Abstract
Aggregation is a common phenomenon in the field of protein therapeutics and can lead to function loss or immunogenic patient responses. Two strategies are currently used to reduce aggregation: (1) finding a suitable formulation, which is labor-intensive and requires large protein quantities, or (2) engineering the protein, which requires extensive knowledge about the protein aggregation pathway. We present a biophysical characterization of the oligomerization and aggregation processes by Interferon alpha-2a (IFNα-2a), a protein drug with antiviral and immunomodulatory properties. This study combines experimental high throughput screening with detailed investigations by small-angle X-ray scattering and analytical ultracentrifugation. Metropolis Monte Carlo simulations are used to gain insight into the underlying intermolecular interactions. IFNα-2a forms soluble oligomers that are controlled by a fast pH and concentration-dependent equilibrium. Close to the isoelectric point of 6, IFNα-2a forms insoluble aggregates which can be prevented by adding salt. We show that monomer attraction is driven mainly by molecular anisotropic dipole–dipole interactions that increase with increasing pH. Repulsion is due to monopole–monopole interactions and depends on the charge of IFNα-2a. The study highlights how combining multiple methods helps to systematically dissect the molecular mechanisms driving oligomer formation and to design ultimately efficient strategies for preventing detrimental protein aggregation.
Highlights
The development of therapeutic protein drugs has increased dramatically in the past decade.[1,2]. Owing to their high specificity, they often show fewer side effects compared with small molecule drugs and open many possibilities for the treatment of diseases.[3]. They are challenging and costly in their development due to their short half-life and low stability compared to small molecules.[4−6] In particular, the formation of oligomers and aggregates remains a challenge as they differ in their characteristics from protein to protein.[7−9] There have been many approaches targeting this problem, but one of the challenges is the diverse behavior of different kinds of protein drugs
IFNα-2a belongs to the type I interferon cytokines, which are part of the innate immune, system and is used in the treatment of, for example, hepatitis, carcinoma, leukemia, and lymphoma.[12−14] Type I interferons show a high structural identity (Figure S1, Table S1)
Because of the sequence and structural similarity among cytokines, our results benefit the study of these powerful protein drugs
Summary
The development of therapeutic protein drugs has increased dramatically in the past decade.[1,2] Owing to their high specificity, they often show fewer side effects compared with small molecule drugs and open many possibilities for the treatment of diseases.[3] they are challenging and costly in their development due to their short half-life and low stability compared to small molecules.[4−6] In particular, the formation of oligomers and aggregates remains a challenge as they differ in their characteristics from protein to protein.[7−9] There have been many approaches targeting this problem, but one of the challenges is the diverse behavior of different kinds of protein drugs. The detailed characterization of oligomers and aggregates formed is essential to understand the mechanisms behind protein aggregation. This knowledge can be used to alter proteins to avoid oligomerization and aggregation.[11] IFNα-2a consists of 165 amino acids (19.24 kDa) with 68% α-helical content. IFNα-2a belongs to the type I interferon cytokines, which are part of the innate immune, system and is used in the treatment of, for example, hepatitis, carcinoma, leukemia, and lymphoma.[12−14] Type I interferons show a high structural identity (Figure S1, Table S1)
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