Stability Comparison Between Microglassification and Lyophilization Using a Monoclonal Antibody.

Producing solid-state formulations of biologics remains a daunting task despite the prevalent use of lyophilization and spray drying technologies in the biopharmaceutical industry. The challenges include protein stability (temperature stresses), high capital costs, particle design/controllability, shortened processing times and manufacturing considerations (scalability, yield improvements, aseptic operation, etc.). Thus, scientists/engineers are constantly working to improve existing methodologies and exploring novel dehydration/powder-forming technologies. Microglassification™ is a dehydration technology that uses solvent extraction to rapidly dehydrate protein formulations at ambient temperatures, eliminating the temperature stress experienced by biologics in traditional lyophilization and spray drying methods. The process results in microparticles that are spherical, dense, and chemically stable. In this study, we compared the molecular stability of a monoclonal antibody formulation processed by lyophilization to the same formulation processed using Microglassification™. Both powders were placed on stability for 3 months at 40 °C and 6 months at 25 °C. Both dehydration methods showed similar chemical stability, including percent monomer, charge variants, and antigen binding. These results show that Microglassification™ is viable for the production of stable solid-state monoclonal antibody formulations.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Ameloblastin peptide encoded by exon 5 interacts with amelogenin N-terminus.

Interactions between enamel matrix proteins are important for enamel biomineralization. In recent in situ studies, we showed that the N-terminal proteolytic product of ameloblastin co-localized with amelogenin around the prism boundaries. However, the molecular mechanisms of such interactions are still unclear. Here, in order to determine the interacting domains between amelogenin and ameloblastin, we designed four ameloblastin peptides derived from different regions of the full-length protein (AB1, AB2 and AB3 at N-terminus, and AB6 at C-terminus) and studied their interactions with recombinant amelogenin (rP172), and the tyrosine-rich amelogenin polypeptide (TRAP). A series of amelogenin Trp variants (rP172(W25), rP172(W45) and rP172(W161)) were also used for intrinsic fluorescence spectroscopy. Fluorescence spectra of rP172 titrated with AB3, a peptide encoded by exon 5 of ameloblastin, showed a shift in λmax in a dose-dependent manner, indicating molecular interactions in the region encoded by exon 5 of ameloblastin. Circular dichroism (CD) spectra of amelogenin titrated with AB3 showed that amelogenin was responsible for forming α-helix in the presence of ameloblastin. Fluorescence spectra of amelogenin Trp variants as well as the spectra of TRAP titrated with AB3 showed that the N-terminus of amelogenin is involved in the interaction between ameloblastin and amelogenin. We suggest that macromolecular co-assembly between amelogenin and ameloblastin may play important roles in enamel biomineralization.

Efficacy of amelogenin-chitosan hydrogel in biomimetic repair of human enamel in pH-cycling systems.

Amelogenin-chitosan (CS-AMEL) hydrogel has shown great potential for the prevention, restoration, and treatment of defective enamel. As a step prior to clinical trials, this study aimed to examine the efficacy of CS-AMEL hydrogel in biomimetic repair of human enamel with erosive or caries-like lesions in pH-cycling systems. Two models for enamel defects, erosion and early caries, were addressed in this study. Two pH-cycling systems were designed to simulate the daily cariogenic challenge as well as the nocturnal pH conditions in the oral cavity. After pH cycling and treatment with CS-AMEL hydrogel, a synthetic layer composed of oriented apatite crystals was formed on the eroded enamel surface. CS-AMEL repaired the artificial incipient caries by re-growing oriented crystals and reducing the depth of the lesions by up to 70% in the pH-cycling systems. The results clearly demonstrate that the CS-AMEL hydrogel is effective at the restoration of erosive and carious lesions under pH-cycling conditions.

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residue and fluorescence resonance energy transfer [FRET], we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions.

Interactions of amelogenin with phospholipids.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles.

Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence, and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modeling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Structure, dynamics, and activity of an all-cysteine mutated human beta defensin-3 peptide analogue.

Human beta defensin-3 (HBD-3) is a unique potent antimicrobial peptide. To explore the importance of the three-dimensional structure of HBD-3 in its activity and selectivity, we have mutated all six cysteine residues of HBD-3 to other amino acids, expressed the mutant (named as Def-A) in Escherichia coli, and analyzed the mutant's activity, structure, and dynamics. Def-A is active against several bacterial strains, but the activity is influenced by the ionic strength of the environment. When subjected to vesicles like POPG or to micelles like SDS, Def-A is changed from a random coil structure to an ordered helical form. We have determined the structure of Def-A in SDS micelle and found that it is folded into two distinct helices separated by a proline kink. We propose that the long N-terminal helix with many hydrophobic residues is inserted inside the micelle while the C-terminal helix with one large positive charge patch is located outside the micelle and interacts with the charged head groups of the micelle. The model is supported by NMR relaxation and H-D exchange data. Our results indicate that in addition to the number of positively charged residues and hydrophobic residues, the arrangement of these residues in the three-dimensional space is important to the antimicrobial selectivity and salt-dependent activity of human beta defensins.