Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubilities mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure, and solvent conditions to minimize protein-protein interactions is key to designing well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of superfolder GFP (sfGFP) and two supercharged sfGFP variants engineered to have formal charges of -18 or +15. Under low cosolute conditions, sfGFP and the -18 variant formed a gel or phase separated at ∼10 mg/mL. Under conditions that screen surface charges, including formulations with high histidine or high NaCl concentrations, all three variants attained concentrations up to 250 mg/mL with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded-volume effects without shielding charge. Collectively, these data demonstrate that charge shielding neutralizes not only long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically relevant proteins.

Use of Debye-Hückel-Henry charge measurements in early antibody development elucidates effects of non-specific association.

The diffusion interaction parameter (kD ) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm ) as a function of the experimentally determined Debye-Hückel-Henry surface charge (ZDHH ) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.

Enhancing Stability and Reducing Viscosity of a Monoclonal Antibody With Cosolutes by Weakening Protein-Protein Interactions.

An understanding of how cosolutes affect the viscosity and storage stability of highly concentrated mAbs as a function of protein-protein interactions (PPIs) would be desirable for improving processing and administration of protein therapeutics. The effects of inorganic and organic cosolutes on the viscosity and stability of mAb5 were determined for concentrations up to 250 mg/mL. Organic electrolytes Arg(HCl) and His(HCl) produced the largest viscosity reductions, indicating screening of local anisotropic short-ranged attractive and hydrophobic interactions. These cosolutes significantly reduced mAb5 aggregate concentration as measured by size-exclusion chromatography after 4 weeks of 40°C storage at 200 mg/mL, with the largest reduction for Arg(Glu). The effects of the cosolutes on storage stability and viscosity are related to their ability to reduce attractive PPIs at high concentration (200 mg/mL), as shown by comparing measurements of structure factor (by small-angle X-ray scattering) and collective diffusion (by dynamic light scattering) with models of hard and attractive spheres. The improved stability of Arg(Glu) over Arg(HCl) despite similar PPI by small-angle X-ray scattering at high concentration is consistent with higher protein conformational stability as determined by differential scanning fluorimetry and differential scanning light scattering.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.