Optimization of candidate monoclonal antibodies (mAb) is an essential step in the development of therapeutic formulations. Injectability is a key property required to assess the safety and efficacy of candidate mAb’s. Viscosity of a formulation is the sole parameter that dictates injectability and is thus critically important to characterize during the development process. In this brief, we demonstrate a method for accurate and repeatable viscosity measurement that reliably predicts injectability and provides important insights about intermolecular interactions involving candidate compounds. Automated measurement of viscosity with VROC® initium can increase the throughput and efficiency of the formulation development pipeline, optimizing use of valuable time and resources.
Material: Bovine γGlobulin from Sigma Aldrich (Lot # SLBO4091V) was used as a model mAb. 10 mM Phosphate Buffered Saline was prepared by dissolving dry powder from Sigma Aldrich (Lot 039K8200) in one liter of deionized water. Bovine serum albumin (BSA) obtained from Sigma Aldrich (Lot 060M1708V) was also used for this study. Sucrose was purchased from Fisher Scientific (Lot 092848).
Sample Preparation: Three media solutions were prepared for this study: PBS buffer, 4 wt% sucrose solution in deionized water, and 30 mg/mL BSA solution in PBS. Viscosity of each media at 25 oC (as measured with initium) is shown in the table below.
Table 1. Measured viscosities of three media.
Media 
PBS buffer 
30 mg/mL BSA in PBS 
4 wt% Sucrose solution in PBS 
Viscosity, mPas 
0.904 
1.029 
1.010 
% RSD 
0.41 
0.36 
0.14 
RSD (relative standard deviation) was calculated from 8 data points. See below for measurement.
Measurement: VROC^{®} initium was used to test γGlobulin solutions in various media using the same measurement protocol, which is as follows: 12 measurements each sample at 600 μL/min (shear rate = 11,786 s1) at 25 oC, with sample retrieval between each measurement. The first four data points from each measurement were discarded because of possible incorporation of small bubbles due to air entrapment. Sample volume was 100 μL in each case. The measurement protocol also works well with 50 μL sample volumes. Contact RheoSense for more information. For all measurements, %RSD was less than 0.5%.
Results: Measured viscosities of γGlobulin solutions in the three different media are summarized in Tables 2–4 below.
Table 2. Viscosities of γGlobulin solutions in PBS buffer
C, mg/mL 
h, mPas 
%RSD 
h_{r} 
h_{sp}/C 
f (vol. fraction) 
48.46 
1.336 
0.17 
1.478 
0.00974 
0.1229 
38.88 
1.222 
0.16 
1.352 
0.00896 
0.0986 
29.34 
1.122 
0.32 
1.241 
0.00816 
0.0744 
19.71 
1.039 
0.24 
1.150 
0.00759 
0.0500 
11.53 
0.978 
0.51 
1.082 
0.00708 
0.0292 
5.092 
0.935 
0.29 
1.035 
0.00684 
0.0129 
0.000 
0.904 
0.41 
1.000 
 
0.0000 
In the table, h_{r}, h_{sp}/C, and f are calculated using the following equations:
Where R_{h} is the hydrodynamic radius, C is the concentration, N_{a} is Avogadro’s number, and M is the molecular weight. The hydrodynamic radius is calculated from the intrinsic viscosity [h] using the following equation for an apparent spherical shape:
The intrinsic viscosity is calculated by extrapolating h_{sp}/c to zero concentration as shown in Figure 1.
Figure 1. h_{sp}/C vs. C plot to obtain [h] and K_{H}. (see equation below for the definition of [h] and K_{H}.
Tables 3 and 4 summarize the analysis of the viscosity data of gGlobulin solutions in 30 mg/mL BSA and 4 wt% sucrose in PBS media.
Table 3. Analysis of the viscosity data of gglobulin in 30 mg/mL BSA in PBS buffer.
C, mg/mL 
h, mPas 
%RSD 
h_{r} 
h_{sp}/C 
f (vol. fraction) 
40.22 
1.448 
0.23 
1.407 
0.01012 
0.1077 
32.43 
1.357 
0.48 
1.319 
0.00982 
0.0868 
23.53 
1.238 
0.32 
1.203 
0.00862 
0.0630 
16.53 
1.174 
0.78 
1.141 
0.00850 
0.0443 
8.030 
1.090 
0.34 
1.059 
0.00737 
0.0215 
4.420 
1.061 
0.25 
1.031 
0.00693 
0.0118 
0.000 
1.029 
0.36 
1.000 
 
0.1077 
Table 4. Analysis of the viscosity data of gglobulin in 4 wt% sucrose in PBS buffer.
C, mg/mL 
h, mPas 
%RSD 
h_{r} 
h_{sp}/C 
f (vol. fraction) 
38.53 
1.360 
0.23 
1.346 
0.00898 
0.0894 
30.91 
1.270 
0.26 
1.257 
0.00830 
0.0717 
23.58 
1.194 
0.17 
1.182 
0.00770 
0.0547 
16.06 
1.124 
0.32 
1.112 
0.00700 
0.0372 
7.960 
1.062 
0.40 
1.051 
0.00645 
0.0185 
5.620 
1.046 
0.35 
1.036 
0.00632 
0.0130 
0.000 
1.010 
0.14 
1.000 
 
0.0000 
Figure 2 compares the change of h_{sp}/C with C, which yields [h] and K_{H} for the gGlobulin solution in the three media. As shown in Table 5, gGlobulin molecules interact with the media differently. The intrinsic viscosity is smallest and K_{H} is the largest in 4 wt% sucrose in PBS medium. The results suggest that gGlobulin molecules are the smallest in 4% sucrose in PBS media in dilute solution. However, higher K_{H}[h]^{2} suggests that viscosity of gGlobulin solution in 30 mg/mL BSA in PBS will increase faster as concentration increases since h_{r }follows the equation below at low concentrations.
Table 5 summarizes the calculated intrinsic viscosities, hydrodynamic radii, and Huggins constants of gGlobulin in three different media.

PBS 
30 mg/mL BSA in PBS 
4 wt% Sucrose in PBS 
[h], mL/mg 
0.00633 
0.00668 
0.00579 
R_{h}, nm 
5.41 
5.51 
5.26 
K_{H} 
1.76 
2.02 
2.43 
Figure 3. h_{r} of the gglobulin solution in various media as a function of f.
In Figure 3, the relative viscosities, h_{r}, of the gglobulin solutions in various media are plotted against the volume fraction and compared to the Batchelor’s calculation of the viscosity of suspensions of hard spheres with hydrodynamic interaction^{1}. As shown in the figure, the relative viscosity of gglobulin deviates from the hard sphere prediction at a volume fraction of 0.03. Also noted is that the viscosity increase relative to volume fraction is most rapid in 4 wt% sucrose in PBS media.
Conclusions:
^{1} Batchelor, G. K. (1977). The effect of Brownian motion on the bulk stress in a suspension of spherical particles. J. Fluid Mech. 83, 97117.
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