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Biomedical subjects

J F Carpenter

Publications and source records attributed to J F Carpenter.

At least 37 records · Page 2Linked to original sources
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

Stability of human serum albumin during bioprocessing: denaturation and aggregation during processing of albumin paste.

PURPOSE: To assess the impact of various bioprocessing steps on the stability of freshly precipitated human serum albumin (HSA) obtained from pooled human plasma. METHODS: After initial precipitation of HSA from plasma, the resultant paste is either (a) lyophilized or (b) washed with acetone and then air-dried in order to obtain a dry powder. The structure of HSA was examined using Fourier transform infrared (IR) spectroscopy. The extent of aggregation of redissolved HSA was measured using both dynamic light scattering and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Both lyophilization and air-drying perturb the secondary structural composition of HSA, as detected by infrared (IR) spectroscopy. Upon dissolution of dried paste, most of the protein refolds to a native-like conformation. However, a small fraction of the protein molecules form soluble aggregates that can be detected by both dynamic light scattering and SDS-PAGE. The level of aggregation is so low that it could not be detected in the bulk by either circular dichroism or IR spectroscopy. The lyophilized protein, which appears to be more unfolded in the solid state than the acetone washed/air-dried material, exhibits a higher level of aggregation upon dissolution. CONCLUSIONS: There is a direct correlation between the extent of unfolding in the solid state and the amount of soluble aggregate present after dissolution. Moreover, the presence of the aggregates persists throughout the remainder of the purification process, which includes dissolution, chromatography, sterile filtration and viral inactivation steps. Analytical methods used to monitor the stability of biopharmaceuticals in the final product can be used to assess damage inflicted during processing of protein pharmaceuticals.

Freeze Drying↗
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↗
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

Use of infrared spectroscopy to assess secondary structure of human growth hormone within biodegradable microspheres.

The purpose of this study was to test the utility of infrared (IR) spectroscopy to determine protein secondary structure in biodegradable microspheres. Encapsulation of proteins within biodegradable polymers, [e.g. poly(lactic-co-glycolic acid) (PLGA)] for controlled drug release has recently been the subject of intense research effort. The ability to assess protein integrity after microsphere production is necessary to successfully produce microspheres that release native proteins. We used IR spectroscopy, a noninvasive method-as opposed to conventional organic solvent extraction or in vitro release at elevated temperature-to assess the secondary structure of recombinant human growth hormone (rhGH) within dry and rehydrated microspheres. PLGA microspheres containing rhGH with different excipients were prepared by a conventional double-emulsion method. The protein IR spectra indicated that the encapsulation process could perturb the structure of rhGH and that excipients could inhibit this damage to varying degrees. A strong positive correlation was found between intensity of the dominant alpha-helical band in the spectra of rhGH in rehydrated microspheres and the percent monomer released from microspheres during incubation in buffer. We also studied microspheres prepared with zinc-precipitated rhGH. The addition of Zn2+ during microsphere processing partially inhibited protein unfolding and fostered complete refolding of rhGH upon rehydration. In conclusion, IR spectroscopy can serve as a valuable tool to assess protein structure within both dried and rehydrated microspheres.

Delayed-Action Preparations↗
PubMed1998

Secondary structure of antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis.

Antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis are among the most active antifreeze proteins known. The Dendroides AFPs (DAFPs) consist of 6 or 7, 12- or 13-mer repeat units with a consensus sequence of -C-T-X3-S-X5-X6-C-X8-X9-A-X11-T-X13-. Nearly all of the Cys residues are in internal disulfide bridges between positions 1 and 7 within the repeats. The study presented here identified the secondary structure of the DAFPs using infrared and circular dichroism (CD) spectroscopies. The eight disulfide bridges impose significant constraints on potential secondary structural features (i.e., a number of three-residue gamma-turns) which may lead to unusual infrared and CD spectra that require special interpretation. At 25 degreesC the DAFPs contain approximately 46% beta-sheet, 39% turn, 2% helix, and 13% random structure. In the presence of ice there is a slight increase in helix and beta-sheet structures and a decrease in both turn and especially random structures. This change in the presence of ice may reflect a certain amount of flexibility in the DAFP structure. These structural changes may permit an improved lattice match between the DAFPs and ice, a requisite for the noncolligative freezing-point-depressing activity of the DAFPs.

Amino Acid Sequence↗
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

Intermolecular beta-sheet results from trifluoroethanol-induced nonnative alpha-helical structure in beta-sheet predominant proteins: infrared and circular dichroism spectroscopic study.

2,2,2-Trifluoroethanol (TFE)-induced nonnative alpha-helical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary information from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonnative alpha-helical structure in two beta-sheet-predominant proteins, beta-lactoglobulin and alpha-chymotrypsin, is unstable in comparison with those found in the alpha-helix-predominant proteins myoglobin and cytochrome c under identical conditions. IR spectra showed that, immediately after dissolution of the beta-sheet proteins in 50% (v/v) TFE, a strong amide I band component appears at 1654 cm-1 in H2O and at 1650 cm-1 in D2O, which is ascribed to alpha-helical structure. However, the intensities of the alpha-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm-1 in H2O and 1612 and 1684 cm-1 in D2O, a typical IR spectral pattern for an intermolecular beta-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the alpha-helical proteins. CD spectra suggested initially that the TFE-induced alpha-helix was retained in the gelled state. However, further analysis of the spectra, and Gaussian function modeling with basic spectra, indicated that the apparent alpha-helix signal was actually due to a combination of signals from intermolecular beta-sheet and residual alpha-helix. These results indicate that the TFE-induced nonnative alpha-helix structure in predominantly beta-sheet proteins is unstable and readily converts to an intermolecular beta-sheet aggregate.

Animals↗