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Biomedical subjects

T W Randolph

Publications and source records attributed to T W Randolph.

47 records · Page 3Linked to original sources
PubMed1996

Effects of phase separating systems on lyophilized hemoglobin.

Polymer liquid-liquid two-phase systems offer a unique opportunity to study the mechanisms of protein stabilization during freezing and freeze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of recombinant hemoglobin frozen and lyophilized in the separated phases of a polyethylene glycol (PEG)-dextran system. Protein in each phase of an equilibrated biphasic PEG-dextran system experiences similar levels of structural protection against freezing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are protected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protection that polymers in equilibrium phases provide to proteins during lyophilization, emphasizing that the mechanisms of protein protection during freezing and drying are fundamentally different. In addition, we provide evidence that phase separation per se occurring during the course of the lyophilization cycle can be detrimental to the structural stability of a protein.

Dextrans↗
PubMed1996

Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers.

PURPOSE: This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. METHODS: Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. RESULTS: Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (Ts), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two Ts regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. CONCLUSIONS: Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.

Calorimetry, Differential Scanning↗
PubMed1995

Stability of protein formulations: investigation of surfactant effects by a novel EPR spectroscopic technique.

Surfactants are known to stabilize proteins and are often employed as additives in protein formulations. We have developed a method to study the interaction of these formulation additives with proteins by using the partitioning behavior of a spin label. In protein-free formulations, 16-doxyl stearic acid partitions into micelles above the critical micelle concentration (CMC) of the surfactant and gives rise to composite electron paramagnetic resonance (EPR) spectra composed of spectra from "free" label and "rotationally hindered" label. We compute the fraction of micelle-associated label by factor analysis and generate a label partition curve. When protein is added to the formulation, surfactant-protein aggregates form at concentrations below the surfactant's CMC. Partitioning of the label into these aggregates causes the EPR spectrum to reflect hindered rotation of the label at lower surfactant concentrations than in the protein-free solutions. A simple model of label partitioning shows that these partitioning shifts can be correlated to the surfactant:protein binding stoichiometry. We have studied the interactions of various non-ionic surfactants like Brij and Tween with recombinant human growth hormone and recombinant human interferon-gamma and obtained corresponding binding stoichiometries. These binding stoichiometries match those obtained by other techniques. This technique offers a new method for estimating the protein:surfactant binding stoichiometries.

Chemistry, Pharmaceutical↗
PubMed1992

Mechanism of polyethylene glycol interaction with the molten globule folding intermediate of bovine carbonic anhydrase B.

Polyethylene glycol has been shown to bind to the molten globule intermediate on the bovine carbonic anhydrase B folding pathway. The mechanism of this interaction has been extensively probed. Polyethylene glycol (PEG) binds weakly to the molten globule first intermediate as measured by hydrophobic interaction chromatography, but PEG does not bind to either the native state or the second intermediate. The binding of PEG to the molten globule has been confirmed with both intrinsic fluorescence and fluorescence quenching experiments which indicate a single PEG-binding site on the molten globule. Electron paramagnetic resonance spectroscopic studies with nitroxide-labeled PEG also indicate a single binding site. Additional electron paramagnetic resonance studies with spin-labeled carbonic anhydrase B suggest that a conformational change occurs in the molten globule intermediate after PEG binds to the surface. The formation of a PEG-molten globule complex results in a reduction in self-association of this compact hydrophobic structure. PEG-molten globule complex formation is analogous to the observed interaction between chaperonins and a molten globule intermediate (Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A.L., and Hartl, F.U. (1991) Nature 352, 36-42).

Animals↗
PubMed1990

Supercritical fluid extractions in biotechnology.

Over the past decade, supercritical fluid (SCF) extraction has been shown to deserve consideration as an alternative to liquid-liquid extraction or distillation. Most current commercial applications of SCF extraction involve biologically produced materials; the technique may be particularly relevant to extraction of biological compounds in cases where there is a requirement for low-temperature processing, high mass-transfer rates and negligible carry over of solvent into the final product. New advances, in which extraction is combined with reaction or crystallization steps, may further increase the attractiveness of SCFs in the bioprocessing industries.

Biotechnology↗
PubMed1988

Cholesterol aggregation and interaction with cholesterol oxidase in supercritical carbon dioxide.

High-pressure EPR spectroscopy indicates that cholesterol forms aggregates in supercritical carbon dioxide. In pure carbon dioxide, changes in cholesterol aggregate size or packing structure are observed with changing pressure. Near the critical point of carbon dioxide, cholesterol solubility is too low to permit significant aggregation, and monomeric cholesterol is observed. Addition of small amounts of dopants to supercritical carbon dioxide strongly affects cholesterol aggregation. Branched butanols (2-methyl-1-propanol and 2-methyl-2-propanol) and ethanol (to a lesser degree) promote cholesterol aggregation, while methanol, acetone, and 1-butanol do not. Cosolvents that promote aggregation also increase the rate at which cholesterol oxidase from Gloeocysticum chrysocreas catalyzes the oxidation of cholesterol. In supercritical carbon dioxide solutions, the EPR spectroscopy reveals little or no conformational change in cholesterol oxidase as 2-methyl-2-propanol or methanol is added. Damp cholesterol oxidase binds multiple cholesterol molecules; dry enzyme loses the ability to bind cholesterol. When molecular oxygen is the oxidizing agent, the rate of enzymatic cholesterol oxidation is greatly reduced in bone-dry carbon dioxide compared to that in water-saturated carbon dioxide.

3-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Stability of subtilisin and lysozyme under high hydrostatic pressure.

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.

Enzyme Stability↗
PubMedDate not supplied

Sub-micrometer-sized biodegradable particles of poly(L-lactic acid) via the gas antisolvent spray precipitation process.

Sub-micrometer-sized particles of poly(L-lactic acid) may be formed by using near-critical or supercritical carbon dioxide as an antisolvent to precipitate poly(L-lactic acid) from droplets of methylene chloride solution sprayed into a carbon dioxide continuous phase. Particle sizes may be controlled by varying the density of the carbon dioxide; at constant temperature in the supercritical region, higher carbon dioxide densities yield larger particles. Two methods (one batch and one continuous) for introducing the poly(L-lactic acid) solutions into carbon dioxide are demonstrated. Although the two methods use very different mechanisms for forming the droplets, similar particle sizes are observed as a function of carbon dioxide density. We suggest that mass transport, rather than jet breakup and hydrodynamics, controls particle sizes in the near-critical and supercritical regions.

Biodegradation, Environmental↗
PubMedDate not supplied

Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants.

We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.

Anilino Naphthalenesulfonates↗
PubMedDate not supplied

Manipulation of lyophilization-induced phase separation: implications for pharmaceutical proteins.

Lyophilization, or freeze-drying, of pharmaceutical proteins is often the only processing method that provides requisite long-term product stability. Freezing and drying, however, can cause acute damage to proteins. To alleviate damage, formulations frequently include protein stabilizers (often polymers and/or sugars), as well as buffering salts and "inert" bulking agents. While great efforts are placed on developing a formulation and suitable lyophilization cycle, incompatibilities among components through freezing and drying have been almost completely ignored. We demonstrate that solutions of poly(ethylene glycol) (PEG) and dextran, initially below critical concentrations for phase separation, do indeed experience a liquid-liquid phase separation induced by freeze concentration during the lyophilization cycle. The separation is shown to evolve with annealing at -7 degrees C and can be effectively inhibited simply by replacing NaCl with KCl in the formulation buffer. In addition, we show that phase separation causes unfolding of a model protein, recombinant hemoglobin, when freeze-dried in the PEG/dextran system. When the phase separation is averted by switching to KCl, the protein structural damage is also avoided. Measurements of pH in the frozen solutions show that the structural damage is not a result of pH changes. We suggest that KCl forms a glass with rapid cooling which kinetically prevents the phase separation and thus the protein structural damage.

Chemical Phenomena↗