Measuring aggregates, self-association, and weak interactions in concentrated therapeutic antibody solutions

Abstract

Monoclonal antibodies are a class of biotherapeutics used for an increasing variety of disorders, including cancer, autoimmune, neurodegenerative, and viral diseases. Besides their antigen specificity, therapeutic use also mandates control of their solution interactions and colloidal properties in order to achieve a stable, efficacious, non-immunogenic, and low viscosity antibody solution at concentrations in the range of 50–150 mg/mL. This requires characterization of their reversible self-association, aggregation, and weak attractive and repulsive interactions governing macromolecular distance distributions in solution. Simultaneous measurement of these properties, however, has been hampered by solution nonideality. Based on a recently introduced sedimentation velocity method for measuring macromolecular size distributions in a mean-field approximation for hydrodynamic interactions, we demonstrate simultaneous measurement of polydispersity and weak and strong solution interactions in a panel of antibodies with concentrations up to 45 mg/mL. By allowing approximately an order of magnitude higher concentrations than previously possible in sedimentation velocity size distribution analysis, this approach can substantially improve efficiency and sensitivity for characterizing polydispersity and interactions of therapeutic antibodies at or close to formulation conditions.

Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations

“…Integration of c NI ( s 0 ) yields weight-average sedimentation coefficients s w ,0 of sedimenting species corrected for hydrodynamic nonideality. We have recently demonstrated application of this method for mAbs of the current panel up to 45 mg/mL, and the end-point of the previous study, i.e., the resulting s w,0 isotherm and the k S -values, will serve as starting point here 24 .…”

“…( d) (wt%) B 1 6.73 6.5 (0.2) ∞ (> 17 mM) 1.13 (0.12) − 4.24 5.7 0.06 (< 0.01) A 1–2 6.67 5.0 (0.3) 7.1 mM (5.9–8.5) − 2.13 (0.11) − 9.26 1.8 0.07 (< 0.01) E 1–2 6.70 0.2 (< 0.01) 550 µM (510–730) − 7.7 (0.2) − 15.6 0.1 0.01 (< 0.01) D 1–2 and 2–4…isoH 6.72 3.5 (0.2) 153 µM (136–169) and 5.3 mM (2.7– 75) 0.01 (0.23) − 3.5 6.4 0.02 (0.03) C 1–2 and 2–4 6.41 (0.16) 3.6 (2.2) 15 µM (14.7–21) and 48 µM (40–51) − 5.9 (0.68) − 15.4 0.4 0.0 (< 0.01) Values in parentheses are confidence intervals or +/− errors, respectively, on a confidence level of 68.3%. (a) The monomer s -value was determined from the c ( s ) analysis of sedimentation under ideal conditions at 0.3 mg/mL and fixed in the analysis, except for mAb C. (b) Nonideal c NI ( s 0 ) analysis was carried out to pre-determine k S 24 . (c) Irreversible dimer with assigned f/f 0 = 1.8, at pre-determined population from SV in dilute solution, except for mAb E where best-fit parameters are shown.…”

“… Values in parentheses are confidence intervals or +/− errors, respectively, on a confidence level of 68.3%. (a) The monomer s -value was determined from the c ( s ) analysis of sedimentation under ideal conditions at 0.3 mg/mL and fixed in the analysis, except for mAb C. (b) Nonideal c NI ( s 0 ) analysis was carried out to pre-determine k S 24 . (c) Irreversible dimer with assigned f/f 0 = 1.8, at pre-determined population from SV in dilute solution, except for mAb E where best-fit parameters are shown.…”

“…In the present application different techniques likewise provide synergistic advantages and limitations: SV has the highest resolution, and in the form of the recently introduced nonideal sedimentation coefficient distribution c NI ( s 0 ) separates oligomerization from nonideal sedimentation k S , the latter extracted from characteristic boundary anomalies 29 , 30 . We have shown recently how it can be applied to mAbs up to 45 mg/mL, which provides high sensitivity for self-association 24 . While SV gives clues to the self-association scheme and the polydispersity of the sample, a drawback is that it does not directly measure the molecular weights of oligomers, and has poorer information content on diffusion and k D , and therefore B 2 29 .…”

“…The goal of the present work is to develop a method for determining nonideality coefficients that are quantitative and self-consistent across different techniques, and to dissect specific oligomeric complex formation in self-association processes from nonideality defined as resulting from volume exclusion and ‘soft’ long-range interactions causing non-uniform macromolecular distance distributions 23 . We test the method with a set of monoclonal antibodies with different degrees of self-association and nonideality, which have been previously used as benchmarks (Supplementary Table S1 ) 12 , 14 , 24 . As experimental foundation we use the classical combination of SLS, DLS, and SV, which all probe size, shape, and motion of macromolecules free in solution.…”