10.2 / 2.8 / 6.90E-03 / 13.1 / 1.90E-08 / 7.46 / 12 / 9.30E-03 / 127.2 / 1.45E-06 / 1.40 ± 0.07 E-06
1.35E-06 / 13.5 / 85.4 / 100 / 1.8 / 2.62E-08 / 9.9 / 14.6 / 4.40E-03 / 92.2 / NA / NA / NA / NA / NA / 1.35E-06
3 / 1.33E-06 / 11.6 / 87.4 / 100 / 1.8 / 3.81E-08 / 10.6 / 12.6 / 1.30E-03 / 63.4 / NA / NA / NA / NA / NA / 1.33E-06 / 1.32 ± 0.02 E-06
1.31E-06 / 19.8 / 85 / 100 / 1.8 / 2.63E-08 / 10.3 / 15 / 4.20E-03 / 91.7 / NA / NA / NA / NA / NA / 1.31E-06
Humalog®
1 / 1.02E-06 / 10.8 / 81.8 / 100 / 2.4 / 9.65E-08 / 12.1 / 8.6 / 1.10E-02 / 25 / 1.72E-08 / 12.17 / 9.6 / 1.60E-02 / 140.5 / 1.02E-06 / 1.02 ± 0.01 E-06
1.02E-06 / 10.3 / 81.1 / 100 / 2.4 / 9.98E-08 / 11.2 / 7 / 9.60E-03 / 24.2 / 2.30E-08 / 11.52 / 12 / 1.60E-02 / 105.1 / 1.02E-06
2 / 9.86E-07 / 20 / 56.2 / 99.8 / 2.5 / 1.42E-07 / 19.8 / 19.6 / 1.10E-01 / 17.1 / 2.07E-08 / 19.25 / 24.2 / 5.10E-02 / 116.5 / 9.84E-07 / 9.23 ± 0.9 E-07
8.64E-07 / 35.7 / 58 / 99.8 / 2.8 / 2.78E-08 / 56 / 39.1 / 5.60E-02 / 86.7 / 4.63E-10 / 28.91 / 2.8 / 1.40E-01 / 5215.7 / 8.62E-07
3 / 9.29E-07 / 8.9 / 54.6 / 99.2 / 2.6 / 1.12E-07 / 14 / 10.4 / 3.90E-02 / 21.6 / 3.62E-08 / 15.07 / 28.2 / 1.50E-02 / 66.7 / 9.22E-07 / 9.31 ± 0.1 E-07
9.42E-07 / 18.1 / 58.5 / 99.9 / 2.6 / 7.24E-08 / 24.1 / 22.1 / 2.60E-02 / 33.4 / 1.42E-08 / 25.13 / 19.3 / 5.60E-02 / 169.8 / 9.41E-07
Novolog®
1 / 1.06E-06 / 12 / 92.2 / 100 / 2.3 / 3.02E-08 / 9.7 / 7.8 / 2.30E-03 / 79.9 / NA / NA / NA / NA / NA / 1.06E-06 / 1.07 ± 0.01 E-06
1.07E-06 / 12.1 / 94.5 / 100 / 2.3 / 3.16E-08 / 1 / 5.5 / 1.20E-03 / 76.4 / NA / NA / NA / NA / NA / 1.07E-06
2 / 9.54E-07 / 17.8 / 88.7 / 100 / 2.5 / 2.47E-08 / 14.6 / 11.3 / 1.20E-02 / 97.7 / NA / NA / NA / NA / NA / 9.54E-07 / 9.96 ± 0.6 E-07
1.04E-06 / 11.4 / 93.6 / 100 / 2.3 / 2.89E-08 / 11.9 / 6.4 / 2.60E-03 / 83.5 / NA / NA / NA / NA / NA / 1.04E-06
3 / 1.05E-06 / 11.6 / 91.9 / 100 / 2.3 / 2.73E-08 / 11.8 / 8.1 / 4.20E-03 / 88.3 / NA / NA / NA / NA / NA / 1.05E-06 / 1.06 ± 0.03 E-06
1.08E-06 / 15.3 / 89.5 / 100 / 2.2 / 2.90E-08 / 11.8 / 10.5 / 3.60E-03 / 83.2 / NA / NA / NA / NA / NA / 1.08E-06
a. Data was collected in duplicate for each lot and the diffusion coefficients were averaged to yield DLS diffusion coefficient.
Bruker pulse program for the 2D DOSY-NMR experiment.
;ledbpgp2s
;avance-version (07/04/03)
;2D sequence for diffusion measurement using stimulated
; echo and LED
;using bipolar gradient pulses for diffusion
;using 2 spoil gradients
;
;D. Wu, A. Chen & C.S. Johnson Jr.,
; J. Magn. Reson. A 115, 260-264 (1995).
;
;$CLASS=HighRes
;$DIM=2D
;$TYPE=
;$SUBTYPE=
;$COMMENT=
#include <Avance.incl
#include <Grad.incl
#include <Delay.incl
"TAU=p1*2/3.1416-(p0-p27)*0.231-de+46u"
define list<gradient> diff=<Difframp
"p2=p1*2"
"DELTA1=d20-p1*2-p2-p30*2-d16*2-d29-d16"
;d29 equals p19
;"DELTA1=d20-p1*2-p2-p30*2-d16*2-p19-d16"
;"DELTA2=d21-p19-d16-4u"
"acqt0=-p1*2/3.1416"
1 ze
2 30m
d12 ;pl9:f1
d1 ;cw:f1 ph29
4u ;do:f1
d12 pl1:f1
50u UNBLKGRAD
p1 ph1
p30:gp6*diff
d16
p2 ph1
p30:gp6*-1*diff
d16
p1 ph2
;p19:gp7
d29
d16
DELTA1
p1 ph3
p30:gp6*diff
d16
p2 ph1
p30:gp6*-1*diff
d16
p1 ph4
;p19:gp8
d16
5u
p1 ph5
50u
p16:gp1
d16 pl18:f1
p27*0.231 ph13
d19*2
p27*0.692 ph13
d19*2
p27*1.462 ph13
d19*2
p27*1.462 ph14
d19*2
p27*0.692 ph14
d19*2
p0*0.231 ph14
TAU
p16:gp1
d16
4u BLKGRAD
go=2 ph31
30m mc #0 to 2 F1QF(igrad diff)
exit
ph1= 0
ph2= 0 0 2 2
ph3= 0 0 0 0 2 2 2 2 1 1 1 1 3 3 3 3
ph4= 0 2 0 2 2 0 2 0 1 3 1 3 3 1 3 1
ph5= 0 0 0 0 2 2 2 2 1 1 1 1 3 3 3 3
ph13={0}*8 {1}*8 {2}*8 {3}*8
ph14={2}*8 {3}*8 {0}*8 {1}*8
ph29=0
ph31=0 2 2 0 2 0 0 2 3 1 1 3 1 3 3 1
;pl1 : f1 channel - power level for pulse (default)
;p1 : f1 channel - 90 degree high power pulse
;p2 : f1 channel - 180 degree high power pulse
;p19: gradient pulse 2 (spoil gradient)
;p30: gradient pulse (little DELTA * 0.5)
;d1 : relaxation delay; 1-5 * T1
;d16: delay for gradient recovery
;d20: diffusion time (big DELTA)
;d21: eddy current delay (Te) [5 ms]
;NS: 8 * n
;DS: 4 * m
;td1: number of experiments
;FnMODE: QF
; use xf2 and DOSY processing
;use gradient ratio: gp 6 : gp 7 : gp 8
; 100 : -17.13 : -13.17
;for z-only gradients:
;gpz6: 100%
;gpz7: -17.13% (spoil)
;gpz8: -13.17% (spoil)
;use gradient files:
;gpnam6: SINE.100
;gpnam7: SINE.100
;gpnam8: SINE.100
;use AU-program dosy to calculate gradient ramp-file Difframp
;$Id: ledbpgp2s,v 1.6 2007/04/11 13:34:30 ber Exp $
2