Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Abstract

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.

Boosting antibody developability through rational sequence optimization

“…In addition, transiently formed associated proteins that retain their native conformation can give rise to an increase in the apparent particle size resulting in an exponential increase of viscosity at high protein concentrations. 14 As viscosity can be a critical parameter in downstream processing, as well as in fill and finish operations and drug delivery, viscosity is a developability attribute. 11 In summary, one can say that none of the above described aggregation pathways occurs isolated and exclusively in a solution of a certain antibody.…”

Short-Time Glassy Dynamics in Viscous Protein Solutions with Competing Interactions

“…[1][2][3][4][5][6] The combination of a long-ranged repulsion with a short-ranged attraction has been demonstrated to cause the formation of equilibrium cluster fluids. [1,4,[7][8][9][10][11] While clusters in SALR systems have been observed in systems of nanoparticles in polymer composites [12] and solutions with polymer depletants [1,7] as well as membrane proteins, [13] recent work has associated cluster formation with large viscosities in highly concentrated lysozyme [11] and monoclonal antibody [14] formulations, making these types of interactions relevant to the biopharmaceutical industry.…”

“…[7,[9][10][11][14][15][16] In particular, the phase diagrams of globular protein solutions with pure short-ranged attraction have been widely studied at large salt concentrations. [17,18] For solutions with low salt concentrations, the presence of additional repulsion is known to shift the liquid-liquid coexistence region of an attractive fluid to lower temperatures, T , [3,5] and the percolation transition to smaller volume fractions, φ.…”

Theranos Session at the 2016 AACC Annual Scientific Meeting & Clinical Lab Expo: Expectations, Impressions, and Takeaways

“…Only two articles were pieces reporting original research data involving Theranos scientists (3,4). One of these was an article reporting the biophysical properties of solutions containing monoclonal antibodies, with no clear link to the Theranos diagnostics platform (4). The other article mentioned two assays, for measuring C-reactive protein (CRP) and protein C, based on the Theranos platform (3).…”

“…One was from a research team unrelated to Theranos highlighting potential concerns on the discrepancies between tests conducted on the Theranos platform compared with those from two established commercial vendors (2). Only two articles were pieces reporting original research data involving Theranos scientists (3,4). One of these was an article reporting the biophysical properties of solutions containing monoclonal antibodies, with no clear link to the Theranos diagnostics platform (4).…”