Abstract
A fundamental step in developing a protein drug is the selection of a stable storage formulation that ensures efficacy of the drug and inhibits physiochemical degradation or aggregation. Here, we designed and evaluated a general workflow for screening of protein formulations based on small-angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling, temperature control, and fast data analysis and provides protein particle interaction information. SAXS, together with different methods including turbidity analysis, dynamic light scattering (DLS), and SDS-PAGE measurements, were used to obtain different parameters to provide high throughput screenings. Using a set of model proteins and biopharmaceuticals, we show that SAXS is complementary to dynamic light scattering (DLS), which is widely used in biopharmaceutical research and industry. We found that, compared to DLS, SAXS can provide a more sensitive measure for protein particle interactions, such as protein aggregation and repulsion. Moreover, we show that SAXS is compatible with a broader range of buffers, excipients, and protein concentrations and that in situ SAXS provides a sensitive measure for long-term protein stability. This workflow can enable future high-throughput analysis of proteins and biopharmaceuticals and can be integrated with well-established complementary physicochemical analysis pipelines in (biopharmaceutical) research and industry.
Highlights
Biopharmaceuticals have revolutionized the treatment of a wide range of diseases and are used in almost all branches of medicine [1]
We present a small-angle X-ray scattering (SAXS) methodology to investigate proteins in a highthroughput formulation screening setup that can be combined with available information derived from well-established techniques such as dynamic light scattering (DLS) and size-exclusion HPLC (SE-HPLC)
We developed a generalizable analytical workflow that can be applied to a wide range of formulations by SAXS (Figure 1)
Summary
Biopharmaceuticals have revolutionized the treatment of a wide range of diseases and are used in almost all branches of medicine [1]. Therapeutic proteins are the fastest growing category of biopharmaceuticals for use in many clinical settings, including cancers, infectious diseases, organ transplantation, chronic inflammatory, and cardiovascular diseases [2]. Biopharmaceutical products represent an increasing percentage in drug development and new drug applications for market approval [3], but their commercial and academic usage is currently limited by their physical stability. In contrast to small-molecule drugs, biopharmaceuticals are potentially immunogenic. Even slight alterations in the structure of the active ingredients in biopharmaceuticals can significantly affect their efficacy and immunogenicity [4,5]. Stabilization of biopharmaceuticals is generally performed during drug development, which involves ensuring their proper function and that their structure is preserved [6]. When the protein is stored in a non-optimal condition, this
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