Biomedical subjects

T W Randolph

Publications and source records attributed to T W Randolph.

At least 19 recordsLinked to original sources

PubMed2006

Multiscale processing of mass spectrometry data.

This work addresses the problem of extracting signal content from protein mass spectrometry data. A multiscale decomposition of these spectra is used to focus on local scale-based structure by defining scale-specific features. Quantification of features is accompanied by an efficient method for calculating the location of features which avoids estimation of signal-to-noise ratios or bandwidths. Scale-based histograms serve as spectral-density-like functions indicating the regions of high density of features in the data. These regions provide bins within which features are quantified and compared across samples. As a preliminary step, the locations of prominent features within coarse-scale bins may be used for a crude registration of spectra. The multiscale decomposition, the scale-based feature definition, the calculation of feature locations, and subsequent quantification of features are carried out by way of a translation-invariant wavelet analysis.

Algorithms↗

PubMed2002

Dissolution and partitioning behavior of hydrophobic ion-paired compounds.

PURPOSE: This study was conducted to determine the effects of counterion hydrophobicity on organic/aqueous partition coefficients for hydrophobic ion paired (HIP) complexes. Furthermore, the coupled dissolution and reverse ion-exchange kinetics for dissolution of HIP complexes into aqueous electrolyte solutions were measured and mathematically modeled. METHODS: HIP complexes of model drugs tacrine and l-phenylephrine were formed using linear sodium alkylsulfates and bis (2-ethylhexyl sodium sulfosuccinate). Equilibrium partition coefficients between chloroform and aqueous solutions for the complexes and the kinetics of dissolution of the complexes in buffered aqueous solutions were measured. RESULTS: The chloroform/aqueous partition coefficients for l-phenylephrine/bis (2-ethylhexyl sodium sulfosuccinate) complexes decrease with increasing molar surface tension increment of salts added to the aqueous solution. The logarithm of the partition coefficient for a homologous series of alkyl sulfate complexes decreases as the hydrophilic-lipophilic balance number increases. Dissolution of HIP complexes in deionized water shows first order kinetics, whereas dissolution in aqueous electrolyte solutions shows biphasic kinetics. A kinetic model explains these dissolution rates. CONCLUSIONS: Solubility and dissolution rates for HIP complexes depend on the hydrophobic-lipophilic balance number of the organic counter ion as well as on the electrolyte composition of aqueous solutions. Reverse ion-exchange kinetics are sufficiently slow to allow HIP complexes to be considered simple prodrugs.

Hydrophobic and Hydrophilic Interactions↗

PubMed2001

Electrochemical determination of dissolution rates of lyophilized pharmaceutical formulations.

We present a method to measure dissolution times for rapidly dissolving lyophilized formulations. K4Fe(CN)6 was lyophilized in formulations containing sucrose, salts, and Tween 20. Dissolution of the lyophilized powders was measured by monitoring the time dependence of the oxidation of Fe(CN)6(4-) ion at the surface of a platinum rotating disk electrode. Reconstitution of lyophilized K4Fe(CN)6 formulations with aqueous solutions of 0.03% Tween 20 altered the time of dissolution for all cases. Salt and sucrose formulations without Tween 20 dissolved more slowly in a Tween 20 solution than in water alone. In contrast, formulations containing Tween 20 dissolved faster in the Tween 20 solution when compared to dissolution in water.

Chemistry, Pharmaceutical↗

PubMed2001

High pressure refolding of recombinant human growth hormone from insoluble aggregates. Structural transformations, kinetic barriers, and energetics.

Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 m guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 m GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 m GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 m GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.

Dose-Response Relationship, Drug↗

PubMed2001

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying.

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.

Animals↗

PubMed2001

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: high-pressure and cosolute studies on recombinant human IFN-gamma.

The equilibrium dissociation of recombinant human IFN-gamma was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was -209 +/- 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 +/- 1.6 nm2/molecule of dimer. The first-order aggregation rate of recombinant human IFN-gamma in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were -41 +/- 9 ml/mol of dimer and 3.5 +/- 0.2 nm2/molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values approximately 4-fold larger for both transitions.

Dimerization↗

PubMed2001

Counteracting effects of renal solutes on amyloid fibril formation by immunoglobulin light chains.

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.

Amyloid↗

PubMed2001

The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf.

The objective of this study was to determine the influence of ice nucleation temperature on the primary drying rate during lyophilization for samples in vials that were frozen on a lyophilizer shelf. Aqueous solutions of 10% (w/v) hydroxyethyl starch were frozen in vials with externally mounted thermocouples and then partially lyophilized to determine the primary drying rate. Low- and high-particulate-containing samples, ice-nucleating additives silver iodide and Pseudomonas syringae, and other methods were used to obtain a wide range of nucleation temperatures. In cases where the supercooling exceeded 5 degrees C, freezing took place in the following three steps: (1) primary nucleation, (2) secondary nucleation encompassing the entire liquid volume, and (3) final solidification. The primary drying rate was dependent on the ice nucleation temperature, which is stochastic in nature but is affected by particulate content and the presence of ice nucleators. Sample cooling rates of 0.05 to 1 degrees C/min had no effect on nucleation temperatures and drying rate. We found that the ice nucleation temperature is the primary determinant of the primary drying rate. However, the nucleation temperature is not under direct control, and its stochastic nature and sensitivity to difficult-to-control parameters result in drying rate heterogeneity. Nucleation temperature heterogeneity may also result in variation in other morphology-related parameters such as surface area and secondary drying rate. Overall, these results document that factors such as particulate content and vial condition, which influence ice nucleation temperature, must be carefully controlled to avoid, for example, lot-to-lot variability during cGMP production. In addition, if these factors are not controlled and/or are inadvertently changed during process development and scaleup, a lyophilization cycle that was successful on the research scale may fail during large-scale production.

Freeze Drying↗

PubMed2001

Annealing to optimize the primary drying rate, reduce freezing-induced drying rate heterogeneity, and determine T(g)' in pharmaceutical lyophilization.

In a companion paper we show that the freezing of samples in vials by shelf-ramp freezing results in significant primary drying rate heterogeneity because of a dependence of the ice crystal size on the nucleation temperature during freezing.1 The purpose of this study was to test the hypothesis that post-freezing annealing, in which the product is held at a predetermined temperature for a specified duration, can reduce freezing-induced heterogeneity in sublimation rates. In addition, we test the impact of annealing on primary drying rates. Finally, we use the kinetics of relaxations during annealing to provide a simple measurement of T(g)', the glass transition temperature of the maximally freeze-concentrated amorphous phase, under conditions and time scales most appropriate for industrial lyophilization cycles. Aqueous solutions of hydroxyethyl starch (HES), sucrose, and HES:sucrose were either frozen by placement on a shelf while the temperature was reduced ("shelf-ramp frozen") or by immersion into liquid nitrogen. Samples were then annealed for various durations over a range of temperatures and partially lyophilized to determine the primary drying rate. The morphology of fully dried liquid nitrogen-frozen samples was examined using scanning electron microscopy. Annealing reduced primary drying rate heterogeneity for shelf-ramp frozen samples, and resulted in up to 3.5-fold increases in the primary drying rate. These effects were due to increased ice crystal sizes, simplified amorphous structures, and larger and more numerous holes on the cake surface of annealed samples. Annealed HES samples dissolved slightly faster than their unannealed counterparts. Annealing below T(g)' did not result in increased drying rates. We present a simple new annealing-lyophilization method of T(g)' determination that exploits this phenomenon. It can be carried out with a balance and a freeze-dryer, and has the additional advantage that a large number of candidate formulations can be evaluated simultaneously.

Crystallization↗

PubMed2001

The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution.

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.

Antibodies, Monoclonal↗

PubMed2000

The excluding effects of sucrose on a protein chemical degradation pathway: methionine oxidation in subtilisin.

The conformational stabilization of proteins by sucrose has been previously attributed to a preferential exclusion mechanism. The present study links this mechanism to stability against a chemical degradation pathway for subtilisin. Oxidation of a methionine residue adjacent to the active site to the sulfoxide form compromises subtilisin's enzymatic activity. In the presence of hydrogen peroxide and borate buffer, a borate-hydrogen peroxide complex binds to subtilisin's active site prior to the formation of methionine sulfoxide. Sucrose decreases the oxidation rate by limiting the accessibility of the complex to the methionine at the partially buried active site. The stabilization mechanism of sucrose is based on shifting the equilibrium of transiently expanding native conformations of subtilisin to favor the most compact states. Enzymatic parameter determination (kcat, KM) and hydrogen-deuterium exchange measurements confirm the limited conformational mobility of the enzyme in the presence of sucrose. Further support for limited mobility as the cause of oxidation inhibition by sucrose comes from the findings that neither viscosity nor possible interactions of sucrose with hydrogen peroxide, hydroxyl radicals, or borate can adequately explain the inhibition. The volume exclusion of sucrose from subtilisin is used to estimate the extent by which the native state of subtilisin must expand in solution to allow oxidation. The surface area of the oxidation-competent state is ca. 3.9% greater than that of the native state.

Borates↗

PubMed2000

Entrapping intermediates of thermal aggregation in alpha-helical proteins with low concentration of guanidine hydrochloride.

Aggregation of proteins is a problem with serious medical implications and economic importance. To develop strategies for preventing aggregation, the mechanism(s) and pathways by which proteins aggregate must be characterized. In this study, the thermally induced aggregation processes of three alpha-helix proteins (myoglobin, cytochrome c, and lysozyme) in the presence and absence of 1.0 m guanidine hydrochloride (GdnHCl) were investigated by means of infrared spectroscopy. In the absence of GdnHCl, intensities of the alpha-helix bands (approximately 1656 cm(-1)) decrease as a function of temperature at above 50 degrees C. With myoglobin and cytochrome c, the loss of helix bands was accompanied by the appearance of two new bands at 1694 and 1623 cm(-1), indicative of the formation of intermolecular beta-sheet aggregates. For lysozyme, bands indicative of intermolecular beta-sheet aggregates did not appear in any significant intensity. In the presence of 1.0 m GdnHCl, two major intermediate states rich in 3(10)-helix (represented by the band at 1663 cm(-1)) and beta-turn structure (represented by the band at 1667 cm(-1)), respectively, were observed. These findings demonstrated that IR spectroscopic studies of protein aggregation using a combination of thermal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates.

Animals↗

PubMed2000

Thermodynamic modulation of light chain amyloid fibril formation.

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Amino Acid Sequence↗

PubMed2000

Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran.

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.

Actins↗

PubMed2000

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor.

Metal-induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc-induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc-induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation.

Binding Sites↗

PubMed2000

Permeability and diffusion in vitreous humor: implications for drug delivery.

PURPOSE: Previous experimental work suggests that convection may be important in determining the biodistribution of drugs implanted or injected in the vitreous humor. To develop accurate biodistribution models, the relative importance of diffusion and convection in intravitreal transport must be assessed. This requires knowledge of both the diffusivity of candidate drugs and the hydraulic conductivity of the vitreous humor. METHODS: Hydraulic conductivity of cadaveric bovine vitreous humor was measured by confined compression tests at constant loads of 0.15 and 0.2 N and analyzed numerically using a two-phase model. Diffusion coefficient of acid orange 8, a model compound, in the same medium was measured in a custom-built diffusion cell. RESULTS: Acid orange 8 diffusivity within vitreous humor is about half that in free solution. When viscous effects are properly accounted for, the hydraulic conductivity of bovine vitreous humor is 8.4+/-4.5 x 10(-7) cm2/Pa x s. CONCLUSIONS: We predict that convection does not contribute significantly to transport in the mouse eye, particularly for low-molecular-weight compounds. For delivery to larger animals, such as humans we conclude that convection accounts for roughly 30% of the total intravitreal drug transport. This effect should be magnified for higher-molecular-weight compounds, which diffuse more slowly, and in glaucoma, which involves higher intraocular pressure and thus potentially faster convective flow. Thus, caution should be exercised in the extrapolation of small-animal-model biodistribution data to human scale.

Animals↗

PubMed1999

High pressure fosters protein refolding from aggregates at high concentrations.

High hydrostatic pressures (1-2 kbar), combined with low, nondenaturing concentrations of guanidine hydrochloride (GdmHCl) foster disaggregation and refolding of denatured and aggregated human growth hormone and lysozyme, and beta-lactamase inclusion bodies. One hundred percent recovery of properly folded protein can be obtained by applying pressures of 2 kbar to suspensions containing aggregates of recombinant human growth hormone (up to 8.7 mg/ml) and 0.75 M GdmHCl. Covalently crosslinked, insoluble aggregates of lysozyme could be refolded to native, functional protein at a 70% yield, independent of protein concentration up to 2 mg/ml. Inclusion bodies containing beta-lactamase could be refolded at high yields of active protein, even without added GdmHCl.

Growth Hormone↗

PubMed1999

Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding.

The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm-1 in infrared spectra of dried protein-sugar samples was examined. For samples in which a high level of native protein structure was retained in the dried solid, the apparent moisture content predicted by the carboxylate band area was greater than the actual moisture content, indicating that protection results from direct sugar-protein hydrogen bonding and not entrapment of water at the protein surface. Further, we show that the degree of structural protection conferred by sucrose and trehalose apparent in second derivative, amide I infrared spectra, correlates with the extent of hydrogen bonding between sugar and protein. The failure of dextran to inhibit dehydration-induced lysozyme unfolding is shown to result from the inability of the polymer to hydrogen bond adequately to the protein. Therefore, formation of an amorphous phase alone is not sufficient to maintain protein structure during dehydration. Glucose hydrogen bonds to a high degree with dried lysozyme, but is incapable of inhibiting lyophilization-induced protein unfolding in the absence of an effective cryoprotectant. However, the addition of polyethylene glycol, which is known to protect proteins during freezing, but not drying, to glucose protected lysozyme structure during lyophilization. Together, these results show that hydrogen bonding between carbohydrate and protein is necessary to prevent dehydration-induced protein damage. However, hydrogen bonding alone is not sufficient to protect proteins during lyophilization in the absence of adequate freezing protection.

Animals↗