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T W Randolph

Publications and source records attributed to T W Randolph.

At least 37 records · Page 2Linked to original sources
PubMed1999

Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation.

Hemoglobin has been previously shown to unfold during freeze drying when lyophilized from formulations that undergo freeze-concentration induced phase separation (Heller et al. 1997. Biotechnol Prog 13:590-596). In this report, we show that such damage may be avoided using kinetic strategies to arrest the phase separation. By rapidly cooling samples during liquid nitrogen spray-freeze drying, the time that the formulation spends in temperature regimes (ca. -3 to -23 degrees C) in which phase separation is both thermodynamically favorable and kinetically realizable is minimized. Increased protein damage with decreasing cooling rates and/or longer annealing periods at -7 degrees C is observed by FTIR spectroscopy. Phase separation and concomitant protein damage may also be avoided by addition of mannitol at concentrations sufficient to cause crystallization. Mannitol crystals segregate the freeze concentrated solution into microscopic domains that block propagation and nucleation of phase separating events. Addition of noncrystallizing sugars, such as sucrose and trehalose, or nonionic surfactants, such as Tween 80 and Triton X-100, has little protective effect against phase separation induced damage during freezing drying.

Calorimetry, Differential Scanning↗
PubMed1999

Application of a thermodynamic model to the prediction of phase separations in freeze-concentrated formulations for protein lyophilization.

Many of the compounds considered for use in pharmaceutical formulations demonstrate incompatibilities with other components at high enough concentrations, including pairs of polymers, polymers and salts, or even proteins in combination with polymers, salts, or other proteins. Freeze concentration can force solutions into a region where incompatibilities between solutes will manifest as the formation of multiple phases. Such phase separation complicates questions of the stability of the formulation as well as labile components, such as proteins. Yet, phase separation events are difficult to identify by common formulation screening methods. In this report, we use the osmotic virial expansion model of Edmond and Ogston (1) to describe phase-separating behavior of ternary aqueous polymer solutions. Second osmotic virial coefficients of polyethylene glycol 3350 (PEG) and dextran T500 were measured by light scattering. Assuming an equilibrium between ice and water in the freeze-concentrated solution, a degree of freeze concentration can be estimated, which, when combined with the phase separation spinodal, describes a "phase separation envelope" in which phase separation tendencies can be expected in the frozen solution. The phase separation envelope is bounded at low temperatures by the glass transition temperature of the freeze-concentrated solution. Scanning electron microscopic images and infrared spectroscopy of protein structure are provided as experimental evidence of the phase separation envelope in a freeze-dried system of PEG, dextran, and hemoglobin.

Dextrans↗
PubMed1999

Conformational stability of lyophilized PEGylated proteins in a phase-separating system.

PEGylation of proteins is of great interest to the pharmaceutical industry as covalent attachment of poly(ethylene glycol) (PEG) molecules can increase protein sera half-lives and reduce antigenicity. Not surprisingly, PEGylation significantly alters the surface characteristics of a protein, and consequently, its conformational stability during freezing and drying. Freeze concentration-induced phase separation between excipients has been previously shown to cause degradation of the secondary structure in lyophilized hemoglobin. In this report we show how PEGylation of two proteins, hemoglobin- and brain-derived neurotrophic factor (BDNF), influences partitioning and protein secondary structure as determined by FTIR spectroscopy in a system prone to freezing-induced phase separation. PEGylation of hemoglobin reduces the loss of structure induced by lyophilization in a PEG/dextran system that phase separates during freezing, perhaps due to altered partitioning. The partition coefficient for native hemoglobin favors the dextran-rich phase (PEG/dextran partition coefficient = 0.3), while PEGylated hemoglobin favors the PEG phase (partition coefficient = 3.1). In addition, we demonstrate that PEGylation alters hemoglobin's stability during lyophilization in the absence of other excipients. In contrast, because native BDNF already partitions into the PEG-rich phase, PEGylation of BDNF has a less dramatic effect on both partition coefficients and conformational stability during lyophilization. This is the first report on the effects of PEGylation on protein structural stability during lyophilization and points out the need to consider modification of formulations in response to changing protein surface characteristics.

Brain-Derived Neurotrophic Factor↗
PubMed1999

Effects of additives on the stability of Humicola lanuginosa lipase during freeze-drying and storage in the dried solid.

The effects of various classes of additives on the stability of a protein with a relatively hydrophobic surface, Humicola lanuginosa lipase (HLL), during lyophilization and storage in the dried solid, were investigated. Prior to lyophilization, it was found that 1 M trehalose or 1% (wt/vol) Tween 20 caused the protein to precipitate. Infrared spectroscopy indicated that trehalose "salted-out" native HLL, whereas Tween 20 induced non-native aggregates. Optimal recovery of native protein in the initial dried solid was obtained in the presence of additives which formed an amorphous phase and which had the capacity to hydrogen bond to the dried protein (e.g., trehalose and sucrose). Additives which crystallized during lyophilization (e.g., mannitol) or which remained amorphous, but were unable to hydrogen bond to the dried protein (e.g., dextran), afforded less stabilization relative to that seen in the absence of additives. Optimal storage stability in the dried solid required that both protein unfolding during lyophilization was minimized and that the formulation was stored at a temperature below its Tg value. Crystallization of sucrose during storage greatly reduced the storage stability of HLL. This was attributed to the increased moisture content and the reduced Tg value in the remaining amorphous phase containing the protein. Sucrose crystallization and the resulting damage to the protein were inhibited by decreasing the mass ratio of sucrose:protein.

Dextrans↗
PubMed1999

Investigation of protein-surfactant interactions by analytical ultracentrifugation and electron paramagnetic resonance: the use of recombinant human tissue factor as an example.

PURPOSE: The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain. As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations. The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique. In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored. METHODS: Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids. Neither of the recombinant proteins, produced in E. coli, contains the cytoplasmic domain. Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences. Recombinant human tissue factor 220 (rhTF 220), however, contains only the first 221 amino acids of the human tissue factor, lacking those of the transmembrane region. EPR and AUC were used to investigate the interactions between these two forms of rhTF and polyoxyethylene 8 lauryl ether, C12E8. RESULTS: Binding of C12E8 to rhTF 243 is detected by both EPR spectroscopy and AUC. Although a unique binding stoichiometry was not determined, EPR spectroscopy greatly narrowed the range of possible solutions suggested by the AUC data. Neither technique revealed an interaction between rhTF 220 and C12E8. CONCLUSIONS: The complementary nature of EPR spectroscopy and AUC make the combination of the two techniques useful in data interpretation when studying the interactions between rhTF and C12E8. By utilizing these techniques in this study, the binding stoichiometry of rhTF 243 to C12E8 ranges from 1.2:1 to 1.3:0.6 based on an aggregation number of 120. This binding is consistent with previously reported activity data that showed an increase in clotting rate when rhTF 243 is in the presence of C12E8 micelles. From the rhTF 220 data, it can further be concluded that the transmembrane domain of rhTF is necessary for interactions with C12E8.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid.

Freeze-drying is often used to improve storage stability of therapeutic proteins. In order to obtain a product with optimal storage stability it is important to understand the mechanisms by which solutes protect the protein against freeze-drying-induced stresses and also against damage induced during subsequent storage. The objective of the current study was to examine the importance of various mechanisms proposed to account for acute and long-term storage stability using recombinant human Factor XIII (rFXIII)4 as a model protein. Initially, for acute stability during freeze-drying, it was found that solutes which formed an amorphous phase stabilized rFXIII to a greater degree than solutes which crystallized during freeze-drying. However, only amorphous solutes which were able to hydrogen bond to the protein, and thus preserve the native protein structure in the dried solid, provided optimal acute stability. Thus, in addition to forming an amorphous phase, it was also important to possess the ability to hydrogen bond to the protein. Long-term storage stability was found to be optimal in the presence of solutes which formed and maintained amorphous phases with Tg values above the storage temperature and which also preserved the native protein structure during freeze-drying. Solute crystallization during storage compromised storage stability.

Dextrans↗
PubMed1998

A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma.

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-gamma (rhIFN-gamma) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-gamma aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-gamma and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-gamma native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-gamma against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.

Chromatography, Gel↗
PubMed1998

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.

Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.

Actins↗
PubMed1998

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis.

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.

Absorption↗
PubMed1998

Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII.

Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.

Circular Dichroism↗
PubMed1998

Aggregation of recombinant human interferon gamma: kinetics and structural transitions.

Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.

Humans↗
PubMed1998

Effects of Tween 80 and sucrose on acute short-term stability and long-term storage at -20 degrees C of a recombinant hemoglobin.

The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to recombinant hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and methemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested methemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in methemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.

Drug Stability↗
PubMed1998

Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions.

In the absence of surfactants, recombinant human growth hormone (rhGH) rapidly forms insoluble aggregates during agitation. The nonionic surfactant Tween 20, when present at Tween:protein molar ratios >4, effectively inhibits this aggregation. Differential scanning calorimetry (DSC) of rhGH solutions showed melting transitions that decreased by ca. 2 degrees C in the presence of Tween. Circular dichroism (CD) studies of the same thermal transition showed that the decrease is specific to the relatively high protein concentrations required for DSC. CD studies showed melting transitions that decreased with lower protein concentrations. Tween has an insignificant effect on the melting transition of rhGH at lower protein concentrations (0.18 mg/mL). Injection titration microcalorimetry showed that the interaction of Tween with rhGH is characterized by a weak enthalpy of binding. For comparison, interferon-g, another protein which has been shown to bind Tween, also shows weak enthalpy of binding. Fluorescent probe binding studies and infrared spectroscopic investigations of rhGH secondary structure support suggestions in the literature (Bam, N. B.; Cleland, J. L., Randolph, T. W. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnol. Prog. 1996. 12, 801-809) that Tween binding is driven by hydrophobic interactions, with little perturbation of protein secondary structure.

Calorimetry↗
PubMed1998

Process variable implications for residual solvent removal and polymer morphology in the formation of gentamycin-loaded poly (L-lactide) microparticles.

PURPOSE: The purpose was to determine the influence of process parameters in the precipitation with a compressed antisolvent (PCA) process on the morphology and residual dichloromethane (DCM) levels in gentamycin-loaded PLA microparticles. METHODS: The three variables studied were the rate of CO2 co-flowed during the polymer and drug post-precipitation, the post-precipitation pure CO2 flush rate, and the post-precipitation CO2 flush volume. Residual DCM levels were determined from headspace gas chromatography-mass spectroscopy (GC-MS) with single ion monitoring. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to estimate the crystallinity within microparticles. DCM was extracted from drug-loaded microparticles by both supercritical CO2 extraction and vacuum drying for up to two days to determine a lower limit for solvent removal. RESULTS: Increasing either the post-precipitations CO2 flow rate or flush volume resulted in lower residual DCM levels in the microparticle. The CO2 co-flow rate showed an opposite trend. Increasing in value resulted in a higher DCM value after precipitation. XRD and DSC analysis on these samples suggest that those produced at lower CO2 co-flow rates have a higher degree of crystallinity, which increases the diffusivity of DCM through the polymer matrix. Finally, samples subjected to extended (48 hr) CO2 extraction resulted in DCM levels on the order of one to three ppm. CONCLUSIONS: Specific PCA process conditions during microparticle formation have a strong influence on the residual solvent levels within the microparticles. Polymer morphology affects the diffusivity of solvent through the polymer matrix, which in turn determines the solvent removal rates.

Gas Chromatography-Mass Spectrometry↗
PubMed1997

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state.

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193-7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein's surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Chemical Phenomena↗