Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data

• Published in: European biophysics journal Vol. 49; no. 8; pp. 687 - 700
• Main Authors: Correia, J. J., Wright, R. T., Sherwood, P. J., Stafford, W. F.
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-12-2020
• Subjects: Biochemistry; Biological and Medical Physics; Biomedical and Life Sciences; Biophysics; Cell Biology; Life Sciences; Membrane Biology; Nanotechnology; Neurobiology; Original; Original Article; Thermodynamic nonideality; Diffusion; 2nd Virial coefficient; Hydrodynamic nonideality; Sedimentation velocity
• ISSN: 0175-7571; 1432-1017
• DOI: 10.1007/s00249-020-01474-5

The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709–722, 2018, Anal Biochem 550:72–83, 2018). This involved fitting both hydrodynamic,
k
s
, and thermodynamic, BM
1
, nonideality where concentration dependence is expressed as
s
 = 
s
o
/(1 + 
k
s
c
) and
D
 = 
D
o
(1 + 2BM
1
c
)/(1 + 
k
s
c
) and
s
o
and
D
o
are values extrapolated to
c
 = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of
k
s
(0–100 ml/g) and BM
1
(0–100 ml/g). This provides a visual understanding of the separate and joint impact of
k
s
and BM
1
on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334–1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1–120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for
k
s
, BM
1
and weak association. Because a typical clinical dose of mAb is 50–200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.