Biomedical subjects

C Russell Middaugh

Publications and source records attributed to C Russell Middaugh.

At least 19 recordsLinked to original sources

PubMed2007

A rapid, three-step process for the preformulation of a recombinant ricin toxin A-chain vaccine.

A systematic, three-step approach was employed to develop a stable, optimized formulation of ricin toxin A-chain V76M/Y80A (rRTA) for use as a vaccine against ricin toxicity. The method first uses spectroscopic techniques to evaluate the stability of rRTA as a function of temperature and pH. To synthesize the data, empirical phase diagrams are generated to display the conditions under which the protein maintains particular conformational states. Following identification of optimal pH conditions, light scattering and fluorescence assays are employed to screen a wide variety of compounds for their abilities to stabilize rRTA. Once stabilizers were identified, the ability of rRTA to adsorb to aluminum salt adjuvants was evaluated. Desorption of the protein from the adjuvant was also analyzed. Using this approach, the optimal formulation conditions for rRTA were determined to be pH 6.0 utilizing glycerol as a stabilizer and Alhydrogel as an adjuvant. Such an approach has the potential to significantly reduce the time it takes to get vaccines into clinical testing.

Adjuvants, Immunologic↗

PubMed2007

Effects of stabilizers on the destabilization of proteins upon adsorption to aluminum salt adjuvants.

Excipients for protein-based vaccines are currently identified by evaluating the stability of the protein in solution. In most cases, however, the protein is adsorbed to the surface of an aluminum salt adjuvant in the final vaccine formulation. Previous studies showed that model protein antigens may be structurally altered and less thermally stable upon adsorption to aluminum salt adjuvants [Jones LS, Peek LJ, Power J, Markham A, Yazzie B, Middaugh CR, 2005, J Biol Chem 280:13406-13414]. The work presented herein provides evidence that compounds that stabilize the protein in solution also stabilize the adsorbed protein; however, the stability of the adsorbed protein in the presence of the stabilizer remains lower than that of the protein in solution. Potential implications of the reduced stability on the approach used to select excipients during formulation development are discussed.

Adjuvants, Immunologic↗

PubMed2007

Effects of solutes on empirical phase diagrams of human fibroblast growth factor 1.

A variety of solutes are commonly used to increase the stability of protein in therapeutic formulations. An empirical phase diagram approach is used to evaluate the effects of different types of additives on the solution behavior of a protein of pharmaceutical interest, human fibroblast growth factor 1 (FGF-1). A specific stabilizer, heparin, and a nonspecific stabilizer, sucrose, were used in this work. The protein was characterized as a function of pH (3-8) and temperature (10-85 degrees C) using Far-UV circular dichroism (Far-UV CD), intrinsic and extrinsic fluorescence as well as second derivative UV absorption spectroscopy. Empirical phase diagrams were constructed to summarize the biophysical characterization data obtained with FGF-1 alone, in the presence of a threefold weight excess of heparin (3x heparin) or 10% sucrose (w/v). Three phases are observed in the low temperature regions at pH 3, 4, and 5-8. Phase boundaries corresponding to major heat-induced transitions are detected in the physiological temperature range. The highest thermal stabilities are observed near neutral pH (pH 6 and 7). Both heparin and sucrose appear to enhance the thermal stability of FGF-1, although their effects on the phase diagram are quite distinct. The greatest stabilization is observed at pH 8. Only heparin appears to protect FGF-1 from acid-induced unfolding to any extent.

Circular Dichroism↗

PubMed2007

Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2).

The interaction of several of the fibroblast growth factors (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -2. The extent of such phenomena throughout the 23-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of keratinocyte growth factor 2 (KGF-2, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein's structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid) fluorescence, differential scanning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-2 by increasing the thermal-unfolding temperature by approximately 9-15 degrees C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-2. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this growth factor. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport.

Calorimetry, Differential Scanning↗

PubMed2006

Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding.

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.

Crystallography, X-Ray↗

PubMed2006

Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations.

The protective effects of amino acids on stabilizing protein secondary structure were evaluated using diffuse reflectance FTIR spectroscopy, and interactions between proteins and arginine were detected using solid-state NMR spectroscopy. Upon freeze-drying, excipient-free anti-CD11a and anti-IgE antibodies underwent significant changes in their secondary structures. For both antibodies, the amount of intermolecular beta-sheet substantially increased and the native conformation of intramolecular beta-sheet content decreased considerably. The addition of amino acids to the formulations reduced protein secondary structure alterations in a concentration-dependent manner. Histidine and arginine appeared to be the most protective excipients (of the amino acids studied) in inhibiting protein secondary structural changes. Solid-state NMR illustrated that non-covalent interactions (e.g., hydrogen bonding, ion-dipole interactions) were formed between the arginine side chain and the protein. Glycine is the least effective additive of those studied in preventing secondary structure changes upon freeze-drying. Despite secondary structural changes, freeze-dried protein in the presence and absence of amino acids refolded back into its native conformation upon reconstitution in water.

Amino Acids↗

PubMed2006

A network-based analysis of polyanion-binding proteins utilizing yeast protein arrays.

The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.

Amino Acid Sequence↗

PubMed2006

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions.

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.

Acetylation↗

PubMed2006

Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains.

Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.

Antigens, Bacterial↗

PubMed2006

Stability of helix-rich proteins at high concentrations.

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.

Animals↗

PubMed2006

A systematic approach to stabilizing EBA-175 RII-NG for use as a malaria vaccine.

Region II of the erythrocyte-binding antigen (EBA-175 RII) has been identified as a promising target for a malaria vaccine. A systematic approach to identify optimal preformulation conditions of a non-glycosylated (NG) antigen, EBA-175 RII-NG, has been developed. This approach consists of development of an empirical temperature/pH phase diagram, high throughput stabilizer screening and aluminum salt adjuvant adsorption studies. Using these physical methods, we developed a stable formulation for EBA-175 RII-NG at pH 6.0 with sucrose and Brij 35 as stabilizers and Adju-Phos as an adjuvant. This approach should be generally applicable to guiding the development of stable vaccine formulations.

Animals↗

PubMed2006

Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature.

Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 degrees C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 degrees C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner.

Animals↗

PubMed2006

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature.

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.

Amino Acid Sequence↗

PubMed2006

Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes.

Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes.

Amino Acid Substitution↗

PubMed2006

Anthrax vaccine powder formulations for nasal mucosal delivery.

Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6-8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7-8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization.

Administration, Intranasal↗

PubMed2006

Effect of pH and ionic strength on the physical stability of adenovirus type 5.

The thermal stability of adenovirus type 5 (Ad5) was investigated over the pH range 3-8 employing a variety of biophysical techniques under conditions of low and high ionic strength. Analysis of the structural stability of Ad5 by dynamic light scattering, intrinsic and extrinsic fluorescence, and second derivative UV absorption spectroscopies suggest that the capsid stability of Ad5 increases with decreasing pH under both ionic strength conditions. Significant aggregation, however, was observed at pH < or = 5 under conditions of low ionic strength. These studies also suggest that the physical stability of Ad5 is significantly enhanced under acidic conditions in the presence of 1 M NaCl. Evaluation of the quaternary structural stability of Ad5 by dynamic light scattering and extrinsic fluorescence spectroscopy suggest that the Ad5 capsid undergoes a two-step dismantling process wherein the viral particles initially expand in size near 50 degrees C and the DNA core is at least partially exposed to the surrounding solvent. Complete capsid disassembly and total exposure of the DNA core follows at higher temperatures. Data generated during these studies were combined employing a multidimensional eigenvector approach that combines data from numerous techniques into a colored representation. This picture, or "empirical phase diagram," provides an intuitive representation of the physical stability of Ad5 over the pH range 4-8 from 10 degrees C to 85 degrees C.

Adenoviridae↗

PubMed2006

Influence of aggregation on immunogenicity of recombinant human Factor VIII in hemophilia A mice.

Recombinant human factor VIII (rFVIII), a multidomain glycoprotein is used in replacement therapy for treatment of hemophilia A. Unfortunately, 15%-30% of the treated patients develop inhibitory antibodies. The pathogenesis of antibody development is not completely understood. The presence of aggregated protein in formulations is generally believed to enhance the immune response. rFVIII has a tendency to aggregate but the effect of such aggregation on the immunogenicity of rFVIII is not known. We have, therefore, characterized aggregated rFVIII produced by thermal stress and evaluated its effect on the immunogenicity of rFVIII in hemophilia A mice. Aggregated rFVIII alone and mixtures of rFVIII with aggregated rFVIII were less immunogenic than native rFVIII. In vitro Th-cell proliferation studies and cytokine analyses conducted on splenocytes obtained from immunized animals suggest that aggregated rFVIII behaves as a unique antigen compared to native monomeric rFVIII. The antigenic properties of the aggregated and native rFVIII were compared using ELISAs (epitope availability) and cathepsin-B (an antigen processing enzyme) digestion. The data suggest significant differences in the antigenic properties of rFVIII and aggregated rFVIII. Overall it appears that aggregated rFVIII does not enhance the immunogenicity (inhibitor development) of rFVIII in hemophilia A mice but rather acts as a distinct antigen.

Animals↗

PubMed2006

Thermal stability of adenovirus type 2 as a function of pH.

Although several recent studies have focused on the characterization and formulation of adenovirus type 5, similar efforts focusing on adenovirus type 2 (Ad2) have been lacking. To this end, multiple biophysical techniques were employed to investigate the thermal stability of Ad2 as a function of pH. Highly cooperative thermally induced changes in capsid conformation were detected near 45 and 65 degrees C under neutral conditions. The first transition is attributed to the loss of the penton bases and their associated fibers followed by more complete physical degradation at higher temperatures. Data in this work as well as previous studies suggest that a common mechanism of icosahedral virus thermal degradation exists. Conformational changes detected in these studies occurred at increasingly higher temperatures with decreasing pH from 8 to 5 suggesting that the physical stability of Ad2 is enhanced under mildly acidic conditions. To consolidate the data generated in these studies, a multi-dimensional Eigenvector approach was employed to generate an empirical phase diagram (EPD) of Ad2. The EPD identifies conditions, or "phase boundaries," where structural integrity is altered, in addition to providing a tool that can be used to identify conditions under which forced degradation and excipient-screening studies can be conducted.

Adenoviridae↗