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.

Danio rerio Oocytes for Eukaryotic In-Cell NMR.

Understanding how the crowded and complex cellular milieu affects protein stability and dynamics has only recently become possible by using techniques such as in-cell nuclear magnetic resonance. However, the combination of stabilizing and destabilizing interactions makes simple predictions difficult. Here we show the potential of Danio rerio oocytes as an in-cell nuclear magnetic resonance model that can be widely used to measure protein stability and dynamics. We demonstrate that in eukaryotic oocytes, which are 3-6-fold less crowded than other cell types, attractive chemical interactions still dominate effects on protein stability and slow tumbling times, compared to the effects of dilute buffer.

Protein-complex stability in cells and in vitro under crowded conditions.

Biology is beginning to appreciate the effects of the crowded and complex intracellular environment on the equilibrium thermodynamics and kinetics of protein folding. The next logical step involves the interactions between proteins. We review quantitative, wet-experiment based efforts aimed at understanding how and why high concentrations of small molecules, synthetic polymers, biologically relevant cosolutes and the interior of living cells affect the energetics of protein-protein interactions. We then address popular theories used to explain the effects and suggest expeditious paths for a more methodical integration of experiment and simulation.

Protein-Peptide Binding Energetics under Crowded Conditions.

Nearly all biological processes, including strictly regulated protein-protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen-activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19F NMR lineshape analysis. We also analyze the temperature dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3-peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically relevant cosolutes. Although most of the protein cosolutes destabilize the SH3-peptide complexes, the effects are nongeneralizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein-protein interactions in cells and show the importance of using physiologically relevant cosolutes for investigating macromolecular crowding effects.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.

Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

Protecting activity of desiccated enzymes.

Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation-, freezing-, and lyophilization-induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.

Enthalpic stabilization of an SH3 domain by D2 O.

The stability of a protein is vital for its biological function, and proper folding is partially driven by intermolecular interactions between protein and water. In many studies, H2 O is replaced by D2 O because H2 O interferes with the protein signal. Even this small perturbation, however, affects protein stability. Studies in isotopic waters also might provide insight into the role of solvation and hydrogen bonding in protein folding. Here, we report a complete thermodynamic analysis of the reversible, two-state, thermal unfolding of the metastable, 7-kDa N-terminal src-homology 3 domain of the Drosophila signal transduction protein drk in H2 O and D2 O using one-dimensional 19 F NMR spectroscopy. The stabilizing effect of D2 O compared with H2 O is enthalpic and has a small to insignificant effect on the temperature of maximum stability, the entropy, and the heat capacity of unfolding. We also provide a concise summary of the literature about the effects of D2 O on protein stability and integrate our results into this body of data.

Cosolutes, Crowding, and Protein Folding Kinetics.

Long accepted as the most important interaction, recent work shows that steric repulsions alone cannot explain the effects of macromolecular cosolutes on the equilibrium thermodynamics of protein stability. Instead, chemical interactions have been shown to modulate, and even dominate, crowding-induced steric repulsions. Here, we use 19F NMR to examine the effects of small and large cosolutes on the kinetics of protein folding and unfolding using the metastable 7 kDa N-terminal SH3 domain of the Drosophila signaling protein drk (SH3), which folds by a two-state mechanism. The small cosolutes consist of trimethylamine N-oxide and sucrose, which increase equilibrium protein stability, and urea, which destabilizes proteins. The macromolecules comprise the stabilizing sucrose polymer, Ficoll, and the destabilizing globular protein, lysozyme. We assessed the effects of these cosolutes on the differences in free energy between the folded state and the transition state and between the unfolded ensemble and the transition state. We then examined the temperature dependence to assess changes in activation enthalpy and entropy. The enthalpically mediated effects are more complicated than suggested by equilibrium measurements. We also observed enthalpic effects with the supposedly inert sucrose polymer, Ficoll, that arise from its macromolecular nature. Assessment of activation entropies shows important contributions from solvent and cosolute, in addition to the configurational entropy of the protein that, again, cannot be gleaned from equilibrium data. Comparing the effects of Ficoll to those of the more physiologically relevant cosolute lysozyme reveals that synthetic polymers are not appropriate models for understanding the kinetics of protein folding in cells.

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase protein stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living Escherichia coli cells before and after hyperosmotic shock in the presence and absence of the osmolyte, glycine betaine. The 7-kDa N-terminal src-homology 3 domain of Drosophila signal transduction protein drk is used as the test protein. We find that hyperosmotic shock decreases SH3 stability in cells, consistent with the idea that transient attractive interactions are important under physiologically relevant crowded conditions. The subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient attractive interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.

Effects of ovalbumin protein nanoparticle vaccine size and coating on dendritic cell processing.

Nanoparticle vaccine delivery platforms are a promising technology for enhancing vaccine immunogenicity. Protein nanoparticles (PNPs), made entirely from antigen, have been shown to induce protective immune responses against influenza. However, the fundamental mechanisms by which PNPs enhance component protein immunogenicity are not understood. Here, we investigate the role of size and coating of model ovalbumin (OVA) PNPs on particle uptake and trafficking, as well as on inflammation and maturation factor expression in dendritic cells (DCs) in vitro. OVA PNPs enhance antigen uptake in a size-independent manner, and experience attenuated endosomal acidification as compared to soluble OVA. OVA PNPs also trigger Fc receptor upregulation. Expression of cytokines IL-1ß and TNF-α were PNP size- and coating-dependent, with small (∼270 nm) nanoparticles triggering greater inflammatory cytokine production than large (∼560 nm) particles. IL-1ß expression by DCs in response to PNP stimulation implies activation of the inflammasome, a pathway known to be activated by certain types of nanoparticulate adjuvants. The attenuated acidification and pro-inflammatory profile generated by PNPs in DCs demonstrate that physical biomaterial properties can modulate dendritic cell-mediated antigen processing and adjuvancy. In addition to nanoparticles' enhancement of DC antigen uptake, our work suggests that vaccine nanoparticle size and coating are uptake-independent modulators of immunogenicity.