Bioprocessors must minimize the aggregation of therapeutic proteins, but that’s not an easy task. Proteins can aggregate during any step from manufacturing to administering a drug. “The aggregation results in an irreversible drug loss and also leads to an increase in the risk of immunogenicity,” says Lalit Pandey, PhD, associate professor of biosciences and bioengineering at the Indian Institute of Technology Guwahati, India. “The aggregated proteins have also been associated with various protein-deposition diseases, like amyloidosis.”
In a recent review article, Pandey explained that chemical, environmental, and mechanical factors—such as salt levels, pH, and shear—can cause aggregation. “These factors induce the unfolding of proteins, which exposes sites for non-specific protein interactions, leading to the formation of higher-order structures, such as dimers, oligomers, and aggregates,” he says.
Aggregation can occur across the entire process of making a therapeutic protein. In upstream bioprocessing, cell growth and protein stability depend on many factors and conditions, including aeration, agitation, antifoam agents, osmolarity, pH, and temperature.
“This poses another challenge in bioprocessing to tune the parameters so as to optimize the product formation without inducing the aggregation,” Pandey explains. “For example, the optimum temperature for cell growth may affect the stability of therapeutic proteins.” A protein’s stability even depends on interactions with surfaces of bioreactors and pipes.
In addition, Pandey points out steps in downstream bioprocessing that can impact aggregation. For example, “elution buffers of different pH and ionic strength are used in chromatographic separations,” he says, and that can trigger aggregation. Bioprocessors also vary pH and ionic strength in viral inactivation and to neutralize antibodies. Aggregation can also arise from mechanical stresses in filtration and thermal stress in freeze-thaw cycles.
The type of ionic bonding can reduce or drive aggregation. “The spontaneous binding of ions with exothermic sites has been found to stabilize the conformation and inhibit aggregation, and the endothermic binding of ions with proteins disrupts the stabilized structure and accelerates the process of aggregation,” Pandey says. “An understanding of the binding behavior of the ions with protein can help in regulating the molar concentrations of ions to control the aggregation.”
A bioprocessor can minimize aggregation in various ways. “A buffer should be carefully selected for a particular therapeutic protein in order to inhibit a significant change in conformation,” Pandey advises. Other processing steps offer other opportunities to reduce aggregation. As examples, Pandey notes that “fast freezing rates inhibit product aggregation,” and “modifications of the contacting surfaces are being applied to inhibit or minimize non-specific protein-surface interactions,” adding that in situ analysis of subvisible particles and removing them can also reduce aggregation.
As scientists learn more about protein aggregation, bioprocessors must address more parameters to minimize the problem. Consequently, “the stability of therapeutic proteins is still a challenge,” states Pandey.