Formation of nanostructures and weakening of interactions between proteins to design low viscosity dispersions at high concentrations

Date
2014-12
Authors
Borwankar, Ameya Umesh
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Abstract

Monoclonal antibodies and other protein therapeutics are rapidly gaining popularity as a favored class of drugs for treatment of various types of diseases and disorders including rheumatoid arthritis, Crohn’s disease, asthma, macular degeneration, different types of cancer. There great lot of interest in development of subcutaneous self-injection methods for administering these therapeutics to enable patient convenience which requires high concentration formulations to deliver the required dosage in the limited volume. At high concentrations, proteins have a propensity to be insoluble, aggregate, unfold, gel or denature due to strong short ranged protein-protein interactions, resulting in highly viscous solutions. Therefore, it is challenging to form highly concentrated, stable protein formulations with low viscosities. Addition of interacting co-solutes like arginine to protein formulations weakens protein-protein interactions through protein charge modification and hydrophilization of hydrophobic surface patches through binding on proteins. Weakened interactions lower the viscosity of protein formulations with 250 mg/ml protein by 5-6 times compared to conventional protein solutions in buffer not containing any co-solutes. Addition of co-solutes can also give rise to depletion attraction between proteins which can assemble them into amorphous nanostructured domains with lowered diffusion coefficients as determined by dynamic light scattering (DLS). A free energy model was developed to explain the formation of nanostructures due to short-ranged depletion attraction and long-ranged electrostatic repulsion, whereby sizes were predicted to range from 30 to 100 nm as a function of co-solute and protein concentrations. The nanostructured domains dissociated to monomeric, active and stable protein upon dilution to about 1 mg/ml. Supplemental sizing techniques, namely, cryogenic scanning electron microscopy (cryo-SEM) and small angle x-ray scattering (SAXS) show evidence of nanostructures larger than the monomer although determining the ratio of the amount of protein in monomeric state to that in the nanostructure state is still a challenge. In order to further understand cluster formation in a simpler system, gold nanoclusters were synthesized via assembly of primary particles by reaction. The morphology of these gold nanoclusters was also controlled by favoring kinetic over thermodynamic control of growth for generating asymmetrical structures thus allowing higher extinction in the near infrared region enabling biomedical imaging.

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