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J F Carpenter

Publications and source records attributed to J F Carpenter.

At least 55 records · Page 3Linked to original sources
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

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

Stability of lipid/DNA complexes during agitation and freeze-thawing.

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Animals↗
PubMed1998

Inhibition of isoproterenol-induced tachycardia by azimilide in the isolated perfused guinea pig heart.

The class III antiarrhythmic agent, azimilide, has been shown to inhibit dihydroalprenolol binding to the beta-adrenergic receptor of rat brain and heart in an in-vitro ligand-binding assay. Azimilide, was assessed for beta-adrenergic activity, either agonist or antagonist, in the isolated perfused guinea pig heart in comparison with class III reference agents and the class II agent, propranolol. Varying concentrations of compound (0.03-100 microM) were retrogradely perfused and the effects on corrected QT interval, baseline heart rate, and isoproterenol-stimulated heart rate were measured. Propranolol, dl-sotalol, azimilide, and d-sotalol inhibited isoproterenol-induced tachycardia with IC50 values (the concentration giving 50% inhibition of isoproterenol-stimulated heart rate) of 0.12, 1.4, 14.6, and 38.0 microM, respectively. Clofilium, dofetilide, and sematilide did not affect the action of isoproterenol. Dofetilide, clofilium, azimilide, sematilide, dl-sotalol, and d-sotalol increased the QTc interval approximately 20 ms at concentrations of 0.1, 0.3, 1.0, 3.0, 30.0, and 100.0 microM, respectively. The class III antiarrhythmic agents also slowed baseline heart rate and exhibited linear R-R and QT-interval relationships of similar slope. Azimilide's antagonism of isoproterenol in this isolated heart model may reflect a direct receptor interaction or a contribution from the bradycardic action of the compound, which distinguishes it from several other pure IKr-blocking class III antiarrhythmic agents.

Animals↗
PubMed1998

The role of vitrification in anhydrobiosis.

Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

Adaptation, Biological↗
PubMed1997

Maintenance of transfection rates and physical characterization of lipid/DNA complexes after freeze-drying and rehydration.

It is well established that cationic liposomes form complexes with DNA and effectively transfect cells in vivo and ex vivo. Lipid/DNA complexes have proven safe and nonimmunogenic in clinical trials; however, they are known to aggregate readily in liquid formulations. This physical instability requires clinicians to prepare lipid/DNA complexes immediately prior to injection. In order to eliminate problems associated with this temporal requirement, we investigated the feasibility of preserving complexes as a dried preparation that could be tested, stored, and rehydrated as needed. To this end, our study evaluated the ability of different stabilizers to preserve transfection rates of complexes during acute freeze-drying stress. Our data show that complexes lyophilized in 0.5 M sucrose or trehalose possessed transfection rates similar to those of fresh preparations. In addition, dried complexes that exhibited full transfection activity upon rehydration had sizes comparable to nonlyophilized controls. Our work demonstrates that lipid/DNA complexes can be stabilized as dried powders that offer significant advantages over current liquid formulations. Furthermore, the correlation of transfection rates with maintenance of complex diameter suggests that size plays a critical role in lipid-based DNA delivery.

Animals↗
PubMed1997

Spectroscopic study of secondary structure and thermal denaturation of recombinant human factor XIII in aqueous solution.

The secondary structure and thermal denaturation (in H2O vs D2O) of recombinant human factor XIII in aqueous solutions were investigated using infrared and circular dichroism (CD) spectroscopies. The infrared amide I spectrum of the protein in H2O solution at 25 degrees C exhibited an absorbance maximum near 1642 cm-1, indicating the presence of a predominantly beta-sheet structure. Quantitative analysis revealed that the native protein contains 13-16% alpha-helix, 41-49% beta-sheet, 29% beta-turn, and 10-14% extended strand structures. The presence of a strong low-wavenumber beta-sheet band at 1641 cm-1 and a weak high-wavenumber beta-sheet band at 1689 cm-1 indicated that the beta-sheet structure of the protein is predominantly antiparallel. Quantitative analysis of the CD spectrum using the SELCON method indicated a secondary structural content of 10% alpha-helix, 40-50% beta-sheet, 20-35% beta-turns, and 20-35% unordered elements, which matches that determined by X-ray crystallography. The apparent discrepancy with the contents of unordered element determined by infrared spectroscopy is reconciled by considering that CD spectroscopy and X-ray crystallography assign extended loops and strands to unordered elements, whereas infrared spectroscopy recognizes these as distinct structured elements. During heating above 60 degrees C, a pair of new infrared bands appeared at 1626 and 1693 cm-1 for the protein in H2O and 1619 and 1683 cm-1 in D2O, indicating a formation of intermolecular beta-sheet aggregates. The intensities of the new bands increased as a function of temperature, concomitant with an intensity decrease in bands for the native protein structural elements. As expected, there was an increase in thermal stability in D2O relative to that in H2O, which was manifested as an increase of about 5 degrees C in the temperature for initial loss of infrared bands assigned to native structural elements and for appearance of bands due to intermolecular beta-sheet. In addition, the midpoint of the thermally induced transitions in infrared spectra were about 2.5 degrees C higher in D2O than in H2O. Based on the infrared analysis, the thermally denatured state of the protein in both H2O and D2O contains predominantly intermolecular beta-sheet. The broad, poorly resolved absorbance that spans the region between the intermolecular beta-sheet bands was assigned to an ensemble of heterogeneous structural elements (including unordered), none of which is populated to a high enough degree to result in a distinct infrared band. Results from CD spectroscopy support these conclusions about the structure of the denatured, aggregated protein.

Circular Dichroism↗
PubMed1997

Hydrophobic ion pairing as a method for enhancing structure and activity of lyophilized subtilisin BPN' suspended in isooctane.

The use of enzymes in low water environments permits reactions to occur that are difficult or impossible in aqueous solution. In this manner, proteases can be used to form, rather than hydrolyze, ester and amide linkages. Presumably, the native-like structure of the enzyme must remain intact for catalysis to transpire. However, little is known regarding the integrity of the overall structure of lyophilized proteins suspended in organic media. In this study, the structural changes that occur during the freeze-drying process and those effected by suspension in the organic solvent were examined. Using Fourier-transform infrared spectroscopy, the secondary structure of lyophilized subtilisin BPN' was monitored and correlated to the level of enzymatic activity when suspended in isooctane. In addition, the ability of ionic detergents to stabilize subtilisin BPN' via ion pairing was evaluated. It was found that subtilisin unfolds to some degree during lyophilization, whether it is ion paired or not. Furthermore, there are structural changes observed when the enzyme is placed in isooctane, although the effects are less with ion-paired subtilisin. This higher level of retention of secondary structure results in increased enzymatic activity.

Detergents↗
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↗
PubMed1997

Secondary structure of recombinant human cystathionine beta-synthase in aqueous solution: effect of ligand binding and proteolytic truncation.

The secondary structural composition and substrate-induced conformational changes of recombinant human cystathionine beta-synthase (CBS) in aqueous solution have been investigated in its full-length form (tetramer of 63-kDa subunits) by Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. In addition, structural comparison of a proteolytic truncated form (dimer of 45-kDa subunits) to that of the full-length enzyme has also been carried out. Second-derivative and Fourier self-deconvolutional enhanced infrared spectra revealed amide I band components ascribed to beta-sheet (1689, 1638, and 1627 cm(-1)), alpha-helix (1658 cm(-1)), beta-turn (1679 and 1668 cm(-1)), and unordered (1651 cm(-1)) structures in the spectra of the full-length enzyme. Quantitative analysis of FT-IR and CD spectra reveals that the full-length enzyme consists of about 48-53% beta-sheet, 25-30% alpha-helix, 8-10% turn, and 10-19% unordered structures. Under constraint of the spectroscopic data, theoretical prediction of locations of these secondary structural elements using Garnier's method shows that human CBS may contain a beta-sheet/alpha-helix/beta-sheet core structure. Second-derivative spectrum of the truncated enzyme exhibited all the major spectral features that are present in the full-length enzyme, indicating a preservation of the core structure of the enzyme. Significant differences were observed between the infrared spectra of the enzymes with or without the substrate, serine, indicating a substrate-induced conformational change in the enzyme, which did not result in a change in overall composition of secondary structural content based on quantitative analysis of FT-IR and far-UV CD spectra.

Amino Acid Sequence↗
PubMed1997

alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.

Adenosine Monophosphate↗
PubMed1997

Drug delivery matrix containing native protein precipitates suspended in a poloxamer gel.

Sustained delivery systems can achieve more constant blood levels of protein therapeutics than those obtained with bolus doses, leading to improved drug efficacy and fewer adverse side effects. Several different polymeric delivery systems have been studied, including poloxamers, which are unique because they can be prepared in aqueous buffers that are compatible with proteins. Poloxamers are nontoxic block copolymers of poly(ethylene oxide) and poly(propylene oxide). Certain poloxamers exhibit reversible thermal gelation. Thus, a solution of protein and poloxamer prepared at low temperatures and injected extravascularly will form a gel as it warms to body temperature. Subsequently, the protein is released slowly from the gel. To date, however, poloxamer gel delivery systems have been limited to relatively low protein concentrations (i.e., < or = 0.4 mg/mL) that produce a completely soluble protein and an optically clear gel. Much higher concentrations of other protein drugs might be needed to obtain an efficacious sustained dose. In the current in vitro study we found that a poloxamer 407 (22% wt/wt) matrix could be prepared containing tens of milligrams/mililiter of the model proteins alpha-chymotrypsin and lactate dehydrogenase. Under these conditions the protein forms a homogeneous suspension. Warming through the poloxamer 407 transition temperature (ca. 18 degrees C) results in a gel that retains a homogeneous distribution of protein precipitates for several days at 37 degrees C. Infrared spectroscopy documented that the precipitated proteins in the suspension have native secondary structure. Furthermore, the fully active protein can be recovered completely when the gel is dissolved in excess buffer. Finally, at the higher protein concentrations used to form the suspensions in poloxamer 407, protein stability during incubation at 37 degrees C was greatly improved over that seen at lower protein concentrations.

Buffers↗
PubMed1997

Real-time in situ monitoring of lysozyme during lyophilization using infrared spectroscopy: dehydration stress in the presence of sucrose.

PURPOSE: First, to investigate the role of sucrose in stabilizing protein structure (as measured by changes in the amide I band of lysozyme) caused by dehydration encountered during lyophilization. Second, to demonstrate the utility of internal reflection spectroscopy as a tool for conducting controlled lyophilization experiments. METHODS: A custom-built internal reflection FTIR accessory was used to follow the entire freeze-drying process of solutions consisting of 49.4 mg/mL lysozyme in the presence and absence of 10% sucrose in real-time. Studies were carried out using D2O as a transparent medium in the infrared region of the protein amide bands. Potential self-association of the protein in the presence of sucrose was investigated using dynamic light scattering. Hydration levels were determined using a multiple regression equation. Differential scanning calorimetry (DSC) permitted characterization of the final lyophilized product. Moisture content was determined using Karl Fischer titration. RESULTS: Throughout freezing and drying, minimal changes were observed both in frequency (1647 +/- 1 cm-1) and bandwidth (46 +/- 1 cm-1) of the amide I band in the presence of sucrose. In contrast, greater changes in frequency and band width were seen in the absence of sucrose. A successfully lyophilized cake was obtained which had properties of a glass as measured by DSC, with a Tg of 50 degree C. The lyophilized product containing sucrose had 4% moisture by weight. Three distinct rates of water desorption were discovered during drying under vacuum (50 mg/hr within the sample temperature range from -35 degrees to -25 degrees C; 30 mg/hr from -10 degrees to 25 degrees C; 1.2 mg/hr from 27 degrees to 38 degrees C). CONCLUSIONS: The inclusion of sucrose served to minimize perturbations of protein structure caused by freezing and dehydration stresses encountered during lyophilization (compared to studies conducted in absence of sucrose). The results support the water replacement hypothesis and underscore the role of the sugar in preserving a native structure in the dried state. This investigation demonstrates the usefulness of infrared spectroscopy in evaluating lyophilization process parameters and formulation design.

Freeze Drying↗
PubMed1996

Effect of secondary structure on the activity of enzymes suspended in organic solvents.

Despite the extensive use and study of enzymes suspended in organic solvents, whether activity differences between different preparations can be accounted for by differences in protein secondary structure is still unknown. To address this issue, in the current study two model enzymes, alpha-chymotrypsin and subtilisin Carlsberg, were lyophilized and suspended in both polar and nonpolar organic solvents. The secondary structures of the proteins in the initial aqueous solution, in the lyophilized powder, and in the subsequent suspensions in organic solvents were determined using infrared spectroscopy. Lyophilization perturbed the secondary structure of both enzymes. With alpha-chymotrypsin, lyophilization from buffer followed by suspension in ethanol, hexane, or pyridine did not alter the unfolded structure observed in the dried powder. In contrast, with subtilisin Carlsberg, suspension of the dried enzyme in ethanol led to further perturbation of structure, whereas in hexane, and more so in pyridine, there was some return toward native structure. Lyophilization of the aqueous protein solutions in the presence of either trehalose or sorbitol led to retention of more native-like structure of both enzymes in the dried solid. However, large structural perturbations arose when these samples were suspended in organic solvents. The only exception was the subtilisin-trehalose mixture, which regained some native structure in ethanol and hexane. The greatest changes were noted in samples suspended in pyridine, in which the infrared spectra indicated extensive intermolecular beta-sheet formation from protein aggregates. There was not any consistent correlation between activity in organic solvents and either the initial structure obtained in the dried powders or the final structure when suspended in organic solvents. Nor could differences in residual water contents in dried samples or the total water content in the organic solvent reaction system account for the activity differences.

Chymotrypsin↗
PubMed1996

Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state.

Enzymes subjected to freeze-thawing are known to be protected by polymers that are preferentially excluded from the hydrated surface of proteins [reviewed in Carpenter et al. (1994) ACS Symp. Ser. 567, 134-147]. Preferentially excluded solutes are also known to stabilize quaternary structure, which enhances the thermostability of multimeric proteins in aqueous systems. Also, it has been suggested that retention of quaternary structure may play a role in the protection of multimeric proteins by polymers during freeze-drying (lyophilization). Although preferential solute exclusion cannot occur in the absence of water, we reasoned that polymers could protect multimeric proteins during freeze-drying by stabilizing quaternary structure in the frozen state. Our results are consistent with this hypothesis and demonstrate that bovine serum albumin and polyvinylpyrrolidone stabilize lactate dehydrogenase by inhibiting dissociation in the frozen solution, during the initial phase of the sublimation step of lyophilization. Dissociation at this critical step correlated directly with decreased recovery of enzyme activity after rehydration. The damage to the protein, under conditions where dissociation was studied, was due to a large decrease in pH in the frozen state (e.g., from pH 7.5 to 4.5), which was attenuated by protective levels of polymers. Thus, inhibition of freezing-induced pH shifts, in addition to stabilization by the preferential exclusion mechanism, plays an important role in the protection conferred by polymers. Furthermore, high concentrations of these polymers were capable of maintaining quaternary structure during subsequent drying and rehydration. We suggest that the proximate cause for increased recovery of active, native protein after lyophilization is that the holoenzyme is more resistant to the stresses of drying/rehydration than unassociated monomers.

Animals↗
PubMed1996

Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation.

The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56 degrees C, during several weeks of storage at temperatures above and below the samples' Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 +/- 2.5 degrees C. With infrared spectroscopy, we determined that the protein lyophilized with </=1% sucrose was unfolded in the initial dried solid. In contrast, in those formulations with >/=5% sucrose, conformational change was inhibited during lyophilization. When stored at 50 degrees C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation.

Amides↗