Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.
The mechanistic understanding of
three key active pharmaceutical
ingredient (API) drying elements: drying kinetics, physical property
control, and stability control, is critical for downstream formulation
development and manufacture. The objective of this study was to collect
drying kinetic data through the use of a laboratory integrated sorption
chamber to elucidate the drying mechanism and thereby establish a
drying model. In addition, the collection of drying kinetic data would
provide a greater understanding of the solvent to API ratio as well
as the API stability to help develop a large-scale drying protocol.
The study material is an ethanol solvate (ethanolate) of the API.
There is no fundamental crystal structure change upon drying of the
solvate in the temperature range from 50 to 90 °C, and the drying
kinetic data show that the molar ratio of ethanol to API is 1:1. This
is confirmed by X-ray crystallography. An analysis of the drying kinetics
showed that the diffusion of ethanol through the crystal lattice provided
the primary mass transfer resistance, which can be simulated using
the Fickian principle of diffusion through a slab. The diffusion coefficients
from 50 to 90 °C at 20 mmHg were found to be strongly temperature-dependent
and fit well using an Arrhenius type relationship. The activation
energy for diffusion was then determined from this relationship. By
using these diffusion parameters, the drying curves at various temperatures
were generated to predict the drying time to meet the final ethanol
specification. The predicted drying time was in good agreement with
experimental drying data at high temperatures (80 and 90 °C)
but shorter than that observed when drying at lower temperatures (between
50 and 70 °C). Thermal stability data showed that the compound
is stable at drying temperatures equal or below 70 °C. Drying
data from this study are generated from a static balance. Hence, the
effect of shear forces imposed from cake depth and dryer agitation
on particle morphology, physical stability of the desolvated API,
and drying time needs to be studied to generate the optimal drying
protocol for large-scale operation.
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