Crystallization and liquid‐liquid phase separation of monoclonal antibodies and fc‐fusion proteins: Screening results

Abstract

Crystallization holds the potential to be used for protein purification and low-viscosity drug substance and drug product formulations. Twenty-two different proteins (20 monoclonal antibodies and two Fc-fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG2 s examined, at least four formed diffraction-quality crystals and four others formed crystal-like particles. At least three of the IgG2 s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high-yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid-liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc-fusions, for example, was concentrated by LLPS to a self-buffering solution at 150 g/L. Crystallization and LLPS in the salting-in region were shown to be feasible.

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

“…While LLPS in solutions of globular proteins is well documented (16,(18)(19)(20)(21), it is often preempted by aggregation or crystallization. Recently, reports have appeared of such LLPS in solutions of antibodies (5)(6)(7)(8)(9)22). Antibodies can be present in blood at relatively high concentrations.…”

“…Recently, LLPS of several pharmaceutical antibodies have been reported (5)(6)(7)(8)(9). There are five isotypes of mammalian antibodies with distinct Fc regions, including IgA, IgD, IgE, IgG, and IgM.…”

Impact of surface interactions on the phase behavior of Y-shaped molecules

“…Although IgGs are typically quite soluble at physiological conditions, sometimes they can become insoluble. In fact, recent studies of protein condensation have been published both for recombinant pharmaceutical IgGs and monoclonal IgGs [10][11][12][13][14][15][16][17]. A detailed discussion of the importance of IgGs in physiological and pharmaceutical situations is given by Wang et al [6], as well as Nezlin [18].…”

Investigating High-Concentration Monoclonal Antibody Powder Suspension in Nonaqueous Suspension Vehicles for Subcutaneous Injection

“…A powder‐based approach was recently described using mAb crystalline particle suspensions 10,11. The approach was based on the perception that the viscosity of a crystal mAb suspension may be lower than that of a liquid formulation at the same mAb concentration.…”