Preformulation Characterization and the Effect of Ionic Excipients on the Stability of a Novel DB Fusion Protein.

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine.

Investigating the dynamics and polyanion binding sites of fibroblast growth factor-1 using hydrogen-deuterium exchange mass spectrometry.

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics.

Improved Comparative Signature Diagrams to Evaluate Similarity of Storage Stability Profiles of Different IgG1 mAbs.

For therapeutic protein analytical studies related to evaluating lot-to-lot variability, different processes and/or formulations, or biosimilars, there is growing interest in applying novel data visualization tools for fingerprint analysis to identify statistically significant differences between 2 samples. Comparative Signature Diagrams (CSDs) were previously developed to display such differences as colored contour plots using a variety of biophysical data sets. In this study, several improvements are proposed to enhance readability and quantitative determinations of CSDs using protein stability data from more commonly used analytical methods such as size exclusion chromatography and capillary isoelectric focusing. To demonstrate the effectiveness of improved CSDs for data visualization, an accelerated and real-time stability study was set up for an IgG1 mAb (mAb A) and its corresponding triple mutant (mAb E). The stability profiles of both mAbs were compared for differences in aggregation (size exclusion chromatography) and charge heterogeneity (capillary isoelectric focusing) profiles over time. Both traditional data analysis and the improved CSDs conclude that the triple mutant mAb E is more susceptible to physicochemical degradation than mAb A under accelerated conditions. The current abilities and limitations of CSDs to provide fingerprint analysis of protein stability profiles to facilitate the determination of similarity versus nonsimilarity between samples is discussed.